cyclin-d1 and Lung-Neoplasms

Our purpose was to provide data regarding relationships between. MEDLINE library was screened for associations between PET parameters and histopathological features in lung cancer up to December 2017. Only papers containing correlation coefficients between PET parameters and histopathological findings were acquired for the analysis. Overall, 40 publications were identified.. SUV may predict microvessel density and expression of VEGF, KI 67, and HIF-1

Topics: B7-H1 Antigen; Cyclin D1; Fluorodeoxyglucose F18; Gene Expression Regulation, Neoplastic; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Ki-67 Antigen; Lung Neoplasms; Microvessels; Positron-Emission Tomography; Proliferating Cell Nuclear Antigen; Tumor Suppressor Protein p53

Evidence indicates CCND1 G870A polymorphisms as a risk factor for a number of cancers. Increasing studies have been conducted on the association of CCND1 G870A polymorphism with lung cancer risk. However, the results were controversial. The aim of the present study was to derive a more precise estimation of the relationship. Meta-analyses examining the association between CCND1 G870A polymorphism and lung cancer were performed. Subgroup analyses regarding ethnicity, smoking status, histological types and source of controls were also implemented. All eligible studies for the period up to May 2012 were identified. The overall data from ten case-control studies including 5,008 cases and 5,214 controls indicated that variant A allele may have an association with increased lung cancer risk (AA vs GG: OR = 1.21; 95 % CI = 1.08-1.36, dominant model: OR = 1.09; 95 % CI = 1.00-1.19, recessive model: OR = 1.23; 95 % CI = 1.01-1.49). In the subgroup analysis by ethnicity, A allele may elevate lung cancer risk among Asians but not Caucasians or Mixed ethnicities. In smoking status subgroup, A allele was shown to associate with increased lung cancer risk among smokers but not non-smokers. In the subgroup analysis by histological types, increased cancer risks were shown in adenocarcinoma but not squamous cell carcinoma, under the homozygote comparison and recessive models. Collectively, the results of the present study suggest that CCND1 G870A polymorphism might be a low-penetrant risk factor for lung cancer, particularly among Asians and smokers. Moreover, homozygous AA alleles might have a correlation with increased lung adenocarcinoma susceptibility.

Topics: Alleles; Case-Control Studies; Cyclin D1; Genetic Predisposition to Disease; Genotype; Humans; Lung Neoplasms; Odds Ratio; Polymorphism, Genetic; Publication Bias; Risk; Smoking

Several studies have investigated the association between Cyclin D1 (CCND1) G870A genetic polymorphism and lung cancer susceptibility, but the results were inconclusive. The aim of this meta-analysis was to summarize available evidence for such a relationship. The reviewers made use of MEDLINE, EMBASE, and BIOSIS databases. The relevant data were independently extracted by two reviewers. The odds ratio (OR) with 95% confidence interval (CI) was selected as the principal outcome measure. The heterogeneity test, the publication bias test, and the sensitivity analysis were performed. Overall, a total of 10 case-control studies were included. Our meta-analysis indicated that CCND1 G870A genetic polymorphism was a risk factor for lung cancer under homozygote model (OR = 1.18; 95% CI = 1.02, 1.37), recessive model (OR = 1.21; 95% CI = 1.03, 1.41), and allele model (OR = 1.11; 95% CI = 1.02, 1.21). In the subgroup analysis by source of ethnicity, a statistical increase of lung cancer risk was found among Asian groups for allele model (OR = 1.11; 95% CI = 1.01-1.22). The present meta-analysis suggests that CCND1 G870A polymorphism may be a risk factor for lung cancer. Besides, allele A may contribute to increased lung cancer risk.

Topics: Asian People; Case-Control Studies; Cyclin D1; Gene Frequency; Genetic Predisposition to Disease; Genotype; Humans; Lung Neoplasms; Odds Ratio; Polymorphism, Single Nucleotide; Risk Factors; White People

T cell adequate function is critical for defense against pathogens. Transient disruption of T cell homeostasis occurs when primarily naive cells undergo antigen-driven expansion and acquire effector functions. Effector T cells then either undergo programmed cell death (PCD, it occurs usually as massive apoptosis during the contraction phase of the immune response) or survive to become memory cells. Two main pathways of effector T cell PCD are discussed in the review: activation induced cell death (AICD), which is a form of extrinsic apoptosis, and neglect-induced death (NID), which is an intrinsic one. Initial studies using in vitro models supported a role of AICD, mostly initiated by TCR receptor triggering in immune contraction. However, it was not finally supported by either recent in vivo experiments or current review authors' clinical studies concerning primed T cell apoptosis in chronic inflammatory lower airway diseases. Actually, Bcl-2 family members seem to be critical for the culling of T cell responses. The antiapoptotic molecule Bcl-2 and its proapoptotic antagonist Bcl-2, both under upstream control of autocrine interleukin-2, are the most probable candidates for regulators of T cell contraction. Other possible mechanisms regulating the process of contraction such as death receptor ligation, the impact of cytokines, as well as the importance of transcription factor NF-κB, are discussed. Additionally, attention is turned to the potential role of T cell survival/apoptosis regulation in future therapies of some diseases, including tumors and lung fibrosis.

Topics: Apoptosis; Apoptosis Regulatory Proteins; Cell Death; Cyclin D1; Humans; Interleukin-2; Lung Neoplasms; NF-kappa B; Pulmonary Disease, Chronic Obstructive; Pulmonary Fibrosis; Signal Transduction; T-Lymphocytes

Many studies have investigated the association between Cyclin D1 (CCND1) G870A polymorphism and lung cancer risk, but the impact of CCND G870A polymorphism on lung cancer is unclear owing to the obvious inconsistence among those studies. This study aimed to quantify the strength of association between CCND1 G870A polymorphism and lung cancer risk. We searched the PubMed, Embase, and Wangfang databases for articles on studies relating the CCND1 G870A polymorphism to the risk of lung cancer in humans. We estimated summary odds ratios (ORs) with their confidence intervals (CIs) to assess the association. Meta-analyses of total studies showed that CCND1 G870A polymorphism was associated with lung cancer risk under three genetic models (OR(A versus G) = 1.13, 95 % CI 1.03-1.24; OR(AA versus GG) = 1.20, 95 % CI 1.07-1.35; OR(AA versus AG + GG) = 1.23, 95 % CI 1.02-1.50). Meta-analyses of studies with high quality showed that CCND1 G870A polymorphism was associated with lung cancer risk under two genetic models (OR(A versus G) = 1.08, 95 % CI 1.02-1.15; OR(AA versus GG) = 1.17, 95 % CI 1.04-1.32). Subgroup analyses by ethnicity and sensitivity analyses further identified the significant association above. No evidence of publication bias was observed. Meta-analyses of available data show a significant association between the CCND1 G870A polymorphism and lung cancer risk, and CCND1 G870A polymorphic variant A contributes to increased risk of lung cancer.

Topics: Alleles; Cyclin D1; Genetic Predisposition to Disease; Genotype; Humans; Lung Neoplasms; Odds Ratio; Polymorphism, Single Nucleotide; Publication Bias; Risk

Cyclin D1 (CCND1) is critical in the transition of the cell cycle from G1 to S phases and unbalanced cell cycle regulation is a hallmark of carcinogenesis. A number of studies conducted to assess the association between CCND1 G870A polymorphism and susceptibility to lung cancer have yielded inconsistent and inconclusive results. In the present study, the possible association above was assessed by a meta-analysis.. Eligible articles were identified for the period up to November 2011. Pooled odds ratios (OR) with 95% confidence intervals (95%CI) were appropriately derived from fixed effects or random-effects models. Sensitivity analysis excluding studies whose genotype frequencies in controls significantly deviated from the Hardy-Weinberg equilibrium (HWE) was performed.. Ten case-control studies with a total of 10,548 subjects were eligible. At the overall analysis the CCND1 870A allele appeared to be associated with elevated lung cancer risk (for allele model, pooled OR=1.24, 95% CI: 1.08-1.44, P=0.004; for homozygous model, pooled OR=1.45, 95% CI: 1.14-1.84, P=0.003; for recessive model, pooled OR=1.29, 95% CI: 1.06-1.58, P=0.013; for dominant model, pooled OR=1.33, 95% CI: 1.08-1.65, P=0.009). Subgroup analyses by ethnicity and sensitivity analysis further pointed to associations, particularly in Asians.. This meta-analysis suggests that the A allele of CCND1 G870A polymorphism confers additional lung cancer risk.

Topics: Alleles; Case-Control Studies; Cyclin D1; Humans; Lung Neoplasms; Polymorphism, Genetic; Risk Factors

To review the evidence implicating the deregulation of cyclin D1 in the pathogenesis of non-small cell lung cancer (NSCLC), and to discuss the opportunities for targeted clinical intervention.. Data published until June 2006 are summarized, and previously unpublished results from our own research are included.. In normal cells, cyclin D1 complexes with and activates cyclin-dependent kinases (CDK) and acts as a transcriptional regulator. The protein is frequently overexpressed in a wide range of cancers, sometimes coincident with CCND1 (cyclin D1) gene amplification (5-20% of tumours). A low level of somatic mutations have been seen in certain tumours. CCND1 is amplified in NSCLC and cyclin D1 is frequently overexpressed in tumours and pre-invasive bronchial lesions, generally from one parental allele. Mutation analyses revealed a frequent CCND1 gene polymorphism (A870G) that modulates alternative splicing and allows expression of an alternative cyclin D1 transcript (transcript cyclin D1b). The encoded cyclin D1b protein lacks a specific phosphorylation site required for nuclear export. Genotype has been correlated with the risk and/or severity of disease or drug response across a range of malignancies, including lung cancer. Together, these findings suggest a strong pathological role for cyclin D1 deregulation in bronchial neoplasia.. Current data indicate that cyclin D1 overexpression is not a consequence of, but rather a pivotal element in the process of malignant transformation in the lung and other tissues. This understanding may open new avenues for lung cancer diagnosis, treatment and prevention.

Topics: Adenoma; Carcinoma, Non-Small-Cell Lung; Cell Transformation, Neoplastic; Cyclin D1; Gene Expression Regulation, Neoplastic; Glioblastoma; Humans; Lung Neoplasms; Polymorphism, Genetic

Lung cancer remains interdisciplinary problem. The genetic alterations in non-small cell lung cancer (NSCLC) are related to tumor suppressor genes and proto-oncogenes. CCND1 gene, coding cyclin DI, in correlation with pRb is involved in regulation of cell cycle arrest in G1 phase. Amplification of CCND1 gene and cyclin D1 over-expression was found in several cancers including head and neck cancers or colorectal cancer, where these alterations were correlated with worse prognosis. The literature addressing the clinical significance of CCND1 gene amplification/expression in NSCLC remains poor and prognostic value of these alterations in that cancer is still controversial.

Topics: Biomarkers, Tumor; Carcinoma, Non-Small-Cell Lung; Cell Cycle Proteins; Cyclin D1; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Neoplasm Staging; Prognosis; Proto-Oncogene Proteins

Lung cancer is the leading cause of cancer mortality. Chemoprevention is an attractive strategy to combat this major public health problem. Pre-clinical and clinical studies have identified diverse candidate chemopreventive agents that affect cellular proliferation, differentiation, apoptosis and tumor angiogenesis, among other pathways. These pharmacological agents are undergoing testing through use of pre-clinical models and clinical trials. These studies have uncovered cyclin D1 as a chemoprevention target and a surrogate marker of chemopreventive response in the lung. Chemoprevention of tobacco-carcinogen transformed human bronchial epithelial (HBE) cells appears to be due at least partly to degradation of cyclin D1. These studies of cultured HBE cells were extended to the in vivo setting by examination of preneoplastic bronchial lesions that established the frequent aberrant expression of cyclin D1 in lung carcinogenesis. Certain retinoids, natural and synthetic derivatives of vitamin A, repress cyclin D1, but activation of the epidermal growth factor receptor (EGFR) induces cyclin D1. Retinoids and specific chemopreventive agents can activate the proteasome-dependent degradation of cyclin D1 and also repress EGFR expression, thereby reducing cyclin D1 levels. These actions oppose the mitogenic effects of cyclin D1. This is hypothesized to trigger G1 arrest and thereby permit repair of carcinogenic damage of genomic DNA. These and other pre-clinical and clinical studies that will be reviewed here indicate that cyclin D1 and perhaps other cyclins are attractive pharmacological targets for lung cancer chemoprevention.

Topics: Cell Transformation, Neoplastic; Chemoprevention; Cyclin D1; DNA Damage; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Retinoids

Topics: Biomarkers, Tumor; Carcinoma, Non-Small-Cell Lung; Cell Cycle; Cyclin D1; Cyclin E; Cyclin-Dependent Kinase Inhibitor p16; Humans; Lung Neoplasms; Prognosis; Retinoblastoma Protein

Topics: Animals; Cell Cycle; Cell Cycle Proteins; Cell Differentiation; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p27; Genes, p16; Genes, p53; Genes, Retinoblastoma; Humans; Lung Neoplasms; Tumor Suppressor Proteins

Studies on cell cycle regulation and cancer genetics have revealed that multiple cell cycle regulatory proteins play key roles in oncogenesis. These can be categorized in three sets. First; p16INK4-Cyclin D1-RB pathway, which controls G1 to S progression of the cell cycle, second; p53 pathway, which is involved in DNA damage repair, and third; p27KIP1 CDK inhibitor, a negative regulator of cell cycle, and decreased expression of which has been correlated to poor prognosis in cancer patients. Among these, p16INK4, RB and p53 are tumor suppressor genes, and p27 has been pointed out to be haplo-insufficient for tumor suppression. Involvement of these cell cycle regulatory proteins in lung cancer will be discussed.

Topics: Animals; Cell Cycle; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p16; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Genes, p53; Humans; Lung Neoplasms; Microfilament Proteins; Muscle Proteins; Mutation; Retinoblastoma Protein; Signal Transduction

An increasing number of reports have shown that diverse microRNAs are involved in tumorigenesis and tumor progression. We performed this study to identify novel miRNAs that may be involved in lung cancer and study on their functions. We tested the expression of 450 miRNAs in lung tumor tissues and adjacent non-cancerous tissues. We found that miRNA-545 was less abundant in cancerous lung tissues than in adjacent non-cancerous tissues. Our further studies showed that miR-545 suppressed cell proliferation in vitro and in vivo. We also found that miR-545 caused cell cycle arrest at the G0/G1 phase and induced cell apoptosis in lung cancer cells by targeting cyclin D1 and CDK4 genes. The effects of cyclin D1 and CDK4 down-regulated by miR-545 were similar to those caused by siRNAs of cyclin D1 and CDK4, and overexpression of cyclin D1 and CDK4 could abolish the miR-545-induced inhibition of cell proliferation. In conclusion, miR-545 suppressed cell proliferation by inhibiting the expression of cyclin D1 and CDK4. Our findings provide new knowledge regarding the role of miR-545 in the development of lung cancer and indicate the potential application of miR-545 in cancer therapy.

Topics: Animals; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase 4; Female; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Male; Mice; Mice, Inbred BALB C; Mice, Nude; MicroRNAs; RNA, Neoplasm

This phase II study investigated dose-intense erlotinib maintenance after dose-dense chemotherapy for patients with metastatic non-small cell lung cancer and examined two cell cycle biomarkers. Patients with newly diagnosed metastatic non-small cell lung cancer received docetaxel 75 mg/m² and cisplatin 75 mg/m² on day 1 and pegfilgrastim on day 2 every 14 days for four cycles. Patients then received erlotinib with initial doses based on smoking status. Doses were increased in 75 mg increments every two weeks depending on toxicities until each patient's maximal tolerable dose (MTD) was achieved. Cyclin D1 and D3 biomarkers were measured by immunohistochemistry. The objectives of the study were to evaluate time to progression (TTP) and overall survival (OS) for the entire population and biomarker subgroups. Forty-five patients were enrolled. Intra-patient erlotinib MTD ranged from 0 to 525 mg. Median MTD achieved in smokers was higher than in non-smokers (300 vs. 150 mg; P=0.019). TTP for the entire cohort was not significantly improved compared to historical controls. Patients with high cyclin D1 expressing tumors demonstrated improved TTP on erlotinib (8.2 vs. 4.7 months; hazard ratio, 4.1; 95% CI, 1.6-0.6; P=0.003) and improved OS (20.5 vs. 8.0 months; hazard ratio 2.8; 95% CI, 1.2-6.3; P=0.016). Intratumoral cyclin D3 expression did not impact clinical outcomes. Current smokers but not former smokers exhibit a higher erlotinib MTD. High cyclin D1 expression was associated with favorable TTP and OS.

Topics: Adult; Aged; Aged, 80 and over; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Biomarkers, Tumor; Carcinoma, Non-Small-Cell Lung; Cisplatin; Cyclin D1; Cyclin D3; Disease-Free Survival; Docetaxel; Drug Administration Schedule; ErbB Receptors; Erlotinib Hydrochloride; Female; Filgrastim; Granulocyte Colony-Stimulating Factor; Humans; Lung Neoplasms; Male; Middle Aged; Polyethylene Glycols; Protein Kinase Inhibitors; Quinazolines; Recombinant Proteins; Survival; Taxoids; Treatment Outcome

ABT-751 is a novel antimitotic agent that exerted cytotoxic effects in preclinical studies. Carboplatin has efficacy in treating advanced non-small cell lung cancer (NSCLC) in combination with other drugs.. Lung cancer cell lines were treated with ABT-751 and/or carboplatin to investigate their impact on cell growth. A phase I study with an expansion cohort was conducted in previously treated NSCLC patients. The primary objective was the maximum tolerated dose (MTD); secondary objectives were objective response rates, median survival, time to tumor progression, dose-limiting toxicities (DLTs), and pharmacodynamic evaluation of buccal swabs.. Combining ABT-751 with carboplatin significantly reduced growth and induced apoptosis of lung cancer cell lines. Twenty advanced NSCLC patients were enrolled. MTD was ABT-751 125 mg orally twice daily for 7 days with carboplatin AUC 6. DLTs included fatigue, ileus, neutropenia and pneumonitis. Two patients had confirmed partial responses. Median overall survival was 11.7 months (95% CI 5.9-27.0). Time to tumor progression was 2.8 months (95% CI 2.0-2.7). Four of 6 patients showed decreased cyclin D1 protein in posttreatment versus pretreatment buccal swabs.. Combining ABT-751 with carboplatin suppressed growth of lung cancer cell lines and had modest clinical antitumor activity in advanced NSCLC previously treated predominantly with carboplatin. Further studies of this combination are not recommended while investigations of biomarkers in different patient populations, alternative schedules and combinations may be pursued.

Topics: Administration, Oral; Aged; Antineoplastic Agents; Apoptosis; Area Under Curve; Carboplatin; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cyclin D1; Drug Therapy, Combination; Fatigue; Female; Humans; Ileus; Lung Neoplasms; Male; Middle Aged; Neutropenia; Pneumonia; Sulfonamides; Survival Rate

The rexinoid bexarotene represses cyclin D1 by causing its proteasomal degradation. The epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) erlotinib represses cyclin D1 via different mechanisms. We conducted a preclinical study and 2 clinical/translational trials (a window-of-opportunity and phase II) of bexarotene plus erlotinib. The combination repressed growth and cyclin D1 expression in cyclin-E- and KRAS/p53-driven transgenic lung cancer cells. The window-of-opportunity trial in early-stage non-small-cell lung cancer (NSCLC) patients (10 evaluable), including cases with KRAS mutations, repressed cyclin D1 (in tumor biopsies and buccal swabs) and induced necrosis and inflammatory responses. The phase II trial in heavily pretreated, advanced NSCLC patients (40 evaluable; a median of two prior relapses per patient (range, 0-5); 21% with prior EGFR-inhibitor therapy) produced three major clinical responses in patients with prolonged progression-free survival (583-, 665-, and 1,460-plus days). Median overall survival was 22 weeks. Hypertriglyceridemia was associated with an increased median overall survival (P = 0.001). Early PET (positron emission tomographic) response did not reliably predict clinical response. The combination was generally well tolerated, with toxicities similar to those of the single agents. In conclusion, bexarotene plus erlotinib was active in KRAS-driven lung cancer cells, was biologically active in early-stage mutant KRAS NSCLC, and was clinically active in advanced, chemotherapy-refractory mutant KRAS tumors in this study and previous trials. Additional lung cancer therapy or prevention trials with this oral regimen are warranted.

Topics: Aged; Animals; Antineoplastic Combined Chemotherapy Protocols; Bexarotene; Biomarkers, Tumor; Carcinoma, Non-Small-Cell Lung; Cyclin D1; Drug Resistance, Neoplasm; ErbB Receptors; Erlotinib Hydrochloride; Female; Humans; Immunoblotting; Immunoenzyme Techniques; Lung Neoplasms; Male; Mice; Mice, Transgenic; Middle Aged; Mouth Mucosa; Mutation; Necrosis; Neoplasm Recurrence, Local; Proto-Oncogene Proteins; Proto-Oncogene Proteins p21(ras); Quinazolines; ras Proteins; Salvage Therapy; Survival Rate; Tetrahydronaphthalenes; Treatment Outcome; Tumor Cells, Cultured

To compare the diagnostic efficacies of (18)F-FLT and (18)F-FDG PET/CT in non-small-cell lung cancer (NSCLC), focusing on the correlation between FLT and FDG tumour uptake and tumour cell proliferation as indicated by the cyclin D1 labelling index.. A total of 31 patients with NSCLC underwent FLT and FDG PET/CT scanning followed by surgery. PET/CT images were compared with the pathology. Tumour cell proliferation was assessed by cyclin D1 immunohistochemistry.. The sensitivities of FLT and FDG PET/CT for the primary lesion were 74% and 94%, respectively (p=0.031). For N staging, 77% patients were correctly staged, 6% overstaged, 16% understaged by FLT, while the values for FDG were 77%, 16% and 6%, respectively. The sensitivity, specificity, accuracy, and positive predictive value with FLT for lymph nodes were 65%, 98%, 93% and 89%, respectively, and 85%, 84%, 84% and 52% with FDG (p<0.01).Tumour SUV of FLT was significantly correlated with the cyclin D1 labelling index (r=0.644; p<0.01), but the SUV of FDG was not significantly correlated (r=0.293; p>0.05).. In terms of N staging, FLT PET/CT resulted in understaging of more patients but overstaging of fewer patients, and for regional lymph nodes showed better specificity, accuracy and positive predictive value than FDG PET/CT in NSCLC. Tumour FLT uptake was correlated with tumour cell proliferation as indicated by the cyclin D1 labelling index, suggesting that further studies are needed to evaluate the use of FLT PET/CT for the assessment of therapy response to anticancer drugs.

Topics: Adult; Aged; Aged, 80 and over; Biological Transport; Carcinoma, Non-Small-Cell Lung; Cell Proliferation; Cyclin D1; Dideoxynucleosides; Female; Fluorodeoxyglucose F18; Humans; Immunohistochemistry; Lung Neoplasms; Male; Middle Aged; Neoplasm Staging; Positron-Emission Tomography; Tomography, X-Ray Computed

The epidermal growth factor receptor (EGFR) and cyclin D1 are overexpressed in lung carcinogenesis. The rexinoid, bexarotene, represses cyclin D1 and EGFR expression in vitro. It was hypothesized that combining bexarotene with the EGFR inhibitor, erlotinib, would augment clinical activity.. In vitro studies and a phase I clinical trial were performed. Twenty-four patients with advanced aerodigestive tract cancers were enrolled; 79% had non-small-cell lung cancer (NSCLC). The primary objective was to determine the maximum-tolerated dose. Clinical activity was a secondary objective.. Combining erlotinib with bexarotene enhanced growth suppression in vitro compared with each single-agent treatment. This cooperatively repressed cyclin D1 expression. Clinically, the most frequent toxicities were mild hypertriglyceridemia and skin rash. Two serious treatment-related adverse events occurred (creatine phosphokinase elevation attributed to antilipid therapy and a case of generalized pain). Five objective responses (four partial and one minor) were observed in NSCLC patients. Responses were observed in males and smokers. EGFR sequence analyses did not reveal activating mutations in tumors from assessable responding patients. Median time to progression was 2.0 months; overall survival time was 14.1 months; and 1-year survival rate was 73.8%.. The recommended phase II doses are erlotinib 150 mg/d and bexarotene 400 mg/m2/d orally. These agents can be administered in combination at the recommended single-agent doses without added toxicity. Overall survival and clinical features of responding patients differ from prior reports of single-agent erlotinib treatment. These findings are encouraging and warrant further investigation of this regimen.

Topics: Aged; Aged, 80 and over; Antineoplastic Combined Chemotherapy Protocols; Bexarotene; Biomarkers, Tumor; Carcinoma, Non-Small-Cell Lung; Cell Proliferation; Cell Survival; Cyclin D1; Digestive System Neoplasms; ErbB Receptors; Erlotinib Hydrochloride; Exanthema; Female; Head and Neck Neoplasms; Humans; Hypertriglyceridemia; Lung Neoplasms; Male; Middle Aged; Mouth Mucosa; Protein Kinase Inhibitors; Quinazolines; Sequence Analysis, DNA; Tetrahydronaphthalenes; Treatment Outcome; Tumor Cells, Cultured

This study aims to investigate cordycepin's effects on the proliferation, migration, and invasion of retinoblastoma (RB) cells and its regulatory mechanism. In this study, it was found that cordycepin inhibited RB cell proliferation, migration, and invasion in vitro, and pulmonary metastasis in vivo. c-Myc was a downstream target of cordycepin, and cordycepin significantly suppressed c-Myc expression, and c-Myc overexpression markedly counteracted the impacts of cordycepin on RB cell proliferation, migration, and invasion. c-Myc was positively correlated with the cell cycle pathway. Cordycepin restrained cyclin D1 expression, and c-Myc overexpression rescued this effect. In conclusion, cordycepin targets the c-Myc/cyclin D1 pathway, thereby suppressing the malignant biological behaviors of RB cells.

Topics: Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin D1; Humans; Lung Neoplasms; Retinal Neoplasms; Retinoblastoma

Topics: Animals; Apoptosis; Carcinoma, Non-Small-Cell Lung; Cell Proliferation; Cisplatin; Cyclin D1; Disease Models, Animal; Drug Resistance, Neoplasm; Humans; Lung Neoplasms; MicroRNAs

Ketamine, a N-methyl-D-aspartate (NMDA) receptor antagonist, is commonly used to induce anaesthesia during cancer surgery and relieve neuropathic and cancer pain. This study was conducted to assess whether ketamine has any inhibiting effects on neuroglioma (H4) and lung cancer cells (A549) in vitro. The cultured H4 and A549 cells were treated with ketamine and MK801 (0.1, 1, 10, 100, or 1000 μM) for 24 h. The expressions of glutamate receptors on both types of cancer cells were assessed with qRT-PCR. In addition, cell proliferation and migration were assessed with cell counting Kit-8 and wound healing assays. Cyclin D1, matrix metalloproteinase 9 (MMP9), phosphorylation of extracellular signal-regulated kinase (pERK), and cleaved-caspase-3 expression together with reactive oxygen species (ROS) were also assessed with Western blot, immunostaining, and/or flowcytometry. NMDA and α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors were expressed on both H4 and A549 cells. Ketamine inhibited cancer cell proliferation and migration in a dose-dependent manner by suppressing the cell cycle and inducing apoptosis. Ketamine decreased cyclin D1, pERK, and MMP9 expression. In addition, ketamine increased ROS and cleaved caspase-3 expression and induced apoptosis. The anti-cancer effect of ketamine was more pronounced in A549 cells when compared with H4 cells. MK801 showed similar effects to those of ketamine. Ketamine suppressed cell proliferation and migration in both neuroglioma and lung cancer cells, likely through the antagonization of NMDA receptors.

Topics: Apoptosis; Caspase 3; Cyclin D1; Dizocilpine Maleate; Humans; Ketamine; Lung Neoplasms; Matrix Metalloproteinase 9; N-Methylaspartate; Reactive Oxygen Species; Receptors, N-Methyl-D-Aspartate

To investigate the effect of licochalcone A (LCA) on the proliferation and cell cycle of human lung squamous carcinoma cells and explore its possible molecular mechanism.. LCA is capable of inhibiting the proliferation and inducing cell cycle arrest in lung squamous carcinoma cells possibility by regulating the PI3K/Akt singling pathway.

Topics: Animals; Carcinoma, Non-Small-Cell Lung; Carcinoma, Squamous Cell; Cell Cycle Checkpoints; Cyclin D1; Humans; Lung; Lung Neoplasms; Mice; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Signal Transduction

Inhibition of Serum Amyloid A-like 1 (SAAL1) expression could inhibit cancer progression and improve the prognosis of cancer patients. At present, the correlation between SAAL1 and lung adenocarcinoma (LAC) remains unclear. Therefore, this study surveyed the worth and pathway of SAAL1 in LAC progression and immunity.. Bioinformatics and immunohistochemistry were used to identify the SAAL1 expression in LAC. The roles of SAAL1 expression in the existence values of LAC patients were explored, and the nomograms were constructed. Clinical values of SAAL1 co-expressed genes were evaluated by COX regression, survival, and Receiver operating characteristic (ROC) analysis. EDU and western blotting methods were used to inquiry the functions and pathways of the SAAL1 in cell growths. The correlation between the SAAL1 level and immune microenvironment was visualized using correlation research.. SAAL1 level was elevated in LAC tissues, and was observed in cancer tissues of dead patients. SAAL1 overexpression had something to do with shorter overall survival, progression-free interval, and disease-specific survival in LAC. The area under the curve of SAAL1 was 0.902 in normal tissues and cancer tissues. Inhibition of SAAL1 expression could inhibit cancer cell proliferation, which may be related to the decreased expression of cyclin D1 and Bcl-2 proteins. In LAC, SAAL1 level had something to do with stromal, immune, and estimate scores, and correlated with macrophages, T cells, Th2 cells, CD8 T cells, NK CD56dim cells, DC, eosinophils, NK CD56bright cells, pDC, iDC, cytotoxic cells, Tgd, aDC cells, B cells, Tcm, and TFH levels. SAAL1 overexpression had something to do with existence values and the immunity in LAC.. Inhibition of SAAL1 expression could regulate cancer growth via cyclin D1 and Bcl-2. SAAL1 is a promising prognostic biomarker in LAC patients.

Topics: Adenocarcinoma of Lung; Biomarkers, Tumor; Cyclin D1; Humans; Lung Neoplasms; Prognosis; Tumor Microenvironment

To explore the mechanism of circular RNA (circRNA)-AnnexinA7 (ANXA7) in non-small cell lung cancer (NSCLC) cisplatin (DDP) resistance through microRNA (miR)-545-3p to target Cyclin D1 (CCND1).. DDP-resistant and non-resistant NSCLC tissues and normal tissues were collected. DDP-resistant cells (A549/DDP and H460/DDP) were constructed. circ-ANXA7, miR-545-3p, CCND1, P-Glycoprotein, and glutathione S-transferase-π in tissues and cells were measured. Analysis of circ-ANXA7 ring structure was performed, as well as detection of circ-ANXA7 distribution in cells. Cell proliferation was detected by MTT and colony formation assay, apoptosis rate was detected by flow cytometry, and cell migration and invasion were evaluated by Transwell assay. The targeting relationship between circ-ANXA7, miR-545-3p and CCND1 was verified. Measurement of tumor volume and quality in mice was performed.. Circ-ANXA7 and CCND1 were elevated, while miR-545-3p was suppressed in DDP-resistant NSCLC tissues and cells. Circ-ANXA7 combined with miR-545-3p, which targeted CCND1 to expedite A549/DDP cell proliferation, migration, invasion, DDP resistance, but inhibited cell apoptosis.. Circ-ANXA7 enhances DDP resistance in NSCLC via absorbing miR-545-3p to target CCND1 and might be a latent therapeutic target for NSCLC.

Topics: Animals; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Proliferation; Cisplatin; Cyclin D1; Drug Resistance, Neoplasm; Lung Neoplasms; Mice; MicroRNAs; RNA, Circular

Non-small-cell lung carcinoma (NSCLC) is a type of epithelial lung cancer accounting for about 85% of all lung cancers. In our research, a novel lupene derivative namely acetoxy-lup-5(6), 20(29)-diene (ALUP), as well as two known triterpenes; lupeol (LUP) and betulinic acid (BA) were isolated through the chromatographic purification of the 95% ethanolic extract of Thymus capitatus. Identification of the compounds was carried out by physicochemical properties as well as spectral 1D and 2D NMR analysis. The anti-cancer activity of the three triterpenes was assessed on non-small cell lung cancer cell line; A549 using MTT assay and cell cycle analysis using annexin V/propidium iodide. The molecular mechanism underlying anti-apoptotic effects was determined by analyzing Let-7 miRNA and miRNA-21 expression, the mRNA gene expression level of Bax, CASP-8, CD95, Bcl2, KRAS, VEGF, Cyclin D1 using qRT-PCR. Our results revealed that the three isolated compounds ALUP, LUP, and BA caused cell cycle arrest at the G2/M phase with an increase in the apoptosis which may be attributed to their significant effect on raising Bax, CASP-8, and CD95 and reducing the mRNA expression levels of Bcl-2, KRAS, VEGF, and Cyclin D1 compared to control cells. RT-PCR results showed that the ALUP, LUP, and BA significantly downregulated miRNA-21 expression. Meanwhile, the three compounds caused significant overexpression of Let-7 miRNA. This is the first report on the anti-cancer activity of acetoxy-lup-5(6), 20(29)-diene (ALUP) in reducing the proliferation and differentiation of the A549 cell line through inducing apoptosis. Finally, by targeting the Let-7 miRNA/Cyclin D1/VEGF cascade, acetoxy-lup-5(6), 20(29)-diene could be a potential therapeutic agent for lung cancer.

Topics: A549 Cells; Apoptosis; bcl-2-Associated X Protein; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cyclin D1; Humans; Lung Neoplasms; MicroRNAs; Proto-Oncogene Proteins p21(ras); RNA, Messenger; Triterpenes; Vascular Endothelial Growth Factor A

Cell-cycle regulators are mutated in approximately 40% of all cancer types and have already been linked to worse outcomes in non-small cell lung cancer adenocarcinomas treated with osimertinib. However, their exact role in osimertinib resistance has not been elucidated.. In this study, we aimed to evaluate how the CDK4/6-Rb axis may affect the sensitivity to osimertinib.. We genetically increased the level of CCND1 (Cyclin D1) and reduced the levels of CDKN2A (p16) in two different adenocarcinoma cell lines, PC9 and HCC827. We also retrospectively evaluated the outcome of patients with epidermal growth factor receptor-mutated advanced non-small cell lung cancer depending on their level of Cyclin D1 and p16.. The modified clones showed higher proliferative capacity, modifications in cell-cycle phases, and higher migratory capacity than the parental cells. Cyclin D1-overexpressing clones were highly resistant to acute osimertinib treatment. CDKN2A knockdown conferred intrinsic resistance as well, although a longer time was required for adaption to the drug. In both cases, the resistant phenotype was epidermal growth factor receptor independent and associated with a higher level of Rb phosphorylation, which was unaffected by osimertinib treatment. Blocking the phosphorylation of Rb using abemaciclib, a CDK4/6 inhibitor, exerted an additive effect with osimertinib, increasing sensitivity to this drug and reverting the intrinsic resistant phenotype. In a group of 32 patients with epidermal growth factor receptor-mutated advanced non-small cell lung cancer, assessed for Cyclin D1 and p16 expression, we found that the p16-deleted group presented a lower overall response rate compared with the control group.. We conclude that perturbation in cell-cycle regulators leads to intrinsic osimertinib resistance and worse patient outcomes.

Topics: Aniline Compounds; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cyclin D1; Drug Resistance, Neoplasm; ErbB Receptors; Humans; Lung Neoplasms; Mutation; Protein Kinase Inhibitors; Retrospective Studies

Changes in microRNAs (miRs) contribute to the alternative chemo-resistance of cancers. Bortezomib (BTZ) is a well-characterized anticancer agent that inhibits proteasome, and its effect is associated with the function of miRs. Based on the data of microarray assay and comprehensive bioinformatics analyses, in the current study, we explored the role of miR-466 and its downstream effector CCND1 in the BTZ-resistance of non-small-cell lung cancer (NSCLC) cells.. miR expression profiles in NSCLC tissues and paratumor tissues were determined with microarray assay. The potential miR involved in the chemo-resistance of NSCLC cells was explored via a series of bioinformatics analyses, and miR-466 was selected. Afterward, levels of miR-466 and CCND1 were investigated in NSCLC samples and analyzed by clinicopathologic parameters, including age, sex, stage of NSCLC, tumor size, tumor differentiation status, and lymphocytic infiltration status. The expression of CCND1 and miR-466 was then modulated in vitro to explore the influence on cell phenotypes, which was then verified with mouse models.. Based on microarray detection, 287 miRs were dysexpressed between NSCLC tissues and paratumor tissues, including 90 upregulated members and 197 downregulated members. After bioinformatics analyses and reverse transcription quantitative PCR validation, miR-466 and CCND1 were selected. Following clinical investigations, miR-466 was downregulated, while CCND1 was upregulated in NSCLC samples, contributing to the advanced cancer progression. The overexpression of CCND1 increased cell viability, suppressed cell apoptosis, decreased p21 and induced N-cadherin, CCND2, and CDK4 under BTZ treatment. The induced expression of miR-466 re-sensitized NSCLC cells to BTZ treatment. In the animal model, the overexpression of CCND1 impaired the inhibitory effect of BTZ on the growth and metastasis of solid tumor, which was restored by miR-466 induction.. The findings showed that the interaction between BTZ, miR-466, and CCND1 determined the antitumor effect of BTZ on NSCLC.

Topics: Animals; Bortezomib; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin D1; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Mice; MicroRNAs

Non-small cell lung cancer (NSCLC) is one of the leading causes of cancer-associated mortality worldwide. Circular RNA (circRNA) circZCCHC6 has been reported to be upregulated in the plasma from NSCLC patients. This study is designed to explore the role and mechanism of circZCCHC6 in NSCLC. CircZCCHC6, microRNA-433-3p (miR-433-3p), and lysophosphatidylcholine acyltransferase 1 (LPCAT1) level were determined by real-time quantitative polymerase chain reaction. Cell viability, cell cycle progression, migration, and invasion were assessed by 3-(4,5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide (MTT), flow cytometry, wound healing, and transwell assays, severally. The binding relationship between miR-433-3p and circZCCHC6 or LPCAT1 was predicted by Circinteractome or Starbase, and then verified by a dual-luciferase reporter, RNA pull-down, or RNA Immunoprecipitation (RIP) assays. Protein levels of LPCAT1, Cyclin D1, E-cadherin, and Vimentin were examined by western blot assay. The biological role of circZCCHC6 on NSCLC tumor growth and epithelial-mesenchymal transition (EMT) was examined by the xenograft tumor model in vivo. CircZCCHC6 was highly expressed in NSCLC serum, tissues, and cells. Moreover, circZCCHC6 knockdown could repress cell viability, cell cycle progression, migration, invasion, and EMT in NSCLC cells in vitro. The mechanical analysis suggested that circZCCHC6 acted as a sponge of miR-433-3p to regulate LPCAT1 expression. CircZCCHC6 silencing hindered cell growth and EMT of NSCLC in vivo. CircZCCHC6 inhibited the progression of NSCLC cells partly by regulating the miR-433-3p/LPCAT1 axis, implying a promising therapeutic target for the NSCLC treatment.

Topics: 1-Acylglycerophosphocholine O-Acyltransferase; Bromides; Cadherins; Carcinogenesis; Carcinoma, Non-Small-Cell Lung; Cell Movement; Cell Proliferation; Cyclin D1; Humans; Lung Neoplasms; MicroRNAs; RNA, Circular; Vimentin

Heterogeneous nuclear ribonucleoprotein K (hnRNPK) is a nucleic acid-binding protein. Reportedly, hnRNPK is overexpressed in many human tumors, and such overexpression is associated with poor prognosis, implicating the role of hnRNPK as an oncogene during tumorigenesis. In this study, hnRNPK expression in lung cancer tissues was investigated.. Briefly, hnRNPK was knocked down in lung cancer cell lines, and effects of knockdown on the cell proliferation, migration, and cell cycle were assessed using a cell counting kit-8 (CCK-8) assay, colony formation assay, transwell assay and flow cytometry. The effects of hnRNPK knockdown on the p53-dependent signaling pathway were examined using western blotting. Finally, the effect of hnRNPK knockdown on tumor growth was verified in vivo using a lung cancer xenograft mouse model.. hnRNPK knockdown inhibited the cell proliferation, migration and cell cycle. In addition to phenotypic changes, hnRNPK knockdown upregulated expressions of pCHK1, pCHK2, and p53，p21，cyclin D1, thereby mediating the DNA damage response (DDR). The regulatory function of hnRNPK during p53/p21/cyclin D1 signaling in hnRNPK-knockdown A549 cells was confirmed by suppressed the protein expression of associated signaling pathways, which inhibited DDR.. hnRNPK plays a crucial role in the progression of lung cancer, ultimately affecting survival rate. Inhibition of progression of lung cancer cells induced by hnRNPK-knockdown is dependent on activation of p53 by the p53/p21/cyclin D1 pathway.

Topics: Animals; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Gene Expression Regulation, Neoplastic; Heterogeneous-Nuclear Ribonucleoprotein K; Humans; Lung Neoplasms; Mice; Signal Transduction; Tumor Suppressor Protein p53

Large-cell neuroendocrine carcinoma (LCNEC) and small-cell carcinoma (SCLC) of lung encompass high-grade neuroendocrine tumour category and share several fundamental features. As both tumours may respond to different treatment modalities and show unique molecular alterations distinction between the two is clinically relevant, but can be challenging due to sampling and fixation issues and shared morphological features.. Surgically resected primary SCLC (n = 129) and LCNEC (n = 27) were immunohistochemically stained with Rb1, cyclin D1 and p16 using tissue microarray (TMA), and expression patterns of the proteins were compared between the two to identify the discriminatory pattern.. All markers had high diagnostic accuracy; Rb1 was the highest followed by p16 and cyclin D1. The majority of SCLC had the pattern Rb1-/p16+/cyclin D1- and more than half of LCNEC had Rb1+/p16-/cyclin D1+. Overall, the expression pattern Rb1- and cyclin D1- was strongly associated with the diagnosis of SCLC, while the pattern Rb1+ and/or cyclin D1+ was strongly associated with LCNEC. The use of this simplified expression pattern leads to a diagnostic accuracy of 97.3%. p16 did not add to further discrimination. The heterogeneity in Rb1, cyclin D1 and p16 expression was insignificant in SCLCs compared with LCNECs.. Use of Rb1, cyclin D1 and p16 immunohistochemistry can distinguish the two with high accuracy. Notably, the Rb1-/cyclin D1- pattern in given tumour sample would confirm the diagnosis of SCLC. Our results could be extrapolated and applied to routine diagnostic samples such as biopsies and cytology samples.

Topics: Carcinoma, Large Cell; Carcinoma, Neuroendocrine; Carcinoma, Small Cell; Cyclin D1; Genes, p16; Humans; Immunohistochemistry; Lung; Lung Neoplasms; Retinoblastoma Binding Proteins; Small Cell Lung Carcinoma; Ubiquitin-Protein Ligases

To elucidate the biological function of BAP18 (BPTF-associated protein of 18 kDa) in non-small-cell lung carcinoma (NSCLC) and the molecular mechanism.. Relative levels of BAP18 in NSCLC tissues were detected by quantitative real-time polymerase chain reaction (qRT-PCR), and its influence on pathological characteristics of NSCLC patients was analyzed. Correlation between BAP18 and Ki67 levels in NSCLC was assessed by Pearson correlation test. Furthermore, Kaplan-Meier curves were depicted for revealing survival difference in NSCLC patients expressing high or low level of BAP18. Relative levels of BAP18, CCND1, CCND2 and CCND3 in A549 and H1299 cells transfected with siBAP18 were determined, as well as colony number. In addition, after knockdown of protein level of BAP18 in A549 and H1299 cells by lentivirus transfection, cell cycle progression was examined. Co-regulation of BAP18 and CCND1/2 on cell growth of NSCLC was finally detected.. BAP18 was upregulated in NSCLC tissues, especially cases with advanced stage (III-IV) or large tumor size (>5 cm). BAP18 was closely linked to tumor size, TNM staging and lymphatic metastasis in NSCLC. Knockdown of BAP18 reduced transcriptional levels of CCND1 and CCND2 in A549 and H1299 cells. Furthermore, knockdown of BAP18 delayed transition from G1 to S phase, and weakened growth of NSCLC cells.. BAP18 triggers the progression of NSCLC by regulating transcriptional activities of CCND1/2, which may be a potential target for the treatment and diagnosis of NSCLC.

Topics: A549 Cells; Carcinoma, Non-Small-Cell Lung; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin D2; DNA-Binding Proteins; Humans; Lung Neoplasms; MicroRNAs; Transcription, Genetic

Topics: Animals; Calcium-Binding Proteins; Cell Line, Tumor; Cell Proliferation; Colorectal Neoplasms; Cyclin D1; Humans; Lung Neoplasms; Mice; MicroRNAs; RNA, Circular; Tumor Suppressor Proteins; Wnt Signaling Pathway

Lung cancer has risen to the top of the list of cancer-related deaths worldwide. Aliskiren is a direct renin inhibitor.. This study aims to investigate the impact of cell signaling of Renin-Angiotensin system (RAS)/NF-κB on lung cancer by investigating the potential therapeutic effects of aliskiren for lung cancer treatment in urethane-induced lung cancer in mice.. Male BALB/c mice were randomly assigned to one of five treatment groups for 150 days, including (1) normal control; (2) aliskiren (25 mg/kg/i.p) daily, (3) urethane at a dose of 1.5 g/kg (i.p) at Day 1 and 60 (nonsmall cell lung cancer[NSCLC] group) (4) NSCLC mice received carboplatin (15 mg/kg/i.p) every other day for the last 4 successive weeks and (5) NSCLC mice treated with aliskiren daily. Tumor size was determined based on blood sampling, and lungs were isolated for biochemical analysis, western blot analysis assay, and histopathological examination.. Urethane demonstrated significant changes in all biochemical and molecular parameters and histological patterns. Aliskiren-treated mice had significantly lower levels of NF-κB p65, Bcl-2, cyclin D1, ICAM-1, MMP-2, and Nrf2, with an increase in the catalytic activity of caspase-3 due to its RAS inhibitory mechanism. The combined urethane administration with aliskiren demonstrated a significant improvement in the histopathological examination.. RAS/NF-B cell signaling is a potential therapeutic target for preventing and treating lung adenocarcinoma, evidenced by the fundamental cytotoxic mechanism and attenuation of metastasis and angiogenesis induced by the treatment of NSCLC mice with aliskiren.

Topics: Amides; Animals; Apoptosis; Carboplatin; Carcinoma, Non-Small-Cell Lung; Caspase 3; Cell Cycle Checkpoints; Cyclin D1; Fumarates; Intercellular Adhesion Molecule-1; Lung Neoplasms; Male; Matrix Metalloproteinase 2; Mice; Mice, Inbred BALB C; NF-E2-Related Factor 2; NF-kappa B; Proto-Oncogene Proteins c-bcl-2; Renin; Renin-Angiotensin System; Signal Transduction; Urethane

Despite the huge efforts employed to implement novel chemotherapeutic paradigms for lung cancer, the disease still remains a major concern worldwide. Targeting molecular pathways as Hedgehog (Hh) and Mitogen-activated protein kinase (MAPK) represent a new hope in lung cancer treatment. This work was undertaken to evaluate the antitumor effects of GANT61 (5 μM), BI-847325(30 μM), and GANT61 (5 μM)/BI-847325(30 μM) combination on A549 adenocarcinoma lung cancer cell line. The growth inhibition 50 (GI50) for both drugs was performed using MTT. The protein levels of Caspase-3, Bcl-2-associated X protein (Bax), Myeloid cell leukemia sequence 1 (MCL-1), cyclin D1, vascular endothelial growth factor (VEGF), extracellular signal-regulated kinases (ERK), p-Akt, and phosphohistone H3 (pHH3) were measured using ELISA. Glioma-associated oncogene homolog 1(Gli1) gene expression was assessed by quantitative real-time PCR. The GI50 for GANT61 and BI-8473255 were 5 µM and 30 µM, respectively. Caspase-3 and Bax protein levels were significantly elevated while MCL-1, cyclin D1, VEGF, ERK 1/2, p-Akt, and pHH3 levels were significantly reduced by both drugs and their combination relative to the control group. Gli1 gene expression was down-regulated in all groups relative to the control group. GANT61, BI-847325 and their combination inhibited proliferation and angiogenesis but activated the apoptotic pathway. Both drugs conferred a profound negative impact on the crosstalk between each of Hh and MAPK pathways and Phosphoinositide 3 -kinases (PI3K)/Akt/Mammalian target of Rapamycin (mTOR). To the best of our knowledge, the antitumor effects of BI-847325/GANT61 combination have not been tested before. Further in-vitro and in-vivo studies are warranted to support the findings.

Topics: Aniline Compounds; Apoptosis; Caspase 3; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Hedgehog Proteins; Humans; Indoles; Lung Neoplasms; Myeloid Cell Leukemia Sequence 1 Protein; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Pyridines; Pyrimidines; Signal Transduction; Vascular Endothelial Growth Factor A; Zinc Finger Protein GLI1

Lung cancer ranking high in the cancer-related list has long perplexed patients, in which glucosamine-phosphate N-acetyltransferase 1 (GNPNAT1) is found to be highly expressed. Besides, DNA methylation is perceived as a biomarker to assess the prognosis of patients with various cancers. However, the correlation between GNPNAT1 and DNA methylation and the role of GNPNAT1 in lung cancer remain vague.. Principal component analysis (PCA), heatmap, volcano map, Venn diagram, gene ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis were used to screen out the candidate genes. The viability, migration, and invasion of lung cancer cells were detected by CCK-8 and Transwell assays. An xenograft tumor mouse model was established. The relative expressions of GNPNAT1, E-cadherin, vimentin, Matrix metalloproteinase-2 (MMP-2), tissue inhibitor of metalloproteinase-2 (TIMP-2), E2F1, and cyclin D1 in cells or xenograft tumor tissues were quantified by Western blot, RT-qPCR, or immunohistochemistry assay.. GNPNAT1 was screened as the research object. GNPNAT1 methylation was downregulated, while GNPNAT1 expression was upregulated in lung cancer tissues. The methylation and mRNA levels of GNPNAT1 were correlated with the patient prognosis. GNPNAT1 increased cell viability, migration and invasion, and promoted the xenograft tumor volume and weight, whereas shGNPNAT1 acted oppositely. Moreover, expressions of Vimentin, MMP-2, E2F1, and cyclin D1 were increased, but E-cadherin and TIMP-2 expressions were decreased by overexpressed GNPNAT1, whilst GNPNAT1 knockdown ran conversely.. GNPNAT1 and methylated GNPNAT1 coverage are biomarkers for the diagnosis and prognosis of lung cancer.

Topics: Animals; Cadherins; Cell Line, Tumor; Cell Proliferation; Cyclin D1; DNA Methylation; Gene Expression Regulation, Neoplastic; Glucosamine 6-Phosphate N-Acetyltransferase; Humans; Lung Neoplasms; Matrix Metalloproteinase 2; Mice; Prognosis; Tissue Inhibitor of Metalloproteinase-2; Vimentin

Analysis of the Gene Expression Profiling Interactive Analysis (GEPIA) database revealed that Kelch-like 17 (KLHL17) is overexpressed in non-small cell lung cancer (NSCLC) including adenocarcinoma (ADC) and squamous cell carcinoma (SCC). We therefore explored the role of KLHL17 in the development and progression of NSCLC. Immunohistochemistry and western blotting showed that KLHL17 expression was significantly higher in the tumor tissues from 173 patients with NSCLC, compared with the corresponding non-neoplastic tissue. In addition, upregulated KLHL17 expression was positively correlated with tumor size, lymph node metastasis and tumor node metastasis (TNM) stage, and affected the overall survival (OS) of patients with NSCLC. Consistent with clinical samples, in vitro studies demonstrated that KLHL17 expression was higher in various cell lines of NSCLC (A549, H1299, H460 and SK cells) as compared to normal human bronchial epithelial cells (HBE cells). Overexpression of KLHL17 in the cell lines of NSCLC with KLHL17-Flag plasmid promoted the proliferation and migration of tumor cells, which was associated with elevated activation of Rat sarcoma/Mitogen-activated protein kinases (Ras/MAPK) signaling and increased expression of cyclin D1, cyclin D-dependent kinases 4 (CDK4), matrix metalloproteinase 2 (MMP2) and Ras homolog gene family member A (RhoA). In contrast, knockdown of KLHL17 in the cell lines of NSCLC using KLHL17 small interfering RNA suppressed the proliferation and migration of tumor cells, in association with reduced activation of Ras/MAPK signaling and decreased expression of cyclin D1, CDK4, MMP2 and RhoA. Moreover, treatment of tumor cells with Ras inhibitor salirasib prevented KLHL17-induced Ras/MAPK activity as well as tumor proliferation and migration. These results suggest that upregulated KLHL17 in NSCLC promotes the proliferation and migration of tumor by activating Ras/MAPK signaling pathway. Therefore, KLHL17 may be a novel therapeutic target for the treatment of NSCLC.

Topics: Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin D1; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Matrix Metalloproteinase 2; Mitogen-Activated Protein Kinases; Signal Transduction; Up-Regulation

Rho guanine nucleotide exchange factor 40 (ARHGEF40) is a member of the Dbl-family of guanine nucleotide factor proteins. However, its expression pattern and biological function in malignant tumors, notably in nonsmall cell lung cancer (NSCLC) are currently unknown. The present study demonstrated that ARHGEF40 was highly expressed in NSCLC specimens and that its expression was significantly associated with advanced TNM stage (p < 0.001), lymph node metastasis (p = 0.002), and poor prognosis (p = 0.0056). In addition, ARHGEF40 accelerated nuclear translocation of the key component β-catenin and increased the expression levels of the Wnt signaling pathway targets c-myc, cyclin D1 and MMP7. Moreover, it promoted lung cancer cell proliferation and invasion in vitro and in vivo. To elucidate the underlying molecular mechanism, the current study demonstrated that ARHGEF40 could induce activation of the Wnt signaling pathway by increasing the phosphorylation levels of AKT and GSK3β via interaction with RhoA. Moreover, the Dbl homology (DH)-pleckstrin homology (PH) domain of ARHGEF40 was responsible for this interaction. Its deletion abolished the binding, which blocked the activation of the Wnt signaling. Taken together, the data indicated that ARHGEF40 promoted the malignant phenotype of lung cancer cells by activating the AKT-Wnt axis. This was achieved by its interaction with RhoA via the DH-PH domain. ARHGEF40 may serve as a novel target for NSCLC treatment.

Topics: beta Catenin; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Glycogen Synthase Kinase 3 beta; Guanine Nucleotides; Humans; Lung Neoplasms; Matrix Metalloproteinase 7; Proto-Oncogene Proteins c-akt; Rho Guanine Nucleotide Exchange Factors; rhoA GTP-Binding Protein; Wnt Signaling Pathway

Ubiquitin-specific protease 22 (USP22), a putative cancer stem cell marker, is frequently upregulated in cancers, and USP22 overexpression is associated with aggressive growth, metastasis, and therapy resistance in various human cancers including lung cancer. However, USP22 gene amplification seldom occurs, and the mechanism underlying USP22 upregulation in human cancers remains largely unknown.. A luciferase reporter driven by a promoter region of USP22 gene was selectively constructed to screen against a customized siRNA library targeting 89 selected transcription factors to identify potential transcription factors (TFs) that regulate USP22 expression in human non-small cell lung cancers (NSCLC). Association of identified TFs with USP22 and potential role of the TFs were validated and explored in NSCLC by biological assays and immunohistochemistry analysis.. Luciferase reporter assays revealed that SP1 and activating transcription factor 3 (ATF3) inhibit USP22 transcription, while transcription factor AP-2 Alpha/Beta (TFAP2A/2B) and c-Myc promote USP22 transcription. Binding site-directed mutagenesis and chromosome immunoprecipitation (ChIP) assays validated AP2α and AP2β are novel TFs of USP22. Furthermore, overexpression of AP2A and AP2B significantly upregulates USP22 expression, and its target: Cyclin D1, concurrently enhances the proliferation, migration, and invasion of NSCLC A549 and H1299 cells in a partially USP22-dependent manner. Moreover, AP2 protein level correlated with USP22 protein in human NSCLC tissues.. Our findings indicate AP2α and AP2β are important transcription factors driving USP22 gene expression to promote the progression of NSCLC, and further support USP22 as a potential biomarker and therapeutic target for lung cancer. Video Abstract.

Topics: Activating Transcription Factor 3; Carcinoma, Non-Small-Cell Lung; Cyclin D1; Gene Expression; Humans; Luciferases; Lung Neoplasms; RNA, Small Interfering; Thiolester Hydrolases; Transcription Factor AP-2; Ubiquitin Thiolesterase; Ubiquitin-Specific Proteases; Up-Regulation

Gefitinib is a widely used therapeutic drug for non-small cell lung cancer (NSCLC), and its acquired resistance has become one of the barriers to the successful use of the drugs to treat NSCLC patients. Long non-coding RNA (lncRNA) has an essential role in developing cancer drug resistance. Hence, this study aimed to investigate the effect and modulatory mechanisms of lncRNA MCF2L-AS1 in Gefitinib resistance in NSCLC.. IBEAS-2B and A549 cells and human NSCLC tissues were used. A549/GR cell line was constructed by continuous exposure to Gefitinib. Cell viability, apoptosis, migration, colony formation, and protein expression studies were done in transfected cells. Interactions of MCF2L-AS1, ELAVL1, and Cyclin D1 (CCND1 was also investigated.. In patients with Gefitinib-resistant NSCLC, MCF2L-AS1 and CCND1 were both up-regulated. Knockdown of MCF2L-AS1 reduced Gefitinib-resistant NSCLC cell progression, indicating that inhibition of MCF2L-AS1 restrained Gefitinib-resistant NSCLC. Mechanically, MCF2L-AS1 enhanced CCND1 mRNA stability via combining with ELAVL1, thereby elevating the resistance of NSCLC cells to Gefitinib. Moreover, E2F1 could transcriptionally up-regulate MCF2L-AS1.. The results manifest that lncRNA MCF2L-AS1, as an oncogene of NSCLC, controls CCDN1 via ELAVL1 to drive the growth of NSCLC cells and Gefitinib resistance.

Topics: Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Proliferation; Cyclin D1; E2F1 Transcription Factor; ELAV-Like Protein 1; Gefitinib; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; MicroRNAs; RNA Stability; RNA, Long Noncoding

Neuro-oncological ventral antigen 1 (NOVA1) is a neuron-specific RNA-binding protein which regulates alternative splicing in the developing nervous system. Recent research has found that NOVA1 plays a significant role in carcinogenesis. In this paper, we examine the role of NOVA1 in non-small cell lung cancer (NSCLC) and its underlying molecular mechanisms.. The expression of NOVA1 in NSCLC was detected by immunohistochemistry and correlations between NOVA1 expression and clinicopathological factors were analyzed by chi-square tests. Kaplan-Meier survival analysis and the Cox regression model were used to evaluate the predictive effect of prognostic factors. Western blotting, Cell Counting Kit-8, colony formation, apoptosis, migration and invasion assays were used to detect the effects of silencing (si)NOVA1 RNA on Wnt/β-catenin signaling and biological behavior in NSCLC cell lines.. Our study showed that expression of NOVA1 was up-regulated and significantly correlated with poor differentiation (p = 0.020), advanced TNM stage (P = 0.001), T stage (P = 0.001) and lymph node metastasis (P = 0.000) as well as the expression of β-catenin (P = 0.012) in NSCLC. The down-regulation of NSCLC by siRNA significantly inhibited proliferation, migration and invasion and promoted apoptosis in NSCLC cells. Expression of Wnt signaling molecules, including β-catenin, activated β-catenin, cyclin D1, matrix metalloproteinase (MMP)-2 and MMP-7, was also significantly reduced by siNOVA1. The inhibition of Wnt/β-catenin signaling in A549 and H1299 cells by siNOVA1 was reversed after treatment with a β-catenin expression plasmid.. The present study suggests that NOVA1 may serve as a potential prognosis biomarker in NSCLC. High NOVA1 expression was associated with poor survival rate. Finally, in vitro experiments verified that NOVA1 promotes NSCLC cell proliferation and invasion by regulating Wnt/β-catenin signaling.

Topics: beta Catenin; Biomarkers; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin D1; Humans; Lung Neoplasms; Matrix Metalloproteinase 7; Neuro-Oncological Ventral Antigen; Prognosis; RNA-Binding Proteins; RNA, Small Interfering; Wnt Signaling Pathway

Acquired and primary resistance to epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) is still the bottleneck of clinical treatment of advanced non-small cell lung cancer (NSCLC). STE029 is a novel anticancer drug which consists of 3-hydroxy-3-methylglutarylcoenzyme A reductase (HMGCR) inhibitor and novel cancer cell membrane targeting molecular. This study aimed to investigate the reversal mechanism of EGFR-TKI resistance by STE029 in lung adenocarcinoma.. CCK8 test was used to test the cell viability and survival rate of EGFR mutated PC9 cell (Gefitinib sensitive), PC9/BB4 cell (acquired Gefitinib resistant), and EGFR wild type A549 cell after treatment of STE029, Gefitinib or combination of both. EdU test was applied to detect changes in cell cycle and Hoechst 33258 was applied to detect apoptosis rate in overcoming the EGFR-TKI resistance. The activity of EGFR/PI3K/Akt, cell cycle and apoptosis signal pathways were examined. In vivo, nude mice were exposed to STE029, Gefitinib and STE029+Gefitinib for 5 wk. And the the tumor volume was measured and tumor weight was obtained on the last day.. (1) PC9 cells was highly sensitive to Gefitinib, while PC9/BB4 and A549 cell showed significant resistance to Gefitinib treatment; (2) STE029+Gefitinib treatment could significantly decrease the 50% inhibitory concentrarion (IC₅₀) of Gefitinib in PC9, PC9/BB4 and A549 cells (P<0.05, respectively); (3) In PC9 and PC9/BB4 cells, STE029+Gefitinib can block cell cycle and inhibit cell proliferation (P<0.001), while there was no significant difference in apoptosis rate among three drug intervention groups (P>0.05); However, apoptosis rate was increased in STE029+Gefitinib group in A549 cell (P<0.01), while no significance detected in cell proliferation (P>0.05). (4) In PC9 and PC9/BB4 cells, the combination of STE029 and Gefitinib could downregulate p-EGFR, p-Akt, p-Cyclin D1 and Cyclin D1 (P<0.001), and upregulate the expression of GSK-3β (P<0.001), and the expression of cleaved caspase-8, caspase-8 cleaved caspase-9, caspase-9 showed no difference among groups (P>0.05). In A549 cells, the combination of STE029 and Gefitinib could downregulate p-Akt (P<0.001) and upregulate cleaved caspase-8 and cleaved caspase-9 (P<0.001); (5)In vivo, the combination of STE029 and Gefitinib effectively inhibited tumor development and progression compared to STE029 alone or Gefitinib alone, with significant difference (P<0.05) in PC9 and PC9/BB4 xenografted tumor.. STE029 could sensitize Gefitinib by inhibiting EGFR/PI3K/Akt pathway, blocking the tumor cell cycle and proliferation and inducing apoptosis through caspase-8 and caspase-9 dependent pathway. STE029 deserves further investigations in overcoming EGFR-TKI resistance in lung cancer.. 【中文题目：STE029逆转肺腺癌EGFR-TKI耐药
及其机制研究】 【中文摘要：背景与目的 表皮生长因子受体酪氨酸激酶抑制剂（epidermal growth factor receptor-tyrosine kinase inhibitor, EGFR-TKI）获得性耐药和原发性耐药至今仍是临床治疗晚期非小细胞肺癌（non-small cell lung cancer, NSCLC）的瓶颈。STE029是一种同时具有羟甲基戊二酸单酰辅酶A还原酶（3-hydroxy-3-methylglutarylcoenzyme A reductase, HMGCR）抑制剂抗肿瘤作用和肿瘤特异性细胞膜靶向功能的新型抗肿瘤药物。本研究旨在探讨STE029逆转肺腺癌EGFR-TKI的耐药机制。方法 STE029、吉非替尼单药及联合用药分别处理PC9、PC9/BB4、A549细胞，检测各细胞株的活性变化、细胞增殖、细胞凋亡，鉴定EGFR/PI3K/Akt信号通路、细胞周期及凋亡相关蛋白的表达；同时观察STE029、吉非替尼单药及联合用药对PC9、PC9/BB4裸鼠皮下移植瘤生长的影响。结果 ①PC9细胞为EGFR突变的敏感细胞，PC9/BB4细胞为EGFR突变的获得性耐药细胞，A549细胞为非EGFR突变的耐药细胞；②STE029联合吉非替尼作用于PC9、PC9/BB4、A549细胞后，吉非替尼的半数抑制浓度（50% inhibitory concentration, IC₅₀）值较对照组均显著降低（P<0.05）；③STE029联合吉非替尼可抑制PC9、PC9/BB4细胞的增殖（P<0.001），诱导A549细胞凋亡增加（P<0.01）；④在PC9、PC9/BB4细胞中，联合用药组较单药组p-EGFR、p-Akt的表达明显下调（P<0.000,1），GSK-3β表达升高（P<0.001），其下游p-Cyclin D1及Cyclin D1表达明显降低（P<0.001），cleaved caspase-8、caspase-8、cleaved caspase-9、caspase-9的表达在各给药组间未见明显差异（P>0.05）；在A549细胞中，联合用药组p-Akt表达明显下调（P<0.001），cleaved caspase-8、cleaved caspase-9表达均升高（P<0.001），而GSK-3β、p-Cyclin D1、Cyclin D1、caspase-8、caspase-9的蛋白表达在各给药组间则无明显差异（P>0.05）；⑤裸鼠体内，联合用药组PC9皮下移植瘤的生长明显受到抑制，差异有明显统计学意义（P<0.01）；联合用药组PC9/BB4皮下移植瘤的生长速率亦明显降低（P<0.05）。结论 STE029可在体内外明显增加非EGFR-T790M突变耐药的人肺腺癌细胞对吉非替尼的敏感性，其机制可能与STE029通过EGFR/PI3K/Akt信号通路调节GSK-3β、Cyclin D1的表达、阻滞细胞增殖、诱导细胞凋亡有关。
】 【中文关键词：STE029；EGFR-TKI；耐药；细胞凋亡；细胞周期；肺腺癌】.

Topics: Adenocarcinoma of Lung; Animals; Carcinoma, Non-Small-Cell Lung; Caspase 8; Caspase 9; Cyclin D1; ErbB Receptors; Gefitinib; Glycogen Synthase Kinase 3 beta; Humans; Lung Neoplasms; Mice; Mice, Nude; Phosphatidylinositol 3-Kinases; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt

OBJECTIVE: Among other types of cancerous lesions, lung cancer is one of the prevalent causes of death. Trigonelline is a plant alkaloid, a significant constituent in coffee, and has shown health benefits in several disorders. The present study aims to investigate the potential therapeutic role of trigonelline in lung cancer. MATERIALS AND METHODS: Seventy-five BALB/C mice were assigned to five groups and treated for 150 days as follows (1): normal control group; (2) trigonelline only (50 mg/kg/ P.O) daily for the last thirty days; (3) urethane (1.5 g/kg B.w/i.p) at day one and sixty; (4) urethane and carboplatin (15 mg/kg i.p) for the last thirty days; and (5) urethane and trigonelline for the last thirty days. Tumor size was measured while blood and lung were collected for biochemical, western blotting analysis, and histological examinations. RESULTS: Urethane demonstrated significant changes in all biochemical and molecular parameters and histological examinations. In animals pretreated with urethane, trigonelline significantly reduced tumor size and restored Nrf2, NF-кB p65, Bcl-2, Cyclin D1, ICAM-1, and MMP-2, along with improving cGMP and active caspase three and refining histological architectures. CONCLUSIONS: Nrf2 signaling may be a promising therapeutic target for adenocarcinoma protection or management. Due to its multiple therapeutic effects on Nrf2, cyclin D1, NF-кB pathways, and the BAX/Bcl2 axis, trigonelline significantly induced cell cycle arrest and apoptosis.. https://www.europeanreview.org/wp/wp-content/uploads/Graphical_Abstract-1.jpg.

Topics: Alkaloids; Animals; Apoptosis; Caspases; Cyclin D1; Enzyme Inhibitors; Lung Neoplasms; Mice; Mice, Inbred BALB C; NF-E2-Related Factor 2; NF-kappa B; Urethane

Baicalin plays important roles in different types of cancer. A previous report showed that baicalin attenuates cisplatin resistance in lung cancer. However, its mechanism remains unclear. In this study, we investigated the effect and mechanism of baicalin on DNA repair and sensitivity of lung cancer cells to cisplatin. A549 and A549/DPP cells were treated with baicalin and cisplatin. A549/DPP cells were transfected with XRCC1 and siXRCC1. Cell viability and DNA damage were detected by MTT and comet assay. Apoptosis rate and cell cycle were detected by flow cytometry assay. The expressions of Bax, Bcl-2, and Cyclin D1 were detected by western blot. XRCC1 expression was detected by reverse transcription quantitative polymerase chain reaction (RT-qPCR) and western blot. Baicalin and cisplatin decreased cell viability in A549 and A549/DPP cells in dose-dependent manner. Baicalin enhanced the effect of cisplatin on promoting apoptosis, arresting cell on S stage and triggering DNA damage accompanied with the upregulation of Bcl-2-associated X protein (Bax) and downregulation of B-cell lymphoma 2 (Bcl-2) and Cyclin D1 in A549/DPP cells. Moreover, baicalin promoted the inhibitory effect of cisplatin on XRCC1 expression in A549 and A549/DPP cells. However, the synthetic effects of baicalin and cisplatin on A549/DPP cells were partially inhibited by XRCC1 overexpression and promoted by XRCC1 knockdown. This study demonstrates that baicalin interferes with XRCC1-mediated cellar DNA repair to sensitize lung cancer cells to cisplatin.

Topics: A549 Cells; Antineoplastic Agents; Apoptosis; bcl-2-Associated X Protein; Cell Line, Tumor; Cisplatin; Cyclin D1; DNA Repair; Drug Resistance, Neoplasm; Flavonoids; Humans; Lung Neoplasms; X-ray Repair Cross Complementing Protein 1

Recently, circular RNAs (circRNAs) have become an intense focus of research and large numbers of circRNAs have been identified, awaiting functional elucidation. Thus, the present study aims to examine the regulation of circRNAs and its molecular mechanism in lung cancer growth. Here, we show that circular RNA circ_0000677 was overexpressed and correlated with poor prognosis in non-small cell lung cancer (NSCLC) patients. Functionally, circ_0000677 knockdown markedly inhibited proliferation of NSCLC cells by observing of immunofluorescence staining of Ki67, clone formation assay, and xenograft experiments. In mechanism, circ_0000677 acted as a sponge of microRNA-106b and further regulated CCDND1 gene expression in NSCLC cells by dual luciferase activity assay and their expression examination. Taken together, these findings suggest a role for circ_0000677/miR-106b/CCND1 regulation axis in promoting NSCLC growth and progression.

Topics: Animals; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Gene Expression Regulation, Neoplastic; Humans; Lung; Lung Neoplasms; Male; Mice; Mice, Inbred BALB C; MicroRNAs; RNA, Circular

Non-small cell lung cancer (NSCLC) is the most common subtype of lung cancer. Ubiquitination is closely related to the development of lung cancer. However, the biological importance of newly discovered ubiquitin-specific peptidase (USP) 52 (USP52) in NSCLC remained unclear. Here, our findings identify USP52 as a novel tumor suppressor of NSCLC, the low expression of USP52 predicts a poor prognosis for NSCLC patients. The present study demonstrates that USP52 inhibits cancer cell proliferation through down-regulation of cyclin D1 (CCND1) as well as AKT/mTOR signaling pathway inhibition. Meanwhile, USP25 also suppresses NSCLC progression via enhancing phosphatase and tensin homolog (PTEN) stability in cancer cells, which further indicates the significance/importance of USP52 in NSCLC suppression.

Topics: Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Enzyme Stability; Exoribonucleases; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Proteolysis; Proto-Oncogene Proteins c-akt; PTEN Phosphohydrolase; Signal Transduction; TOR Serine-Threonine Kinases; Ubiquitination

Aldehyde dehydrogenases (ALDHs) are considered as markers for normal and cancer stem cells (CSC) and are involved in cell metabolism, proliferation, differentiation, stemness, and retinoic acid (RA) biosynthesis. The aim of the present study was to identify the ALDH isoforms that are associated with the CSC phenotype in non-small cell lung and hepatocellular carcinomas.. We utilized lung (A549) and hepatocellular (HepG2) cancer cells and generated tumor spheres to isolate the CSC sub-population.. The CSC enrichment was confirmed by the up-regulation of various CSC-related genes. Comparative qPCR analysis indicated the up-regulation of several ALDH isoforms in A549 and HepG2 spheres. Interestingly, cyclin D1 and Akt, down-stream targets of the RA signaling pathway, were also shown to be significantly up-regulated in both sphere populations.. Specific ALDH isoforms appear to be important mediators for the acquisition of an CSC phenotype and thus, are potential promising targets for CSC-based therapeutic approaches in lung and hepatocellular carcinomas.

Topics: A549 Cells; Aldehyde Dehydrogenase; Carcinoma, Hepatocellular; Cyclin D1; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Hep G2 Cells; Humans; Isoenzymes; Liver Neoplasms; Lung Neoplasms; Neoplastic Stem Cells; Phenotype; Proto-Oncogene Proteins c-akt; Signal Transduction; Spheroids, Cellular

Topics: Animals; Apoptosis; Biomarkers, Tumor; Breast Neoplasms; Cell Movement; Cell Proliferation; Cyclin D1; Female; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Mice; Mice, Inbred BALB C; Mice, Nude; Phosphorylation; Prognosis; RNA Stability; RNA-Binding Proteins; Tumor Cells, Cultured; Ubiquitin-Protein Ligases; Xenograft Model Antitumor Assays; Zebrafish

Cullin1 is a representative member of the Cullin family, and it plays an important role in the ubiquitination of cell cycle, transcription and signal transduction related proteins. Cullin1 is closely related to the occurrence and development of a variety of malignant tumors. The aim of this study is to investigate the effects of Cullin1 on biological function of lung adenocarcinoma A549 and H1395 Cells.. The expression of Cullin1 mRNA was detected by quantitative Real-time polymerase chain reaction in lung adenocarcinoma cells (A549, H358, H1395, H1650) and human normal lung epithelial cells BEAS-2B, siRNA technology was used to interfere with lung adenocarcinoma cells with relatively high expression of Cullin1 mRNA; cell proliferation, cell cycle distribution, early cell apoptosis, invasion and migration ability were detected by methyl thiazolyl tetrazolium assay (MTT), flow cytometry and Transwell experiment; Western blot was used to detect the expression levels of matrix metalloproteinase-2 (MMP-2), matrix metalloproteinase-9 (MMP-9), tissue inhibitor of metalloproteinase-1 (TIMP-1), Cyclin D1, Cyclin E2, p21 and p27.. Compared with the BEAS-2B cell, Cullin1 mRNA was highly expressed in lung adenocarcinoma cells, especially in lung adenocarcinoma A549 and H1395 cells (P<0.05). The proliferation ability of lung adenocarcinoma cells was inhibited after interference with Cullin1, and the number of cells in G1 phase increased, the number of cells in S phase decreased, and the early apoptosis rate of lung adenocarcinoma cells is significantly increased (P<0.05); The invasion and migration ability of lung adenocarcinoma cells decreased (P<0.05). After interference with Cullin1, the protein expression of MMP-9, MMP-2, CyclinD1 and CyclinE2 decreased (P<0.05), while the expression of TIMP-1, p21 and p27 protein increased (P<0.05).. Interference with Cullin1 inhibits the proliferation, invasion and migration of lung adenocarcinoma A549 and H1395 cells, Cullin1 plays a role in promoting cancer in lung adenocarcinoma.. 【中文题目：Cullin1对肺腺癌A549和H1395细胞生物学
特性的影响】 【中文摘要：背景与目的 Cullin1是Cullin家族中有代表性的一员，对细胞周期、转录和信号转导相关蛋白泛素化起重要作用，Cullin1与多种恶性肿瘤的发生发展有着密切的联系。本研究旨在探讨Cullin1对肺腺癌A549和H1395细胞生物功能的影响。方法 实时荧光定量聚合酶链式反应（polymerase chain reaction, PCR）检测肺腺癌细胞（A549、H358、H1395、H1650）及人正常肺上皮细胞BEAS-2B中Cullin1 mRNA表达，采用siRNA技术干扰Cullin1 mRNA相对高表达的肺腺癌细胞；采用四甲基偶氮唑盐比色法（methyl thiazolyl tetrazolium assay, MTT）、流式细胞术及Transwell实验检测细胞增殖、细胞周期分布、细胞早期凋亡及侵袭和迁移能力；采用Western blot检测基质金属蛋白酶-2（matrix metalloproteinase-2, MMP-2）、基质金属蛋白酶-9（matrix metalloproteinase-9, MMP-9）、组织基质金属酶抑制剂-1（tissue inhibitor of metalloproteinase-1, TIMP-1）、细胞周期蛋白D1（Cyclin D1）、细胞周期蛋白E2（Cyclin E2）、p21和p27蛋白的表达水平。结果 与BEAS-2B细胞相比，肺腺癌细胞中Cullin1 mRNA均呈高表达，尤其在肺腺癌A549和H1395细胞中相对表达量较高（P<0.05）；干扰Cullin1后肺腺癌细胞的增殖能力受到了抑制，G1期细胞增多，S期细胞数目减少，肺腺癌细胞早期凋亡率明显升高（P<0.05）；肺腺癌细胞的侵袭及迁移能力下降（P<0.05）；干扰Cullin1后MMP-9、MMP-2、Cyclin D1及Cyclin E2蛋白表达量减少（P<0.05），而TIMP-1、p21和p27蛋白表达量增多（P<0.05）。结论 干扰Cullin1后可抑制肺腺癌A549和H1395细胞的增殖、侵袭和迁移，Cullin1在肺腺癌中发挥促癌作用。】 【中文关键词：Cullin1；肺肿瘤；生物学特性】.

Topics: A549 Cells; Adenocarcinoma of Lung; Apoptosis; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cullin Proteins; Cyclin D1; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Neoplasm Invasiveness; Tissue Inhibitor of Metalloproteinase-1

To explore the expression of microribonucleic acid-340 (miR-340) and cyclin D1 (CCND1) in lung cancer (LC) tissues and its relationship with the clinicopathological characteristics and prognosis of LC.. Cancer tissues and paracancerous normal lung tissues of 65 patients with LC admitted to our hospital from January 2014 to March 2015 were included as the LC group, and the paracancerous group, respectively.. The relative expression levels of miR-340 mRNA and miR-340 protein in the LC group were lower than those in the paracancerous group, while the relative expression levels of CCND1 mRNA and CCND1 protein in the LC group were higher than those in the paracancerous group (P < 0.05). Pearson correlation analysis results showed that the mRNA and protein expression of both miR-340 and CCND1 in LC tissues was negatively correlated (r < 0, P < 0.05).The high expression rate (HER) of miR-340 and high expression rate (PER) of CCND1 were related to the tumor size, lymph node metastasis, TNM staging, and degree of differentiation (P < 0.05). The patients with high expression (HE) of miR-340 showed increased 5-year SR compared with the patients with low expression of miR-340, and that of patients positive for CCND1 was lower than that of the patients negative for CCND1 (P < 0.05).. miR-340 was downregulated, whereas CCND1 was upregulated in LC tissues, and the expression levels of the two genes were closely related to the prognosis and clinicopathological characteristics of LC.

Topics: Cyclin D1; Humans; Lung Neoplasms; MicroRNAs; Prognosis

Lung cancer is one of the most frequent types of cancers that lead to death. Sildenafil is a potent inhibitor of phosphodiesterase-5 and showed potential anticancer effects, which has not yet been fully evaluated. Thus, this study aims to investigate the potential anticancer effect of sildenafil in urethane-induced lung cancer in BALB/c mice.. Five-week-old male BALB/c mice were treated with either (i) normal saline only, (ii) sildenafil only 50 mg kg-1/ P.O every other day for the last four successive weeks, (iii) urethane 1.5 gm kg-1 i.p (at day 1 and day 60), (iv) carboplatin after urethane induction, or (v) sildenafil after urethane induction.. It was shown that sildenafil significantly increased the levels of cGMP and Caspase-3 with a reduction of NF-κB, Bcl-2, Cyclin D1, intercellular adhesion molecule 1, matrix metalloproteinase-2 levels and normalisation of Nrf2 along with pronounced improvement in the histological patterns.. These results indicated that sildenafil markedly induces cell cycle arrest, apoptosis and inhibits the metastatic activity through activation of cyclic guanosine monophosphate/protein kinase G pathway and down-regulation of cyclin D1 and nuclear factor kappa light chain enhancer of activated B cells with downstream anti-apoptotic gene Bcl-2, which underscores the critical importance of future using sildenafil in the treatment of lung cancer.

Topics: Animals; Antineoplastic Agents; Apoptosis; Cell Cycle Checkpoints; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Cyclin D1; Drug Repositioning; Lung Neoplasms; Matrix Metalloproteinase 2; Mice; Mice, Inbred BALB C; NF-kappa B p50 Subunit; Phosphodiesterase 5 Inhibitors; Proto-Oncogene Proteins c-bcl-2; Sildenafil Citrate; Treatment Outcome

Topics: Animals; Biomarkers, Tumor; Cell Cycle; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cell Shape; Cortactin; Cyclin D1; Extracellular Matrix; Focal Adhesions; Humans; Hyaluronic Acid; Lumican; Lung Neoplasms; Melanoma; Mice, Inbred C57BL; Neoplasm Metastasis; Phosphorylation; Podosomes; Signal Transduction; Skin Neoplasms; Snail Family Transcription Factors; Vinculin

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) (eg, gefitinib) exert potent therapeutic efficacy in non-small-cell lung cancer (NSCLC) harboring EGFR-activating mutations. However, the resistance to EGFR TKIs limits their clinical therapeutic efficacy. TIP30, a newly identified tumor suppressor, appears to be involved in the regulation of cytoplasmic and nuclear EGFR signaling in NSCLC. Our previous study demonstrated that TIP30 regulated EGF-dependent cyclin D1 transcription in human lung adenocarcinoma and suppressed tumorigenesis. In the present study, the involvement of TIP30 in combating gefitinib resistance in NSCLC was determined for the first time in vitro and in vivo. Gain and loss of function studies showed that overexpression of TIP30 effectively sensitized cells to gefitinib in vitro, whereas TIP30 inhibition promoted gefitinib cell resistance. Moreover, TIP30 negatively regulated the activation of the p-AKT and p-MEK signaling pathways in PC9/GR. Importantly, PC9/GR harbored high levels of nuclear EGFR, and overexpression of TIP30 restored irregular EGFR trafficking and degradation from early endosomes to the late endosomes, decreasing the nuclear accumulation of EGFR, which may partly or totally inhibit EGFR-mediated induction of c-Myc transcription. Xenographic tumors induced by overexpression of TIP30 by PC9/GR cells in nude mice were suppressed compared with their original counterparts. Overall, it was revealed that TIP30 overexpression restored gefitinib sensitivity in NSCLC cells and attenuated the cytoplasmic and nuclear EGFR signaling pathways and may be a promising biomarker in gefitinib resistance in NSCLC.

Topics: Acetyltransferases; Animals; Antineoplastic Agents; Carcinoma, Non-Small-Cell Lung; Cell Nucleus; Cyclin D1; Cytoplasm; Drug Resistance, Neoplasm; Endosomes; ErbB Receptors; Gefitinib; Humans; Lung Neoplasms; Lysosomes; MAP Kinase Kinase 1; Mice; Neoplasm Proteins; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-myc; Transcription Factors; Tumor Suppressor Proteins; Up-Regulation; Xenograft Model Antitumor Assays

THeterogeneous nuclear ribonucleoprotein (HNRNP) A1 is the most abundant and ubiquitously expressed member of the HNRNP protein family. In recent years, it has become more evident that HNRNP A1 contributes to the development of neurodegenerative diseases. However, little is known about the underlying role of HNRNP A1 in cancer development. Here, we report that HNRNP A1 expression is significantly increased in lung cancer tissues and is negatively correlated with the overall survival of patients with lung cancer. Additionally, HNRNP A1 positively regulates vaccinia-related kinase 1 (VRK1) translation via binding directly to the 3' untranslated region (UTR) of

Topics: 3' Untranslated Regions; Base Sequence; Cell Cycle; Cell Line; CRISPR-Cas Systems; Cyclin D1; Eukaryotic Initiation Factor-3; Gene Expression Regulation, Neoplastic; Genes, Reporter; Heterogeneous Nuclear Ribonucleoprotein A1; Humans; Intracellular Signaling Peptides and Proteins; Lung Neoplasms; Neoplasm Proteins; Protein Binding; Protein Biosynthesis; Protein Domains; Protein Interaction Mapping; Protein Serine-Threonine Kinases; Recombinant Proteins; RNA Interference; RNA, Messenger; Sequence Deletion; Up-Regulation

Cyclin D1 (CCND1) has been identified as a metastatic promoter in various tumors including lung adenocarcinoma (LUAD), a subtype of non small cell lung cancer (NSCLC). The previous observation revealed that CCND1 was upregulated in NSCLC and predicted poor prognosis of LUAD patients. In this study, we examined a chaperonin containing TCP1 subunit 5 (CCT5) protein interacts with CCND1 in LUAD. Immunofluorescence demonstrated the co-localization of CCT5 and CCND1 protein in LUAD cells. CCT5 expression was detected with both immunohistochemistry (IHC) and bioinformatics analyses. Similar with the expression pattern of CCND1, CCT5 displayed a high level in LUAD tissues compared to non cancerous lung specimens. Patients with high CCT5 expression showed a significant shorter overall survival relative to those with low expression level. Furthermore, upregulated CCT5 exhibited significant positive correlation with TNM stage of LUAD patients in both IHC analyses and bioinformatics. Knocking down CCT5 remarkably inhibited LUAD cell migration and invasion in vitro by inactivating PI3K/AKT and its downstream EMT signals, which could abrogated the accelerated migration and invasion caused by CCND1 overexpression. In summary, our study discovered a highly expressed protein CCT5 in LUAD which interacted with CCND1 and promoted migration and invasion of LUAD cells by positively moderating PI3K/AKT-induced EMT pathway.

Topics: Adenocarcinoma of Lung; Cell Line, Tumor; Cell Movement; Chaperonin Containing TCP-1; Cyclin D1; Humans; Lung Neoplasms; Neoplasm Invasiveness; Protein Interaction Maps

Cell proliferation and cell death abnormalities are strongly linked to the development of cancer, including lung cancer. The purpose of this study was to investigate the effect of pterostilbene on cell proliferation and cell death via cell cycle arrest during the transition from G1 to S phase and the p53 pathway. A total of 24 female Balb/C mice were randomly categorized into four groups (n = 6): N-nitroso-tris-chloroethyl urea (NTCU) induced SCC of the lungs, vehicle control, low dose of 10 mg/kg PS + NTCU (PS10), and high dose of 50 mg/kg PS + NTCU (PS50). At week 26, all lungs were harvested for immunohistochemistry and Western blotting analysis. Ki-67 expression is significantly lower, while caspase-3 expression is significantly higher in PS10 and PS50 as compared to the NTCU (p < 0.05). There was a significant decrease in cyclin D1 and cyclin E2 protein expression in PS10 and PS50 when compared to the NTCU (p < 0.05). PS50 significantly increased p53, p21, and p27 protein expression when compared to NTCU (p < 0.05). Pterostilbene is a potential chemoprevention agent for lung SCC as it has the ability to upregulate the p53/p21 pathway, causing cell cycle arrest.

Topics: Animals; Anticarcinogenic Agents; Apoptosis; Carcinoma, Squamous Cell; Cell Cycle Checkpoints; Cell Proliferation; Cyclin D1; Cyclins; Disease Models, Animal; Down-Regulation; Female; G1 Phase Cell Cycle Checkpoints; Gene Expression Regulation, Neoplastic; Lung Neoplasms; Mice, Inbred BALB C; Stilbenes; Tumor Suppressor Protein p53; Up-Regulation

LncRNA DLGAP1-AS2 plays an oncogenic role in glioma, while its role in other cancers is unknown. This study aimed to study the role of DLGAP1-AS2 in non-small cell lung cancer (NSCLC).. Expression of DLGAP1-AS2 in NSCLC and paired non-tumor tissues from 64 NSCLC patients and the prognostic value of DLGAP1-AS2 for NSCLC were analyzed by performing a 5-year follow-up study. The interaction between DLGAP1-AS2 and miR-503 was confirmed by dual luciferase reporter assay, and their relationship was explored in NSCLC cells transfected with DLGAP1-AS2 expression vector or miR-503 mimic. The roles of DLGAP1-AS2 and miR-503 in regulating cyclin D1 expression were analyzed by RT-qPCR and Western blot. Cell proliferation was analyzed by CCK-8 assay.. DLGAP1-AS2 was upregulated in NSCLC and predicted poor survival. Interaction between DLGAP1-AS2 and miR-503 was confirmed by dual luciferase activity assay. Overexpression experiments showed that DLGAP1-AS2 and miR-503 overexpression failed to significantly affect the expression of each other. Interestingly, DLGAP1-AS2 overexpression upregulated cyclin D1, a target of miR-503, increased cell proliferation and reduced the effects of miR-503 overexpression on cyclin D1 expression and cell proliferation.. DLGAP1-AS2 may regulate miR-503/cyclin D1 to promote cell proliferation in NSCLC.

Topics: Aged; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Female; Follow-Up Studies; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Male; MicroRNAs; Middle Aged; Prognosis; RNA, Long Noncoding; SAP90-PSD95 Associated Proteins

The Sry-related high-mobility group box6 (SOX6) has been implicated in the development of cancer, but its role in lung cancer is incompletely understood. Here, we report that SOX6 expression is frequently down-regulated in lung adenocarcinoma tissues. Moreover, SOX6 can inhibit the proliferation and invasion of lung adenocarcinoma cells, which may occur through cell cycle arrest at G1/S due to up-regulation of p53 and p21

Topics: A549 Cells; Adenocarcinoma of Lung; beta Catenin; Cell Cycle; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Female; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Male; Middle Aged; Multivariate Analysis; RNA, Messenger; SOXD Transcription Factors; Tumor Cells, Cultured; Tumor Suppressor Protein p53

Methylparaben is most frequently used as an antimicrobial preservative in pharmaceuticals and foods. Methylparaben has been subjected to toxicological studies owing to the increasing concern regarding its possible impact on the environment and human health. However, the cytotoxicity and underlying mechanisms of methylparaben exposure in human lung cells have not been explored. Here, we investigated the effect of methylparaben on cell cycle, apoptotic pathways, and changes in the transcriptome profiles in human lung cells. Our results demonstrate that treatment with methylparaben causes inhibition of cell growth. In addition, methylparaben induced S- and G2/M-phase arrest as a result of enhanced apoptosis. Transcriptome analysis using RNA-seq revealed that mRNA expression of ER stress- and protein misfolding-related gene sets was upregulated in methylparaben-treated group. RNA splicing- and maturation-related gene sets were significantly down-regulated by methylparaben treatment. Interestingly, RNA-seq analysis at the transcript level revealed that alternative splicing events, especially retained intron, were markedly changed by a low dose of methylparaben treatment. Altogether, these data show that methylparaben induces an early phase of apoptosis through cell cycle arrest and downregulation of mRNA maturation.

Topics: Alternative Splicing; Apoptosis; Caspase 3; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cyclin B1; Cyclin D1; Humans; Lung Neoplasms; Parabens; Transcriptome

It is well recognized that competing endogenous RNA (ceRNA) regulatory network is linked to the development and progression of cancer, including non-small cell lung cancer (NSCLC). Herein, we aimed to explore the functional role of circ-CMPK1/miR-302e/cyclin D1 ceRNA signaling in NSCLC.. GEO database (GSE102287) was utilized to screen differentially expressed miRNAs in NSCLC. Quantitative reverse transcription PCR (qRT-PCR) and western blotting assays were used to determine gene expression. Cell proliferation analysis was performed with Cell Counting Kit-8 (CCK-8) and cell cycle assays. Luciferase reporter and RNA pull-down assays were conducted to identify the interaction among circ-CMPK1, miR-302e, and cyclin D1. Xenograft tumor model was established to evaluate the role of circ-CMPK1/miR-302e/cyclin D1 axis in vivo.. miR-302e expression was significantly down-regulated in NSCLC cell lines and tissues and its decrease was closely associated with aggressive clinicopathological features and unfavorable outcome. Overexpression and knockdown of miR-302e obviously retarded and enhanced the growth of NSCLC, respectively. Furthermore, we found that miR-302 was sponged by circular RNA CMPK1 (circ-CMPK1, hsa_circ_0012384), which was remarkably up-regulated in NSCLC and predicted poor prognosis. Circ-CMPK1 was capable to promote NSCLC cells proliferation by increasing the expression of cyclin D1 via inhibiting miR-302 activity. Moreover the miR-302e-mediated tumor inhibition could be effectively counteracted by ectopic expression of circ-CMPK1 or cyclin D1 both in vitro and in vivo.. Our data demonstrate for the first time that circ-CMPK1/miR-302e/cyclin D1 signaling plays an essential regulatory role in NSCLC and targeting this axis may be an efficacious avenue for treatment of NSCLC patients.

Topics: A549 Cells; Animals; Carcinoma, Non-Small-Cell Lung; Cell Proliferation; Cyclin D1; Female; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Male; Mice; Mice, Inbred BALB C; Mice, Nude; MicroRNAs; Middle Aged; Nucleoside-Phosphate Kinase; RNA, Circular; Up-Regulation

Non-small-cell lung cancer (NSCLC) is the main pathological type of lung cancer and has a low overall five-year survival rate. miR-187 has been reported to play major roles in various tumor types. In this study, we explored the impact of miR-187 on NSCLC. qRT-PCR results demonstrated that miR-187 expression is lower in NSCLC and cancer cells than normal tissues and normal lung cells. miR-187 expression levels are associated with tumor size, TNM stage and overall survival rate. MTS and colony formation assays showed that high miR-187 expression inhibits NSCLC cell proliferation and colony formation ability, and flow cytometry showed that miR-187 overexpression induces cell cycle arrest at the G0/G1 phase. A luciferase reporter assay showed that FGF9 is a target of miR-187. miR-187 overexpression reduces the expression of FGF9, cyclin D1 CDK4 and CDK6. Therefore, miR-187 may present a new NSCLC treatment target by regulates cyclins-related protein expression.

Topics: Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase 6; Fibroblast Growth Factor 9; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; MicroRNAs

It has been reported that tripartite motif containing 26 (TRIM26) is involved in the tumorigenesis of some cancers, but its function in non-small cell lung cancer (NSCLC) is still unclear. In this study, we found that TRIM26 was markedly down-regulated in both of NSCLC tumor tissues and cell lines. Additionally, high expression of TRIM26 in NSCLC patients predicted a positive index for patients' overall survival. What is more, overexpression of TRIM26 significantly suppressed NSCLC cell growth. Our further studies indicated that overexpression of TRIM26 inhibited the phosphorylation of PI3K p85 and AKT. And overexpressed TRIM26 regulated cell cycle-related genes' expression, including downregulating CDK4, Cyclin A, Cyclin D1, Cyclin D3, and Cyclin E, and upregulating p27 expression. Finally, we found that TRIM26 up-regulated PTEN expression by stabilizing PTEN protein in NSCLC cells. Collectively, our present study indicated that TRIM26 was decreased in NSCLC and overexpression of TRIM26 inhibited NSCLC cell growth by suppressing PI3K/AKT pathway, which suggested that TRIM26 could be as a potential target for the treatment of NSCLC in the future.

Topics: A549 Cells; Carcinogenesis; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Movement; Cell Proliferation; Class Ia Phosphatidylinositol 3-Kinase; Cyclin A; Cyclin D1; Cyclin D3; Cyclin E; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase Inhibitor p27; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Proto-Oncogene Proteins c-akt; PTEN Phosphohydrolase; Signal Transduction; Survival Analysis; Tripartite Motif Proteins; Ubiquitin-Protein Ligases

Several different mechanisms are implicated in the resistance of lung cancer cells to epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs), and only few have been functionally investigated. Here, using genetically knocked out EGFR and TKI-resistant lung cancer cells, we show that loss of wild-type EGFR attenuates cell proliferation, migration and 3D-spheroid formation, whereas loss of mutant EGFR or resistance to TKIs reinforces those processes. Consistently, disruption of wild-type EGFR leads to suppression of HER2/HER3, while mutant EGFR ablation or resistance to TKIs increases HER2/HER3 expression, compensating for EGFR loss. Furthermore, HER2/HER3 nuclear translocation mediates overexpression of cyclin D1, leading to tumor cell survival and drug resistance. Cyclin D1/CDK4/6 inhibition resensitizes erlotinib-resistant (ER) cells to erlotinib. Analysis of cyclin D1 expression in patients with non-small cell lung carcinoma (NSCLC) showed that its expression is negatively associated with overall survival and disease-free survival. Our results provide biological and mechanistic insights into targeting EGFR and TKI resistance.

Topics: Antineoplastic Agents; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase 6; Drug Resistance, Neoplasm; Epithelial Cells; ErbB Receptors; Erlotinib Hydrochloride; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Protein Kinase Inhibitors; Receptor, ErbB-2; Receptor, ErbB-3; Signal Transduction; Spheroids, Cellular; Survival Analysis; Transcriptional Activation

The aim of this study was to detect the expression of linc00703 in non-small cell lung cancer (NSCLC), and to explore the biological function and potential molecular mechanism of linc00703 in NSCLC using in vitro experiments.. The carcinoma tissues and para-carcinoma tissues were collected from 32 patients diagnosed with NSCLC, from which the RNA was extracted. The relative expression of linc00703 in NSCLC tissues was detected via quantitative reverse transcription-polymerase chain reaction (qRT-PCR). The NSCLC cells and normal human bronchial epithelial cells were selected, in which the relative expression of linc00703 was determined via qRT-PCR. Next, the linc00703 overexpression plasmids were designed and synthesized, and then transiently transfected into NSCLC cells. After 48 h, the overexpression efficiency was detected. Finally, the changes in cell proliferation, apoptosis, cycle distribution and expressions of downstream molecular markers were determined using cell counting kit-8 (CCK8) assay, colony formation assay, flow cytometry and Western blotting, respectively, after overexpression of linc00703 in NSCLC cells.. The results of qRT-PCR revealed that the expression of linc00703 was down-regulated by 5.14 times on average in 29 out of 32 cases of NSCLC tissues, and it was also down-regulated in NSCLC cells. Besides, it was found through CCK-8 assay, colony formation assay and flow cytometry that after overexpression of linc00703 in NSCLC cells, the cell proliferation was inhibited, the apoptosis was enhanced, and the cell cycle was arrested in G1/G0 phase. Furthermore, the results of Western blotting showed that after overexpression of linc00703, the protein expressions of cyclinD1 and cyclin-dependent kinase 4 (CDK4) declined, while those of cyclinE1 and CDK2 did not change.. The expression of linc00703 is down-regulated in NSCLC, and it suppresses the occurrence and development of NSCLC via mediating the expression of cyclinD1/CDK4.

Topics: Apoptosis; Carcinoma, Non-Small-Cell Lung; Cell Cycle Checkpoints; Cell Proliferation; Cells, Cultured; Cyclin D1; Cyclin-Dependent Kinase 4; Disease Progression; Humans; Lung Neoplasms; RNA, Long Noncoding

Puerarin has recently been demonstrated to play anti-cancer roles in a series of human cancers, including non-small cell lung cancer (NSCLC), possibly through regulation of cancer-related microRNAs (miRNAs). The purpose of the present study was to further investigate the detailed role and underlying mechanism of puerarin on NSCLC progression. Cell viability and apoptosis were assessed using the Cell Counting kit-8 (CCK-8) assay and flow cytometry, respectively. Transwell assays were performed to determine cell migration and invasion abilities. The qRT-PCR assay was employed to detect the expression of miR-342 and cyclin D1 (CCND1) mRNA, and CCND1 protein expression was evaluated by western blotting. The targeted interaction between miR-342 and CCND1 was verified by dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay. We found that our data demonstrated that puerarin repressed cell viability, migration, invasion, and cell cycle progression, and enhanced the apoptosis of NSCLC cells. miR-342 overexpression hindered the migration, invasion and cell cycle progression, and accelerated the apoptosis of NSCLC cells. miR-342 inhibited CCND1 expression by directly binding to the 3'-UTR of CCND1. Moreover, miR-342 overexpression-mediated anti-migration, anti-invasion, anti-cell cycle progression, and pro-apoptotic effects were abated by co-transfection of pcDNA-CCND1. More importantly, puerarin inhibited CCND1 expression by upregulating miR-342. Additionally, puerarin hampered NSCLC cell progression in vitro and tumor growth in vivo by upregulating miR-342. In conclusion, our study suggested that puerarin hampered NSCLC progression in vitro and in vivo at least partly through regulating miR-342/CCND1 axis, highlighting a novel mechanism of puerarin exerting anti-cancer property in NSCLC.

Topics: Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin D1; Gene Expression Regulation, Neoplastic; Humans; Isoflavones; Lung Neoplasms; MicroRNAs

The aim of this study was to investigate the effect of long non-coding ribonucleic acid (lncRNA)-bladder cancer associated transcript 1 (BLACAT1) on the drug resistance of non-small cell lung cancer (NSCLC) cells in cisplatin (DDP) chemotherapy by regulating the expression of Cyclin D1.. The analysis of the lncRNA expression profiles in 483 cases of NSCLC tissues and 347 cases of cancer-adjacent tissues in Gene Expression Omnibus (GEO) database revealed that lncRNA-BLACAT1 was differentially expressed in NSCLC and related to prognosis. In order to further study its mechanism of action on DDP-resistant cells, the expression level of lncRNA-BLACAT1 in normal human lung bronchial epithelial cell line BEAS-2B, NSCLC cell line A549, and DDP-resistant cell line A549 (A549/DDP) was detected by quantitative Polymerase Chain Reaction (qPCR). LncRNA-BLACAT1 small interfering RNA (siRNA) (si-BLACAT1) and lncRNA-BLACAT1 negative control (si-NC) were transfected into A549/DPP cells. Then, qPCR was carried out to detect the changes in the expression of lncRNA-BLACAT1 before and after transfection. Thereafter, cell cycle and cell growth rate were detected by flow cytometry and the cell growth curve. Besides, the changes in cell migration, cell apoptosis, and Cyclin D1 were detected via wound healing assay, flow cytometry, and Western blotting (WB).. In GEO database, lncRNA-BLACAT1 was significantly overexpressed in NSCLC (p<0.05), and the prognosis of NSCLC in BLACAT1 low-expression group was better than that in the BLACAT1 high-expression group (p<0.0001). Compared with that in BEAS-2B cells, BLACAT messenger RNA (mRNA) was notably highly expressed in A549 cells (p<0.05), and compared with that in A549 cells, BLACAT1 mRNA in A549/DPP was significantly highly expressed in A549/DDP cells (p<0.05). Additionally, in comparison with that in the si-NC group, the content of lncRNA-BLACAT1 in si-BLACAT1 group was remarkably decreased (p<0.01). Moreover, flow cytometry detection of cell cycle revealed that compared with those in si-NC group, G0/G1 phase was markedly prolonged and S phase was shortened in si-BLACAT1 group. MTS assay manifested that the absorbance at 450 nm in si-BLACAT1 group was evidently decreased on the 3rd day compared with that in the si-NC group (p<0.05), and the difference between the two groups was the most significant on the 5th day (p<0.001). According to wound healing assay, compared with those in si-NC group, the distance between cells became larger, the cell migration ability was remarkably weakened (p<0.05), and cell apoptosis was prominently reduced in si-BLACAT1 group (p<0.05). WB results showed that compared with si-NC group, si-BLACAT1 group had significantly reduced Cyclin D1 (p<0.05) CONCLUSIONS: LncRNA-BLACAT1 regulates the expression of Cyclin D1, reduces the malignant phenotype of drug-resistant cells, and increases the sensitivity of lung cancer cells to DDP.

Topics: Antineoplastic Agents; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cisplatin; Cyclin D1; Drug Resistance, Neoplasm; Humans; Lung Neoplasms; RNA, Long Noncoding

Matrine is one of the major alkaloids extracted from Sophora flavescens Ait of the traditional Chinese medicine, was the main chemical ingredient of compounds of Kushen injection. The Matrine is considered as a promising therapeutic agent for curing nonsmall cell lung cancer (NSCLC), used either alone or combined with chemotherapeutic agents. In the present study, we focused on the possible roles of Matrine exerted on the self-renewal ability of stem-like cells of the NSCLC group, as well as the cytotoxicity of chemotherapeutic agents, in vitro and in vivo. Here we reported that Matrine inhibits cancer stem-like cell (CSC) properties through upregulation of Let-7b and suppression of the Wnt pathway. Overexpression of Let-7b suppressed the ability of tumorsphere formation, decreased Wnt pathway activation through inhibiting its transcriptional activity in lung CSCs. Further studies revealed that Let-7b directly targeted CCND1 and decreased its expression, whereas Matrine increased Let-7b levels and followed by inactivation of the CCND1/Wnt signaling pathway and inhibition of EMT, which was characterized by loss of epithelial markers and acquisition of a mesenchymal phenotype in lung CSCs. What is more, we found that Matrine increased Let-7b level in an endoribonuclease DICER1-dependent manner. And xenografts in nude mice evidenced that Matrine increased the sensitivity of lung CSCs to 5-FU and inhibited the accumulation of CCND1 in tumor tissues induced by 5-FU. Taken together, these data illustrate the role of Let-7b in regulating lung CSCs traits and DICER1/let-7/CCND1 axis in Matrine or in combination with 5-FU intervention of lung CSCs' expansion, helping to fulfill the anti-cancer action of Matrine.

Topics: A549 Cells; Alkaloids; Animals; Antimetabolites, Antineoplastic; Carcinoma, Non-Small-Cell Lung; Cyclin D1; Down-Regulation; Drug Resistance, Neoplasm; Fluorouracil; Humans; Lung Neoplasms; Male; Matrines; Mice; Mice, Inbred BALB C; Mice, Nude; MicroRNAs; Neoplastic Stem Cells; Quinolizines

Tumorigenesis is closely related to the disorder of the cell cycle. The cell cycle progression includes the interphase (G0/G1, S, and G2 phase) and mitosis (M phase). CCND1 is a key protein that regulates the entry of the G0/G1 phase into the S phase. In our study, we found that the short form of Fas Apoptosis Inhibitory Molecule 1 (FAIM-S) could regulate the expression of CCND1 and had a tumor-suppressing role in non-small cell lung cancer (NSCLC). Overexpressing FAIM-S significantly inhibited the proliferation and cell cycle progression in NSCLC cells. Further studies demonstrated that FAIM-S could interact with IKK-α, reducing its protein stability. This effect led to the suppression of the NF-κB pathway, resulting in the decreased expression of CCND1. Thus, our study demonstrated that FAIM-S functioned as a negative regulator of the NF-κB pathway and played a tumor-suppressing role through blocking cell cycle progression in NSCLC cells.

Topics: A549 Cells; Apoptosis; Apoptosis Regulatory Proteins; Carcinoma, Non-Small-Cell Lung; Cell Cycle Checkpoints; Cell Proliferation; Cyclin D1; Genes, Tumor Suppressor; Humans; I-kappa B Kinase; Lung Neoplasms; NF-kappa B; Proteolysis; Signal Transduction; Transfection

Malignant mesothelioma (MM) is an asbestos-induced cancer arising on the mesothelial surface of organ cavities. MM is essentially incurable without a means of early diagnosis and no successful standard of care. These facts indicate a deep chasm of knowledge that needs to be filled. Our group recently delved into MM tumor biology from the perspective of exosome-contained microRNAs (miRNAs). We discovered that the most abundant miRNAs in MM cancer exosomes were tumor suppressors, particularly miR-16-5p. This observation lead us to hypothesize that MM cells preferentially secreted tumor-suppressor miRNAs via exosomes. Through separate avenues of potential therapeutic advance, we embarked on an innovative strategy to kill MM tumor cells. We employed small molecule inhibitors to block exosome secretion, thereby reducing miR-16-5p exosome loss and replenishing cellular miR-16-5p leading to reduced tumorigenic capacity and miR-16-5p target oncoproteins CCND1 and BCL2. Additionally, we force-fed MM tumor exosomes back to MM tumor cells, which led to cell death, and a reduction in the same oncoproteins. We recapitulated these results with direct transfection of miR-16-5p, confirmed that this is a cancer-cell specific effect, and elucidated a part of the miR-16-5p mechanism of exosome loading.

Topics: Aniline Compounds; Antineoplastic Agents; Benzylidene Compounds; Cell Death; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Exosomes; Gene Expression Regulation, Neoplastic; Humans; Indoles; Lung Neoplasms; Maleimides; Mesothelioma; Mesothelioma, Malignant; MicroRNAs; Molecular Targeted Therapy; Ornithine; Proto-Oncogene Proteins c-bcl-2; Signal Transduction; Small Molecule Libraries; Transfection

DNA replication is a vital process in cell division where anomalies can lead to tumorigenesis. Minichromosome maintenance complex component 10 (MCM10) plays a crucial role in this process. However, the role of MCM10 in lung cancer pathogenesis remains to be elucidated. In current study, using the publicly available lung cancer Gene Expression Omnibus (GEO) datasets, and Oncomine and the Cancer Genome Atlas databases, an increased expression of MCM10 was found in lung cancer tissues compared to normal lung tissues. The high expression of MCM10 was subsequently validated in clinical specimens by reverse transcription‑quantitative PCR and immunohistochemistry. Analysis of the GEO datasets revealed that the high MCM10 expression was significantly associated with early and late recurrence, pathological stage and worse overall survival (OS). Cox's proportional hazards regression analyses revealed that MCM10 expression was an independent risk factor for poor OS and worse recurrence‑free survival both in univariate and multivariate analysis. Furthermore, the increased expression of MCM10 was enriched in cell cycle‑related processes, while in vitro transfection with small interfering RNA targeting MCM10 significantly suppressed cell viability, clone formation and induced G1 phase arrest in A549 and H661 cell lines by regulating the expression of cyclin D1 (CCND1). In addition, the current results indicated a combined effect of MCM10‑CCND1 in predicting the prognosis of lung cancer patients. Altogether, the present study provided a novel potential molecular mechanism of lung cancer progression and may aid in development of novel treatment strategies.

Topics: A549 Cells; Adult; Aged; Biomarkers, Tumor; Cell Proliferation; Computational Biology; Cyclin D1; Datasets as Topic; Disease-Free Survival; Female; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Humans; Kaplan-Meier Estimate; Lung; Lung Neoplasms; Male; Middle Aged; Minichromosome Maintenance Proteins; Neoplasm Recurrence, Local; Neoplasm Staging; Prognosis; RNA, Small Interfering

Hypoxia occurs naturally at high-altitudes and pathologically in hypoxic solid tumors. Here, we report that genes involved in various human cancers evolved rapidly in Tibetans and six Tibetan domestic mammals compared to reciprocal lowlanders. Furthermore, m

Topics: Adaptation, Physiological; Adenocarcinoma; Altitude; Animals; Animals, Domestic; Carcinoma, Non-Small-Cell Lung; Case-Control Studies; Cattle; Cyclin D1; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase 4; Dogs; Evolution, Molecular; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Genome; Goats; Horses; Humans; Hypoxia; Lung; Lung Neoplasms; Mice; Neoplasm Transplantation; RNA-Binding Proteins; Sheep; Sus scrofa; Swine; Tibet; Tumor Hypoxia

Smoking is one of the major environmental risk factors for lung cancer. In recent years, the role of long-chain noncoding RNAs (lncRNAs) in chemical carcinogenesis has attracted extensive research attention. In this study, we treated human bronchial epithelial cells with cigarette smoke extract (CSE) at a dose of 2 μg/ml to establish a malignantly transformed cellular model (16HBE-M). Screening of lncRNAs highly expressed in transformed cells via differential analysis revealed a crucial role of linc00152 in CSE-induced malignant transformation. The linc00152 serum level in CSE-exposed individuals was increased in a dose-dependent manner and its high expression associated with metastasis and proliferation of lung cancer tissue. In malignantly transformed 16HBE-M cells, linc00152 was involved in regulation of cell adhesion, epithelial transition and other malignant phenotypes, which in turn, affected in vivo metastasis. Interference with linc00152 expression led to G1/S arrest and inhibition of proliferation of 16HBE-M and H1299 cells. Furthermore, linc00152 promoted cyclin D1 expression and G1/S transition by functioning as an endogenous competitive RNA targeting miR-193b. Our collective findings supported a critical regulatory role of linc00152 in cell cycle alterations and abnormal proliferation in CSE-induced malignant transformation of human bronchial epithelial cells.

Topics: Animals; Cell Cycle; Cell Line; Cell Transformation, Neoplastic; Cigarette Smoking; Cyclin D1; Epithelial Cells; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Mice, Inbred BALB C; Mice, Nude; RNA, Long Noncoding; Smoke; Tobacco Products

CircRNAs are reported to be implicated in the development of lung cancer. This study focused on assessing the expression, functions and molecular mechanism of circPUM1 in lung adenocarcinoma. Here, it showed that circPUM1 is significantly upregulated in both lung adenocarcinoma cell lines and tissues. Furthermore, silencing of circPUM1 impaired the proliferation, migration and invasion ability, and increased apoptosis in A549 cells. Nevertheless, overexpression of circPUM1 in SPC-A1 cells has the opposite effect. Silencing of circPUM1 inhibits the tumorigenesis in nude mice. Mechanistically, circPUM1 could sponge miR-326 and promote the expression of its downstream proteins Cyclin D1 and Bcl-2. In summary, this present study revealed that circPUM1 functions as an oncogene to promote the tumorigenesis of lung adenocarcinoma through circPUM1/miR-326/Cyclin D1 and Bcl-2 axis. This indicates that circPUM1 may act as a potential therapeutic target for lung adenocarcinoma.

Topics: Adenocarcinoma of Lung; Animals; Apoptosis; Carcinogenesis; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin D1; Female; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Mice, Inbred BALB C; Mice, Nude; MicroRNAs; Neoplasm Invasiveness; Proto-Oncogene Proteins c-bcl-2; RNA; RNA, Circular

Sulforaphane (SFN) was first isolated from broccoli sprout and it is present at high concentrations in plants belonging to the Cruciferae family. The chemotherapeutic and anti‑cancerogenic capacities of SFN have been demonstrated by inhibition of cancer cell proliferation in several cancer cell lines. The aim of the present study was to evaluate the effect of SFN on apoptosis, cell cycle and expression of selected cell cycle‑associated proteins: Cyclin B1, cyclin D1 and cyclin K in the H1299 cell line. The non‑small cell lung cancer cell line H1299 was treated with increasing concentrations of SFN (5, 10 and 15 µM) for two days. After incubation, the percentage of cells in the individual cell cycle phases, as well as the percentage of necrotic and apoptotic cells, were estimated using flow cytometry. The expression of cyclins was examined by immunofluorescence staining, flow cytometry, western blot analysis and qRT‑PCR. Cyclin K was characterized by nuclear localization and increased expression after treatment with SFN. The expression data were confirmed by qRT‑PCR. SFN‑induced cell cycle arrest was associated with a decrease in cyclin B1 expression. Cells treated with SFN were also characterized by higher cyclin D1 and cyclin K expression. These data suggest the involvement of cyclin K in response to SFN. Moreover, we investigated the prognostic value of cyclin K, CDK12 and CDK13 in adenocarcinoma patients using 'The Kaplan‑Meier plotter' (KM plotter) database. It was shown that high expression of CDK12 and CDK13 but no cyclin K proteins is associated with worse overall survival among adenocarcinoma patients.

Topics: Anticarcinogenic Agents; Apoptosis; Carcinoma, Non-Small-Cell Lung; Cell Cycle Checkpoints; Cell Division; Cell Line, Tumor; Cyclin B1; Cyclin D1; Cyclins; Humans; Isothiocyanates; Lung Neoplasms; Sulfoxides

Tetracenomycin X (Tcm X) has been reported to have antitumour activity in various cancers, but there have not been any studies on its activity with respect to lung cancer to date. Therefore, this study aims to investigate the anti-lung cancer activity of Tcm X. In this study, we found that tetracenomycin X showed antitumour activity in vivo and selectively inhibited the proliferation of lung cancer cells without influencing lung fibroblasts. In addition, apoptosis and autophagy did not contribute to the antitumour activity. Tetracenomycin X exerts antitumour activity through cell cycle arrest induced by the downregulation of cyclin D1. To explore the specific mechanism, we found that tetracenomycin X directly induced cyclin D1 proteasomal degradation and indirectly downregulated cyclin D1 via the activation of p38 and c-JUN proteins. All these findings were explored for the first time, which indicated that tetracenomycin X may be a powerful antimitotic class of anticancer drug candidates for the treatment of lung cancer in the future.

Topics: A549 Cells; Actinobacteria; Antibiotics, Antineoplastic; Apoptosis; Aquatic Organisms; Cell Proliferation; Cyclin D1; Down-Regulation; Drug Screening Assays, Antitumor; Fibroblasts; Humans; Lung; Lung Neoplasms; MAP Kinase Signaling System; Naphthacenes; Proteolysis

Src homology region 2 (SH2)-containing protein tyrosine phosphatase 2 (SHP2) is ubiquitously expressed in cytoplasmic localization, which in turn confers tumor malignancy and poor prognosis in various human cancers. YAP1 interacts with SHP2 to promote translocation of SHP2 to nucleus, which consequently promotes Wnt target activation. However, the oncogenic role of the nuclear localization of SHP2 in human cancers remains unclear. We hypothesized that nuclear SHP2 localization, in combination with nuclear YAP1 expression, could be associated with poor overall survival (OS) and relapse free survival (RFS) due to an increase in cyclin D1 and c-Myc mRNA expression following activation of Wnt/ß-catenin signaling. Immunohistochemical analysis of SHP2 and YAP1 protein expression in 102 tumors resected from patients with NSCLC revealed that nuclear SHP2 expression was well correlated with nuclear YAP1 expression (P < 0.001). Evaluation of cyclin D1 and c-Myc mRNA levels by the real-time reverse-phase polymerase chain reaction (RT-PCR) revealed that patients with high cyclin D1 and high c-Myc mRNA expressing tumors more commonly showed high nuclear YAP1 and high nuclear SHP2 (high/high) rather than the high/low, low/high, or low/low combinations (P < 0.001 for cyclin D1 and c-Myc). Kaplan-Meier and Cox-regression models showed OS and RFS to be poorer in patients in the high/high subgroup than in the low/low subgroup (OS: HR = 2.85, 95% CI, 1.52-5.35, P = 0.001; RFS: HR = 2.55, 95% CI, 1.37-4.72, P = 0.003). No prognostic significance was observed for the other two subgroups (low/high and high/low) when compared to the low/low subgroup in this study population. Therefore, we suggest that the prognostic value of SHP2 could reflect the nuclear localization of SHP2 and its interaction with nuclear YAP1, which led to subsequent upregulation of cyclin D1 and c-Myc mRNA expression via activation of the Wnt/ß-catenin signaling pathway.

Topics: Adaptor Proteins, Signal Transducing; Aged; beta Catenin; Carcinoma, Non-Small-Cell Lung; Cell Nucleus; Cyclin D1; Female; Humans; Immunohistochemistry; Lung Neoplasms; Male; Middle Aged; Phosphoproteins; Prognosis; Protein Tyrosine Phosphatase, Non-Receptor Type 11; Proto-Oncogene Proteins c-myc; Signal Transduction; Survival Rate; Transcription Factors; YAP-Signaling Proteins

Long noncoding RNAs have been reported to be essential regulators in several human diseases, including tumorigenesis. A recent report revealed that FLVCR1-AS1 promotes the progression of hepatocellular carcinoma. However, whether FLVCR1-AS1 is involved in lung cancer remains unclear. In this study, we found that the expression of FLVCR1-AS1 was increased in lung cancer tissues according to The Cancer Genome Atlas database. Similarly, FLVCR1-AS1 was significantly upregulated in lung cancer cell lines. Knockdown of FLVCR1-AS1 dramatically reduced the cell proliferation, migration, and invasion of SPCA1 and A549. Mechanistically, we found that the expression levels of CTNNB1, SOX4, CCND1, CCND2, c-MYC, as well as nucleus β-catenin were decreased in lung cancer cells after FLVCR1-AS1 silencing. Thus, FLVCR1-AS1 positively regulates the activation of the Wnt/β-catenin pathway. Overexpression of CTNNB1 reversed the effect of FLVCR1-AS1 knockdown on A549 cells. In sum, FLVCR1-AS1 silencing inhibited the proliferation, migration, and invasion of lung cancer cells by inhibiting the activity of the Wnt/β-catenin signaling pathway.

Topics: A549 Cells; beta Catenin; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin D1; Cyclin D2; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Humans; Lung Neoplasms; RNA, Antisense; RNA, Long Noncoding; SOXC Transcription Factors; Wnt Signaling Pathway

Ubiquitin specific peptidase 49 (USP49) has been reported as a tumor suppressor in several tumors, but its function and molecular mechanism in non-small cell lung cancer (NSCLC) are still unknown. In this study, USP49 was found downregulated in NSCLC primary tissues and cell lines, and high USP49 predicted a positive index for the overall survival of NSCLC patients. Overexpression of USP49 downregulated the expression levels of Cyclin D1, and upregulated p53 expression. Further flow cytometry analysis showed that overexpressed USP49 induced cell cycle arrest at G0/G1 phase. As a result, overexpression of USP49 significantly inhibited cell growth of NSCLC cells. In mechanism, overexpression of USP49 inhibited PI3K/AKT signaling, but knockdown of USP49 enhanced this signaling. Further studies indicated that USP49 deubiquitinated PTEN and stabilized PTEN protein, which suggested that USP49 inhibited PI3K/AKT signaling by stabilizing PTEN in NSCLC cells. In conclusion, we demonstrated that USP49 was functional in NSCLC cells, and inhibited NSCLC cell growth by suppressing PI3K/AKT signaling, suggesting that USP49 could be as a novel target for NSCLC therapy.

Topics: Carcinoma, Non-Small-Cell Lung; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Gene Expression Regulation, Neoplastic; HEK293 Cells; Humans; Lung; Lung Neoplasms; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; PTEN Phosphohydrolase; Resting Phase, Cell Cycle; RNA, Small Interfering; Signal Transduction; Survival Analysis; Tumor Suppressor Protein p53; Ubiquitin Thiolesterase

Circular RNAs (circRNAs) that form through non-canonical backsplicing events of pre-mRNA transcripts are evolutionarily conserved and abundantly expressed across species. However, the functional relevance of circRNAs remains a topic of debate.. We identified one of the highly expressed circRNA (circANKRD12) in cancer cell lines and characterized it validated it by Sanger sequencing, Real-Time PCR. siRNA mediated silencing of the circular junction of circANKRD12 was followed by RNA Seq analysis of circANKRD12 silenced cells and control cells to identify the differentially regulated genes. A series of cell biology and molecular biology techniques (MTS assay, Migration analysis, 3D organotypic models, Real-Time PCR, Cell cycle analysis, Western blot analysis, and Seahorse Oxygen Consumption Rate analysis) were performed to elucidate the function, and underlying mechanisms involved in circANKRD12 silenced breast and ovarian cancer cells.. In this study, we identified and characterized a circular RNA derived from Exon 2 and Exon 8 of the ANKRD12 gene, termed here as circANKRD12. We show that this circRNA is abundantly expressed in breast and ovarian cancers. The circANKRD12 is RNase R resistant and predominantly localized in the cytoplasm in contrast to its source mRNA. We confirmed the expression of this circRNA across a variety of cancer cell lines and provided evidence for its functional relevance through downstream regulation of several tumor invasion genes. Silencing of circANKRD12 induces a strong phenotypic change by significantly regulating cell cycle, increasing invasion and migration and altering the metabolism in cancer cells. These results reveal the functional significance of circANKRD12 and provide evidence of a regulatory role for this circRNA in cancer progression.. Our study demonstrates the functional relevance of circANKRD12 in various cancer cell types and, based on its expression pattern, has the potential to become a new clinical biomarker.

Topics: Biomarkers, Tumor; Breast; Breast Neoplasms; Cell Movement; Cyclin D1; Exons; G1 Phase Cell Cycle Checkpoints; Gene Expression Regulation, Neoplastic; Gene Silencing; Humans; Lung; Lung Neoplasms; MCF-7 Cells; Neoplasm Invasiveness; Nuclear Proteins; Phenotype; RNA, Circular; RNA, Small Interfering; Transfection

Lung cancer is the most common cause of cancer-related mortality worldwide despite diagnostic improvements and the development of targeted therapies, notably including epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs). The phosphoinositide 3-kinase (PI3K)/AKT/mechanistic target of rapamycin (mTOR) signaling has been shown to contribute to tumorigenesis, tumor progression, and resistance to therapy in most human cancer types, including lung cancer. Here, we explored the therapeutic effects of co-inhibition of PI3K and mTOR in non-small-cell lung cancer (NSCLC) cells with different EGFR status.. The antiproliferative activity of a dual PI3K/mTOR inhibitor BEZ235 was examined by the WST-1 assay and the soft agar colony-formation assay in 2 normal cell lines and 12 NSCLC cell lines: 6 expressing wild-type EGFR and 6 expressing EGFR with activating mutations, including exon 19 deletions, and L858R and T790 M point mutations. The combination indexes of BEZ235 with cisplatin or an EGFR-TKI, BIBW2992 (afatinib), were calculated. The mechanisms triggered by BEZ235 were explored by western blotting analysis. The anti-tumor effect of BEZ235 alone or combined with cisplatin or BIBW2992 were also studied in vivo.. BEZ235 suppressed tumor growth in vitro and in vivo by inducing cell-cycle arrest at G1 phase, but without causing cell death. It also reduced the expression of cyclin D1/D3 by regulating both its transcription and protein stability. Moreover, BEZ235 synergistically enhanced cisplatin-induced apoptosis in NSCLC cells by enhancing or prolonging DNA damage and BIBW2992-induced apoptosis in EGFR-TKI-resistant NSCLC cells containing a second TKI-resistant EGFR mutant.. The dual PI3K/mTOR inhibition by BEZ235 is an effective antitumor strategy for enhancing the efficacy of chemotherapy or targeted therapy, even as a monotherapy, to restrict tumor growth in lung cancer treatment.

Topics: A549 Cells; Afatinib; Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Carcinoma, Non-Small-Cell Lung; Cell Cycle Checkpoints; Cisplatin; Cyclin D1; Cyclin D3; Drug Resistance, Neoplasm; ErbB Receptors; Humans; Imidazoles; Lung Neoplasms; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Quinolines; Signal Transduction; TOR Serine-Threonine Kinases

Emerging studies suggest that microRNAs (miRNAs) play multiple roles in cancer malignancy, including proliferation and acquisition of metastatic potential. Differentially expressed miRNAs responsible for the malignancy of lung cancer were searched by miRNA microarray using a previously established brain metastatic lung cancer model. Twenty-five miRNAs were down-regulated in brain metastatic lung cancer cells. Among those, miR-193b-3p and -5p were chosen for further studies. Their function in metastatic potential and proliferation was examined using Transwell invasion, wound healing, and colony forming assays. The underlying mechanism of tumor-suppressor miR-193b-3p and -5p was explored using reverse transcriptase quantitative polymerase chain reaction (RT-qPCR), Western blot, Argonaute 2-RNA immunoprecipitation (Ago2-RIP), and reporter assays. Both strands of miR-193b were down-regulated in brain metastatic lung cancer cells and in tissues from lung cancer patients. Overexpression of miR-193b-3p and -5p inhibited invasive and migratory activities and diminished clonogenic ability. Conversely, inhibition of miR-193b-3p or -5p increased the metastatic potential and colony forming ability. Cyclin D1 (

Topics: Argonaute Proteins; Cell Line, Tumor; Cyclin D1; Genes, Tumor Suppressor; Humans; LIM Domain Proteins; Lung Neoplasms; MicroRNAs; Neoplasm Metastasis; RNA, Neoplasm

Transcription Elongation Factor A-like 7 (TCEAL7) was first reported as a candidate tumor suppressor gene because of its inactivation in ovarian cancer as a result of promoter methylation. Down-regulation of the TCEAL7 gene expression was also associated with other cancers such as endometrial, breast, brain, prostate, gastric cancers, glioblastoma and linked to tumor phenotypes and clinical outcomes. However, there is no report in the literature investigating the role of TCEAL7 in non-small cell lung cancer. Cyclin D1 is an important molecule in the transition from G1 to S phase of the cell cycle, and is frequently deregulated in cancers. Cylin D1 (CCND1) gene is amplified or overexpressed in a variety of tumors. In our previous study we reported that CCND1 over-expression was not associated with amplification in non-small cell lung cancer. Recently, it has been reported that TCEAL7 regulates CCND1 expression through myc-binding E-box sequences. The aim of this study was to investigate the expression of TCEAL7 gene in non-small cell lung cancer and to determine its effect on the CCND1 expression level. For this purpose, expression levels of TCEAL7 and CCND1 genes were investigated in 50 patients with non-small cell lung cancer by quantitative real time polymerase chain reaction (qRT-PCR). TCEAL7 was under-expressed (68%) in non-small cell lung cancer tumor tissues while CCND1 was over-expressed (42%). The TCEAL7 levels negatively correlated with increased CCND1 expression (p = 0.002).

Topics: Binding Sites; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cyclin D1; Down-Regulation; Female; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Male; Nuclear Proteins; Up-Regulation

To evaluate the anti-tumor activity of ethyl acetate fraction (EFA), extracted with ethanol from the root of ""Dai-Bai-Jie"" in A549 cancer cells and its underlying mechanism.. ""Dai-Bai-Jie"" was extracted with 95% ethanol-aqueous (DBJ-1), 50% ethanol-aqueous (DBJ-2), and water (DBJ-3) by reflux method. 95% ethanol-aqueous extract was separated byethyl acetate (EFA) and n-butyl alcohol (DBJ-5), consecutively. The SRB method was used to evaluate the cytotoxic activity. Annexin V-FITC staining was applied to observe the apoptosis and analyze the cell cycle activated by EFA in A549 tumor cell. Western blot was used to detect the apoptosis/related proteins expressions. A549 tumor cellsbearing nude mice model was employed to measure the tumor volume, mice weight, and tumor inhibition ratio in order to verify the antitumor activity in vivo.. DBJ-1 and EFA showed better cytotoxic activity on A549 tumor cells with IC50 25 and 3.5 ¦Ìg/mL, respectively. EFA can exhibit the proliferation, arrest cell cycle at G0/G1 phase, and induce apoptosis in A549 tumor cells in vitro. The mechanisms of apoptosis induced by EFA may be associated with decreasing Bcl-2 protein expression and increasing p53, Bax, Caspase-3, and Caspase-8 proteins expression. EFA also possessed significant anti-tumor efficacy in nude mice, and little toxicity was observed in the host.. EAF could induce A549 tumor cells apoptosis and G0/G1 cell cycle arrest. A549 tumor cells apoptosis induced by EAF may be associated with the decrease in the ratio of Bcl-2 and Bax mRNA levels, and increase in the expression of p53, Caspase-3, and Caspase-8 proteins.

Topics: A549 Cells; Acetates; Animals; Antineoplastic Agents, Phytogenic; bcl-2-Associated X Protein; Caspase 3; Caspase 8; Cell Proliferation; Cyclin D1; Drugs, Chinese Herbal; Humans; Lung Neoplasms; Male; Marsdenia; Mice; Mice, Inbred BALB C; Mice, Nude; Plant Roots

Esculetin was identified to inhibit cell proliferation and induce apoptosis or cell cycle arrest in several cancer cell lines. However, the effect of esculetin on lung cancer remains elusive.. The anti-proliferative role of esculetin in murine Lewis lung carcinoma (LLC) cells was evaluated by 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) and colony formation assays. BALB/c mice were subcutaneously injected with LLC cells to investigate the inhibitory effect of esculetin on the growth of lung cancer xenograft. Invasive ability was detected in esculetin treated and untreated LLC cells by transwell assay. The association between esculetin and Wnt/β-catenin signaling, as well as nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), was confirmed by testing the expression of c-myc, Cyclin D1 and NF-κB using Western blot.. Esculetin treatment in LLC cells led to significant decrease of cell proliferation in a time- and dose-dependent manner. After injection of LLC cells into mice, reduced size and weight of tumors were observed in esculetin treated mice compared to untreated mice. However, no difference in cell invasion was observed between the treated and untreated LLC cells. Notably decreased expression of c-myc, Cyclin D1 and NF-κB were observed in LLC cells with esculetin treatment compared to untreated cells.. Esculetin plays an inhibitory role in the growth of lung cancer by down-regulating c-myc, Cyclin D1 and NF-κB.

Topics: Animals; Apoptosis; beta Catenin; Carcinoma, Lewis Lung; Cell Proliferation; Coloring Agents; Cyclin D1; Down-Regulation; Female; Heterografts; Lung Neoplasms; Mice; Mice, Inbred BALB C; Neoplasm Invasiveness; NF-kappa B; Salivary alpha-Amylases; Tetrazolium Salts; Thiazoles; Tumor Stem Cell Assay; Umbelliferones; Wnt Proteins

This study is aimed at detecting the anti-tumor efficacy of a new berberine (BBR) derivative 8-cetylberberine (HBBR), which has a significant improvement in hydrophobicity and pharmacological effects compared to BBR.. The human non-small lung cancer cell line A549 and normal human lung epithelial cells (MRC-5) were cultured to observe inhibition in vitro. Cell viability was analyzed via 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The effect of HBBR on cell cycle arrest and apoptosis were assessed by flow cytometry and western blotting. In animal studies, BALB/c nude mice were subcutaneously injected with A549 cells in the armpit and administrated with different dose of HBBR and BBR. The body weight, organ coefficient and tumor inhibitory rate were recorded to evaluate the effect of HBBR in vivo.. The data showed that HBBR induced G1-phase cycle arrest by interfering with the expression of Cyclins D1 and Cyclin E1, increased apoptosis by inducing caspase pathway, and probably inhibited the PI3K/Akt pathway in A549 cells. In addition, animal experiments proved that oral administration of HBBR at a dose of 10mg/kg could significantly inhibit tumor growth, which is stronger than the 120mg/kg dose of BBR treatment.. Our results suggest that HBBR showed a significantly higher anti-tumor efficacy than BBR in vitro and in vivo and could be a potential therapy for lung cancers.

Topics: A549 Cells; Animals; Antineoplastic Agents, Phytogenic; Apoptosis; Berberine; Biomarkers, Tumor; Body Weight; Carcinoma, Non-Small-Cell Lung; Cell Cycle; Cell Proliferation; Cell Survival; Cyclin D1; Dose-Response Relationship, Drug; Humans; Lung Neoplasms; Male; Mice; Mice, Inbred BALB C; Signal Transduction; Tumor Burden; Xenograft Model Antitumor Assays

MiR-374a appears to play a complex role in non-small-cell lung cancer (NSCLC). Here, we demonstrate a dual role for miR-374a in NSCLC pathogenesis. The effects and modulatory mechanisms of miR-374a on cell growth, migration, invasion, and in vivo tumorigenesis and metastasis in nude mice were also analyzed. The expression of miR-374a was examined in NSCLC and non-cancerous lung tissues by quantitative real-time reverse transcription-PCR (qRT-PCR), and in situ hybridization, respectively. miR-374a directly targets CCND1 and inactivates PI3K/AKT and Ras-mediated cell cycle signalings, as well as epithelial-mesenchymal transition (EMT). This not only dramatically suppressed cell growth, migration, invasion,and metastasis, but also elevated A549 and pc-9 NSCLC cell sensitivity to cisplatin (DDP) while increasing survival time of tumor-bearing mice. Interestingly, miR-374a serves an inverse function in SPCA-1 and H1975 NSCLC cells by directly targeting PTEN to activate Wnt/β-catenin and Ras signalings and its downstream cascade signals. Surprisingly, transcription factor c-Jun bound to the promoter region of human miR-374a and suppressed miR-374a in A549 and pc-9 cells while inducing it in SPCA-1 and H1975 cells. Increased levels of miR-374a appeared to serve a protective role by targeting CCND1 in early-stage NSCLC (Stages I and II). Inversely, increased miR-374a was an unfavorable factor when targeting PTEN in more advanced staged NSCLC patients. Our studies are the first to demonstrate that miR-374a plays divergent roles in NSCLC pathogenesis at different stages of the disease and implicate the potential application of miR-374a targeting for cancer therapy.

Topics: 3' Untranslated Regions; Animals; Base Sequence; Carcinoma, Non-Small-Cell Lung; Cell Cycle; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin D1; Epithelial-Mesenchymal Transition; Humans; Lung Neoplasms; Male; Mice, Inbred BALB C; Mice, Nude; MicroRNAs; Neoplasm Invasiveness; Neoplasm Metastasis; Phosphatidylinositol 3-Kinases; Promoter Regions, Genetic; Protein Binding; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-jun; PTEN Phosphohydrolase; Wnt Signaling Pathway

In this study, a peptide-peptide co-administration therapy between hybrid peptide kla-TAT and cationic anticancer peptide HPRP-A1 was designed to increase the anticancer activity of the combination peptides through synergistic effect. kla is a pro-apoptotic peptide which could induce rapid cancer cell apoptosis by disruption the mitochondrial membrane when internalized the cells. To enhance more kla peptides pass through cell membrane, a double improvement strategy was designed by chemically conjugation with cell penetration peptide TAT as well as co-administration with cationic membrane active peptide HPRP-A1, and the double anticancer mechanism of the kla-TAT peptide and HPRP-A1 including membrane disruption and apoptosis induction was verified through in vitro experiments. The CompuSyn synergism/antagonism analysis showed that kla-TAT acted synergistically with HPRP-A1 against a non-small cell lung cancer (NSCLC) A549 cell line. The anticancer activities of the two peptides were dramatically increased by co-administration, under the mechanism of cell membrane disruption, caspase-dependent apoptosis induction, as well as cyclin-D1 down-regulation based G1 phase arrest. We believe that the synergic therapeutic strategy would be a meaningful method for the anticancer peptides used in cancer treatment.

Topics: A549 Cells; Antineoplastic Agents; Apoptosis; Carcinoma, Non-Small-Cell Lung; Cyclin D1; Drug Synergism; G1 Phase Cell Cycle Checkpoints; Humans; Intercellular Signaling Peptides and Proteins; Lung Neoplasms; Peptides

Despite the development of numerous therapeutics targeting the epithelial growth factor receptor (EGFR) for non-small cell lung carcinoma (NSCLC), the application of these drugs is limited because of drug resistance. Here, we investigated the antitumor effect of calcium-mediated degradation of EGFR pathway-associated proteins on NSCLC. First, lactate calcium salt (LCS) was utilized for calcium supplementation. Src, α-tubulin and EGFR levels were measured after LSC treatment, and the proteins were visualized by immunocytochemistry. Calpeptin was used to confirm the calcium-mediated effect of LCS on NSCLC. Nuclear expression of c-Myc and cyclin D1 was determined to understand the underlying mechanism of signal inhibition following EGFR and Src destabilization. The colony formation assay and a xenograft animal model were used to confirm the in vitro and in vivo antitumor effects, respectively. LCS supplementation reduced Src and α-tubulin expression in NSCLC cells. EGFR was destabilized because of proteolysis of Src and α-tubulin. c-Myc and cyclin D1 expression levels were also reduced following the decrease in the transcriptional co-activation of EGFR and Src. Clonogenic ability and tumor growth were significantly inhibited by LSC treatment-induced EGFR destabilization. These results suggest that other than specifically targeting EGFR, proteolysis of associated molecules such as Src or α-tubulin may effectively exert an antitumor effect on NSCLC via EGFR destabilization. Therefore, LCS is expected to be a good candidate for developing novel anti-NSCLC therapeutics overcoming chemoresistance.

Topics: Animals; Antineoplastic Agents; Calcium Compounds; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cyclin D1; Dipeptides; ErbB Receptors; Female; Humans; Lactates; Lung Neoplasms; Mice; Mice, Inbred BALB C; Mice, Nude; Protein Stability; Proteolysis; src-Family Kinases; Tubulin

The chronic inflammatory microenvironment within or surrounding the primary renal cell carcinoma (RCC) site promotes oncogenic transformation as well as contributes to the development of metastasis. G3BP stress granule assembly factor 1 (G3BP1) was found to be involved in the regulation of multiple cellular functions. However, its functions in RCC have not been previously explored. Here, we first showed that the expression of G3BP1 is elevated in human RCC and correlates with RCC progression. In cultured RCC cells, knockdown of G3BP1 results in inhibition of tumor cell proliferation, migration, and invasion, consistently with the alteration of epithelial-mesenchymal transition (EMT) and cell proliferative markers, including Cadherins, Vimentin, Snail, Slug, c-Myc, and cyclin D1. Remarkably, knockdown of G3BP1 dramatically impaired the signaling connection of pro-inflammatory cytokine IL-6 stimulation and downstream STAT3 activation in RCC, thus eventually contributing to the disruption of IL-6-elicited RCC migration and metastasis. In addition, in vivo orthotopic tumor xenografts results confirmed that knockdown of G3BP1 suppressed RCC tumor growth and metastasis in mice. Collectively, our findings support the notion that G3BP1 promotes tumor progression and metastasis through IL-6/G3BP1/STAT3 signaling axis in RCC.

Topics: Aged; Animals; Cadherins; Carcinoma, Renal Cell; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin D1; DNA Helicases; Female; Gene Expression Regulation, Neoplastic; Humans; Interleukin-6; Kidney Neoplasms; Liver Neoplasms; Lung Neoplasms; Male; Mice; Middle Aged; Neoplasm Grading; Nephrectomy; Poly-ADP-Ribose Binding Proteins; RNA Helicases; RNA Recognition Motif Proteins; RNA, Small Interfering; Signal Transduction; Snail Family Transcription Factors; STAT3 Transcription Factor; Tumor Burden; Vimentin; Xenograft Model Antitumor Assays

Mutant EGFR Non-small cell lung cancer has benefit from gefitinib, but it has limited effect for wild-type EGFR tumors. Shikonin, a natural naphthoquinone isolated from a traditional Chinese medicine, the plant Lithospermum erythrorhizon (zicao), not only can inhibit the tumor growth, but also overcome cancer drug resistance. Our aim is to investigate whether shikonin can enhance antitumor effect of gefitinib in EGFR wild-type lung cancer cells in vitro and in vivo.. CCK-8 was used to determine the proliferation of EGFR wild-type non-small cell lung cancer. Apoptosis and cell cycle were detected by flow cytometry. PKM2, STAT3, p-STAT3 and cyclinD1 were detected by Western blot. A549 tumor model was established to observe the antitumor effect of shikonin combination with gefitinib in vivo.. The results showed that combination of shikonin with gefitinib exhibited synergistic antitumor effect in vitro and in vivo. Its potential molecular mechanisms may be associated with inhibition of PKM2/STAT3/cyclinD1.. These results provide a promising therapeutic approach for the treatment of wild-type EGFR non-small cell lung cancer.

Topics: A549 Cells; Animals; Antineoplastic Agents; Antineoplastic Agents, Phytogenic; Carcinoma, Non-Small-Cell Lung; Carrier Proteins; Cell Line, Tumor; Cell Survival; Cyclin D1; Drug Synergism; ErbB Receptors; Gefitinib; Humans; Immunohistochemistry; Lung Neoplasms; Membrane Proteins; Mice; Mice, Nude; Naphthoquinones; Quinazolines; Signal Transduction; Sincalide; STAT3 Transcription Factor; Thyroid Hormone-Binding Proteins; Thyroid Hormones

The identification and targeting of key molecular drivers of melanoma and breast and lung cancer have substantially improved their therapy. However, subtypes of each of these three common, lethal solid tumors lack identified molecular drivers, and are thus not amenable to targeted therapies. Here we show that pleckstrin homology domain-interacting protein (PHIP) promotes the progression of these "driver-negative" tumors. Suppression of PHIP expression significantly inhibited both tumor cell proliferation and invasion, coordinately suppressing phosphorylated AKT, cyclin D1, and talin1 expression in all three tumor types. Furthermore, PHIP's targetable bromodomain is functional, as it specifically binds the histone modification H4K91ac. Analysis of TCGA profiling efforts revealed PHIP overexpression in triple-negative and basal-like breast cancer, as well as in the bronchioid subtype of nonsmall cell lung cancer. These results identify a role for PHIP in the progression of melanoma and breast and lung cancer subtypes lacking identified targeted therapies. The use of selective, anti-PHIP bromodomain inhibitors may thus yield a broad-based, molecularly targeted therapy against currently nontargetable tumors.

Topics: Animals; Breast; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Female; Gene Expression Regulation, Neoplastic; Humans; Intracellular Signaling Peptides and Proteins; Lung Neoplasms; Melanoma; Pleckstrin Homology Domains; Proto-Oncogene Proteins c-akt; Triple Negative Breast Neoplasms

Spirulina is a well-described and popular dietary supplement derived from Arthrospira algae. In the present study, the anticancer potential of a water extract of a commercial Spirulina product (SE) against the human non-small-cell lung carcinoma A549 cell line was evaluated. After qualitative analysis, we investigated the effect of SE on cell viability, proliferation, and morphology. Furthermore, the influence of SE on regulation of the cell cycle, induction of apoptosis in lung cancer cells, and expression of cell cycle/apoptosis-related proteins was evaluated. Additionally, we examined the cytotoxic effect of SE on normal human skin fibroblasts (HSF). Our studies revealed that SE significantly reduced cancer cell viability and proliferation, which was accompanied by cell cycle inhibition in the G

Topics: A549 Cells; Antineoplastic Agents; Apoptosis; bcl-2-Associated X Protein; Cell Proliferation; Cell Survival; Cyclin D1; Cyclin-Dependent Kinase 4; Dose-Response Relationship, Drug; Fibroblasts; G1 Phase Cell Cycle Checkpoints; Humans; Lung Neoplasms; Necrosis; Phosphorylation; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-bcl-2; Retinoblastoma Protein; Signal Transduction; Solvents; Spirulina; Water

Lung cancer is the most common cause of cancer‑associated mortality. MicroRNAs (miRNAs), as oncogenes or tumor suppressor genes, serve crucial roles not only in tumorigenesis, but also in tumor invasion and metastasis. Although miRNA‑let‑7a (let‑7a) has been reported to suppress cell growth in multiple cancer types, the biological mechanisms of let‑7a in lung adenocarcinoma are yet to be fully elucidated. In the present study, the molecular roles of let‑7a in lung adenocarcinoma were investigated by detecting its expression in lung adenocarcinoma tissues and exploring its roles in the regulation of lung cancer cell proliferation. Let‑7a expression was identified to be downregulated in lung adenocarcinoma tissues compared with normal tissues. Overexpression of let‑7a effectively suppressed cancer cell proliferation, migration and invasion in H1299 and A549 cells. Let‑7a also induced cell apoptosis and cell cycle arrest. Furthermore, let‑7a significantly inhibited cell growth by directly regulating cyclin D1 signals. This novel regulatory mechanism of let‑7a in lung adenocarcinoma provides possible avenues for future targeted therapies of lung cancer.

Topics: Adenocarcinoma; Adult; Aged; Apoptosis; Biomarkers, Tumor; Case-Control Studies; Cell Cycle Checkpoints; Cell Movement; Cell Proliferation; Cyclin D1; Female; Follow-Up Studies; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Male; MicroRNAs; Middle Aged; Neoplasm Invasiveness; Prognosis; Signal Transduction; Survival Rate; Tumor Cells, Cultured

The purpose of this study was to investigate the potential role of the cyclin D1 gene G870A polymorphism in the likelihood of the development of lung cancer and the overall survival of lung cancer patients in the North Indian population.. The study consisted of 353 lung cancer cases and 351 age- and gender-matched healthy controls. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLPP) was done for the CCND1 gene. The association analysis was done using the multiple linear regression, and the survival analysis was done using the Kaplan-Meier and the Cox regression models.. The GA genotype was associated with an increased risk for overall lung cancer (odds ratio [OR] = 1.63; p = 0.01). Combined variant genotype showed a significant association for overall lung cancer (OR 1.50; p = 0.03). In addition, smokers with the carrier genotype of CCND1 were found to have a significantly higher risk for lung cancer (OR 1.57; p = 0.04). No significant correlation was observed between the overall survival of lung cancer patients and CCND1 polymorphism. However, on stratifying the subjects on the basis of histology, it was evident that small-cell lung cancer (SCLC) patients carrying the mutant (AA) genotype showed nearly a fivefold increased mortality rate compared to the wild (GG) genotype (p = 0.03).. Our results suggest that polymorphic CCND1 may increase the risk of lung cancer in smokers from North India, and it may be associated with the overall survival of SCLC patients.

Topics: Adult; Aged; Aged, 80 and over; Asian People; Case-Control Studies; Cyclin D1; Female; Genetic Predisposition to Disease; Genotype; Humans; India; Lung Neoplasms; Male; Middle Aged; Polymorphism, Restriction Fragment Length; Polymorphism, Single Nucleotide; Risk; Small Cell Lung Carcinoma; Young Adult

Dysregulation of epidermal growth factor receptor (EGFR) signaling is responsible for the resistance to EGFR tyrosine kinase inhibitors (TKIs), such as gefitinib and erlotinib, and is thereby associated with the progression of tumors in non‑small cell lung cancers (NSCLCs). Immunoblotting results revealed that geranylgeranyl transferase 1 inhibitor (GGTI)‑298, a geranylgeranyl transferase 1 inhibitor with potential antitumor effects, effectively inhibited the phosphorylation of EGFR and its downstream target protein kinase B (AKT). A combination of gefitinib and GGTI‑298 amplified the inhibition of the EGFR‑AKT signaling pathway. In addition, GGTI‑298 treatment produced a synergistic effect on the inhibition of proliferation as indicated by the combination index values of <1 when combined with gefitinib in the NSCLC cell lines HCC827 and A549. These synergistic effects were also observed to induce apoptosis and migration inhibition. Further mechanistic studies demonstrated that GGTI‑298 inhibited the activity of Ras homolog family member A (RhoA), and downregulation of RhoA with small interfering RNA impaired the phosphorylation of EGFR, which suggested that EGFR inhibition by GGTI‑298 may be exerted mainly through RhoA mediation. These results presented a novel, promising therapeutic strategy involving a combination of two drugs for targeting EGFR signaling in lung cancer.

Topics: Alkyl and Aryl Transferases; Antineoplastic Agents; Apoptosis; Benzamides; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin D1; Drug Synergism; Enzyme Inhibitors; ErbB Receptors; Gefitinib; Humans; Lung Neoplasms; Models, Biological; Phosphorylation; Proto-Oncogene Proteins c-akt; rhoA GTP-Binding Protein; Signal Transduction

Lung cancer remains one of the most aggressive human malignancies with a low survival rate. Hyperoside (quercetin-3-O-β-d-galactopyranoside) is a flavonol glycoside with an anti-cancer activity. The microRNA-let-7 was widely regarded as a tumor suppressor in human tumors. Here, we investigated the role of hyperoside and let-7a-5p on the lung cancer cell proliferation, cell cycle and apoptosis in A549 cells in vitro. Our results showed that hyperoside could inhibit the proliferation of A549 cells through inducing apoptosis and G1/S phase arrest. Let-7a-5p could inhibit the proliferation of A549 cells via inhibiting the process of G1/S phase. Additionally, hyperoside and let-7a-5p had a synergetic effect on suppressing the proliferation of A549 cells; microRNA-let-7a-5p directly regulated the expression of CCND1 in A549 cells. Our study illustrated that hyperoside and microRNA-let7a-5p might provide a synergistic effect on anti-cancer, which may provide a new idea for lung cancer treatment.

Topics: 3' Untranslated Regions; A549 Cells; Cell Proliferation; Cell Survival; Cyclin D1; G1 Phase Cell Cycle Checkpoints; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; MicroRNAs; Quercetin

Overexpression of the deubiquitylase ubiquitin-specific peptidase 22 (USP22) is a marker of aggressive cancer phenotypes like metastasis, therapy resistance, and poor survival. Functionally, this overexpression of USP22 actively contributes to tumorigenesis, as USP22 depletion blocks cancer cell cycle progression in vitro, and inhibits tumor progression in animal models of lung, breast, bladder, ovarian, and liver cancer, among others. Current models suggest that USP22 mediates these biological effects via its role in epigenetic regulation as a subunit of the Spt-Ada-Gcn5-acetyltransferase (SAGA) transcriptional cofactor complex. Challenging the dogma, we report here a nontranscriptional role for USP22 via a direct effect on the core cell cycle machinery: that is, the deubiquitylation of the G1 cyclin D1 (CCND1). Deubiquitylation by USP22 protects CCND1 from proteasome-mediated degradation and occurs separately from the canonical phosphorylation/ubiquitylation mechanism previously shown to regulate CCND1 stability. We demonstrate that control of CCND1 is a key mechanism by which USP22 mediates its known role in cell cycle progression. Finally, USP22 and CCND1 levels correlate in patient lung and colorectal cancer samples and our preclinical studies indicate that targeting USP22 in combination with CDK inhibitors may offer an approach for treating cancer patients whose tumors exhibit elevated CCND1.

Topics: Colorectal Neoplasms; Cyclin D1; Epigenesis, Genetic; G1 Phase; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; MCF-7 Cells; Protein Stability; Proteolysis; Thiolester Hydrolases; Ubiquitin Thiolesterase; Ubiquitination

p27 plays critical roles in cell proliferation, differentiation, and apoptosis, which have been well studied in mammals and Drosophila. However, the mechanisms underlying p27 regulation of the cell cycle have not been thoroughly researched. In this study, Genevestigator, Kaplan-Meier Plotter, and the Human Protein Atlas databases were used to analyze the expression of p27, cell division protein kinase 6 (CDK6), and cyclin D1 (CCND1), as well as its prognostic value in different tumor tissues and corresponding normal tissues. Quantitative PCR and immunohistochemistry were used to detect the expression of p27, CDK6, and CCND1 in the tissues of cancer patients. The effects of p27, CDK6, and CCND1 on the proliferation of lung cancer cells were examined by the MTT assay, and flow cytometry was used to investigate the mechanism by which p27 affected cell proliferation. Immunofluorescence, co-immunoprecipitation, and Western blotting were used to determine if p27 interacted with CDK and CCND1 to regulate the cell cycle. The results showed that p27, CDK6, and CCND1 played different roles in tumorigenesis and development, which are in accordance with CDK6 and CCND1 in affecting the cell cycle and cell proliferation. p27 regulated the cell cycle and inhibited cell proliferation by affecting formation of the cell cycle-dependent complex CDK6/CCND1, but did not directly affect the expression of CDK6 and CCND1. Moreover, CCND1 did not regulate the cell cycle alone, but rather, functioned together with CDK6. This study provides insights into the effects of p27 on tumor formation and development, and the underlying regulatory mechanisms.

Topics: A549 Cells; Animals; Cell Cycle Checkpoints; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase 6; Cyclin-Dependent Kinase Inhibitor p27; Drosophila; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Mice; Neoplasms; Protein Binding

GCNT3 is a member of N-acetylglucosaminyltransferase family involved with mucin biosynthesis. GCNT3 aberrant expression is known to promote the progression of several human cancers. However, its role in tumorigenesis and the progression of non-small cell lung cancer (NSCLC) has not been well-characterized. Our study investigated the functional mechanisms of GCNT3 regulated by microRNAs (miRNAs) in NSCLC.. The differential expression of mRNAs in NSCLC tissues and matched adjacent non-cancerous lung tissues from patients in Xuanwei, Yunnan province, China, was screened via mRNA microarray. The expression of GCNT3 and its correlation with NSCLC progression was measured in 92 paired tumor tissues and adjacent normal tissues. The functions of GCNT3 in NSCLC cells and its underlying mechanisms were measured using siRNA and GCNT3-expression vectors. The miRNA immunoprecipitation (miRIP) method was used to identify the miRNAs targeting GCNT3. The protein were measured using western blot assay, and the mRNAs were measured by quantitative real-time PCR (qRT-PCR) assay. Cell proliferation was measured using Cell Counting Kit-8 (CCK-8) and a colony forming assays; cell migration and invasion assays were performed using 24-well Transwell chambers with 8-μm pores filter, and analyses of the cell cycle and apoptosis were performed via flow cytometric analysis. The dual luciferase reporter assay was performed to confirm whether GCNT3 gene was a direct target of miR-302b-3p.. GCNT3 was found to be highly expressed in both NSCLC tissues and cell lines, and higher expression correlated significantly with advanced tumor-node-metastasis (TNM) stage, positive lymph node metastasis, and poor overall survival. Knockdown of GCNT3 inhibited the proliferation, migration and invasion ability of NSCLC cells, while overexpression facilitated these activities. Further mechanistic experiments using miRIP and dual luciferase reporter assays revealed that GCNT3 was a direct target of miR-302b-3p. Low expression of miR-302b-3p was found in NSCLC cells and negatively correlated with GCNT3 levels, while miR-302b-3p overexpression inhibited the proliferation, migration and invasion of NSCLC cells. Co-transfection with miR-302b-3p and the expression vector of GCNT3 abrogated the effects of mir-302b-3p, confirming that miR-302b-3p inhibited NSCLC progression by targeting GCNT3. Western blotting revealed that E-cadherin, N-cadherin, vimentin, p-Erk and cyclin D1 were downstream molecules of miR-302b-3p/GCNT3 pathway.. miR-302b-3p/GCNT3 axis regulated cell proliferation, migration, and invasion by activating the Erk signaling pathway and epithelial-mesenchymal transition (EMT), which was identified as a potential therapeutic target for NSCLC.

Topics: 3' Untranslated Regions; Antagomirs; Carcinoma, Non-Small-Cell Lung; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin D1; Down-Regulation; Epithelial-Mesenchymal Transition; Female; Humans; Kaplan-Meier Estimate; Lung Neoplasms; Male; MicroRNAs; Middle Aged; N-Acetylglucosaminyltransferases; RNA Interference; RNA, Small Interfering

Non-small cell lung cancer (NSCLC) is the most common type of lung tumor. Deregulation of microRNA may be involved in the occurrence of NSCLC and we aimed to find the potential prognostic biomarkers for NSCLC. The microRNA microarray expression profiles were downloaded from GEO dataset and then generated by applying robust multi-array average (RMA). The normalized data was analyzed with a Bioconductor package linear model for microarray data and an independent dataset was used to inspect the results. Then, the differentially expressed genes were identified using the limma package. Besides, in order to investigate the function of the differentially expressed microRNA in NSCLC, the GO and KEGG functional enrichment analysis were applied, and the GSEA analysis was performed for mining the therapeutic candidates. A total of 160 differentially expressed microRNAs were identified, among which 37 microRNAs showed significant expression changes (up-regulated and down-regulated) with the same method in the validation dataset GSE74190. Multiple cancer-related pathways, such as AMPK signaling pathway, AMPK signaling pathway, non-small cell lung cancer signaling pathway, were determined by performing the functional enrichment analysis. Besides, the results of GSEA analysis showed that the CCND1 was mostly enriched in lung cancer group. In conclusion, a set of differentially expressed microRNAs in NSCLC was identified and the CCND1 gene was determined as the potential prognostic biomarkers for NSCLC, providing useful information for discovery of future therapeutic targets and candidates in the clinical management of NSCLC.

Topics: Biomarkers, Tumor; Carcinoma, Non-Small-Cell Lung; Cyclin D1; Gene Regulatory Networks; Humans; Lung Neoplasms; MicroRNAs; RNA, Messenger

The telomerase/telomere interacting protein PinX1 has been suggested as a tumor suppressor. However, the clinical and biological significance of PinX1 in human non-small cell lung cancer (NSCLC) is unclear.. PinX1 gene/expression pattern and its association with NSCLC patient survival were analyzed in cBioportal Web resource and two cohorts of NSCLC samples. A series of in vivo and in vitro assays were performed to elucidate the function of PinX1 on NSCLC cells proliferation and underlying mechanisms.. More frequency of gene PinX1 homozygous deletion and heterozygote deficiency was first retrieved from cBioportal Web resource. Low expression of PinX1 correlated with smoking condition, histological type, T stage, N stage, M stage and TNM stage, and was an independent predictor for overall survival in a learning cohort (n = 93) and a validation cohort (n = 51) of NSCLC patients. Furthermore, knockdown of PinX1 dramatically accelerated NSCLC cell proliferation and G1/S transition, whereas ectopic overexpression of PinX1 substantially inhibited cell viability and cell cycle transition in vitro and in vivo. p15/cyclin D1 pathway and BMP5 might contribute to PinX1-associated cell proliferation and cell cycle transition.. The cost-effective expression of PinX1 could constitute a novel molecular predictor/marker for NSCLC management.

Topics: Adult; Aged; Animals; Biomarkers, Tumor; Bone Morphogenetic Protein 5; Carcinoma, Non-Small-Cell Lung; Cell Cycle; Cell Cycle Proteins; Cell Line, Tumor; Cell Proliferation; Cell Transformation, Neoplastic; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p15; Databases, Nucleic Acid; Disease Models, Animal; Female; Gene Deletion; Gene Silencing; Humans; Lung Neoplasms; Male; Mice; Middle Aged; Neoplasm Grading; Neoplasm Staging; Prognosis; Signal Transduction; Tumor Suppressor Proteins; Xenograft Model Antitumor Assays

Farnesoid X receptor (FXR), a nuclear receptor for maintaining bile acid homeostasis, has been recognized as a tumor suppressor in enterohepatic tissues. However, its expression and functional role in non-small cell lung cancer (NSCLC) remain unclear. We report that FXR is significantly increased in NSCLC and that it predicts poor clinical outcomes in NSCLC patients. FXR knockdown in NSCLC cells inhibited in vitro cell proliferation, blocked xenograft growth in nude mice, and delayed the G1/S transition of the cell cycle, whereas ectopic overexpression of FXR promoted NSCLC cell proliferation. Mechanistic analysis demonstrated that FXR could directly bind to an inverted repeat-0 sequence in the CCND1 promoter and activate its transcription. Cyclin D1 overexpression rescued NSCLC cells from the delayed G1/S transition and the impaired cell proliferation induced by FXR knockdown. Importantly, a positive correlation between the expression of FXR and cyclin D1 was confirmed in NSCLC samples, and patients with high expression of both FXR and cyclin D1 had the worst prognosis. In summary, our results suggest that FXR has oncogenic potential in NSCLC development, providing mechanistic insights that could be exploited for both prognostic and therapeutic purposes.

Topics: Adult; Aged; Carcinoma, Non-Small-Cell Lung; Cell Cycle Checkpoints; Cyclin D1; Female; Fragile X Mental Retardation Protein; Gene Expression Regulation, Neoplastic; Humans; Kaplan-Meier Estimate; Lung Neoplasms; Male; Middle Aged; Neoplasm Grading; Neoplasm Metastasis; Neoplasm Staging; Oncogene Proteins; Prognosis; Promoter Regions, Genetic; Proportional Hazards Models; Protein Binding; Proto-Oncogene Mas; Trans-Activators; Transcriptional Activation

Kctd20 (potassium channel tetramerization protein domain containing 20) is a positive regulator of Akt signaling. However, the role of Kctd20 during the course of tumorigenesis and development is unclear. Using immunohistochemistry, we demonstrated that, in non-small cell lung cancer (NSCLC) patients, Kctd20 protein expression significantly correlates with advanced TNM stage (P < 0.001), positive status for regional lymph node metastasis (P = 0.019), and poor overall survival (P = 0.013). Proliferation and invasion assays showed that Kctd20 dramatically promotes the proliferation and invasion of NSCLC cells (P = 0.007 and P < 0.001, respectively). Subsequent Western Blot and qPCR experiments revealed an upregulation of Cyclin D1 and downregulation of E-cadherin in Kctd20-overexpressing cells. After depleting Kctd20, downregulaton of Cyclin D1, and upegulation of E-cadherin was observed. After overexpressing Kctd20, the levels of phosphorylated Fak (Tyr397) and Akt (Thr308) increased, while after transfection with Kctd20-siRNA these phosporylated proteins were downregulated. Moreover, in Kctd20-overexpressing cells, treatment with an Akt inhibitor reduced expression of p-Akt and Cyclin D1, enhanced E-cadherin expression, and did not impact p-Fak levels. When Kctd20-overexpressing cells were treated with a Fak inhibitor, the same effects were seen, and the level of p-Akt was reduced. Our results suggest that Kctd20 impacts proliferation and invasion of NSCLC through enhancing Fak (Tyr397) and Akt (Thr 308) phosphorylation. Kctd20 may predict prognosis and be targeted therapeutically in NSCLC.

Topics: Cadherins; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Proliferation; Cohort Studies; Cyclin D1; Focal Adhesion Kinase 1; Humans; Intracellular Signaling Peptides and Proteins; Lung Neoplasms; Neoplasm Invasiveness; Nuclear Proteins; Phosphorylation; Prognosis; Proto-Oncogene Proteins c-akt; Real-Time Polymerase Chain Reaction; Signal Transduction; Survival Analysis; Wound Healing

Rab11a, an evolutionarily conserved Rab GTPases, plays important roles in intracellular transport and has been implicated in cancer progression. However, its role in human non-small cell lung cancer (NSCLC) has not been explored yet. In this study, we discovered that Rab11a protein was upregulated in 57/122 NSCLC tissues. Rab11a overexpression associated with advanced TNM stage, positive nodal status and poor patient prognosis. Rab11a overexpression promoted proliferation, colony formation, invasion and migration with upregulation of cyclin D1, cyclin E, and downregulation of p27 in NSCLC cell lines. Nude mice xenograft demonstrated that Rab11a promoted in vivo cancer growth. Importantly, we found that Rab11a induced YAP protein and inhibited Hippo signaling. Depletion of YAP abolished the effects of Rab11a on cell cycle proteins and cell proliferation. Furthermore, immunoprecipitation showed that Rab11a interacted with YAP in lung cancer cells. In conclusion, the present study suggestes that Rab11a serves as an important oncoprotein and a regulator of YAP in NSCLC.

Topics: Adaptor Proteins, Signal Transducing; Animals; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin D1; Cyclin E; Cyclin-Dependent Kinase Inhibitor p27; Down-Regulation; Female; Hippo Signaling Pathway; Humans; Immunohistochemistry; Lung; Lung Neoplasms; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Middle Aged; Neoplasm Invasiveness; Neoplasm Staging; Oncogene Proteins; Phosphoproteins; Prognosis; Protein Serine-Threonine Kinases; rab GTP-Binding Proteins; Signal Transduction; Transcription Factors; Up-Regulation; Xenograft Model Antitumor Assays; YAP-Signaling Proteins

Overexpression of Annexin A2 (Anxa2) is positively correlated with breast cancer progression, drug resistance, and poor prognosis of patients with breast cancer. Tyr23 Phosphorylation by Src-family tyrosine kinase is an important post-translational modification of Anxa2. This modification regulates the subcellular localization and functions of Anxa2 and has significant effects on cell proliferation, migration, and invasion. This study aims at revealing the association of Anxa2-Tyr23 phosphorylation in Anxa2-mediated acceleration of breast cancer progression and their elaborate molecular mechanisms.. Cell biological function experiments were performed to determine the effects of Anxa2-Tyr23 Phosphorylation on breast cancer cell proliferation and invasion in vitro and metastasis in vivo. The interaction of Tyr23 phosphorylated Anxa2 and STAT3 was verified by co-immunoprecipitation assay. Related mRNA and protein expression levels of cyclin D1 and MMP2/9 and phosphorylation level of STAT3 were detected.. Anxa2-Tyr23 phosphorylation is necessary for proliferation, invasion, and metastasis of breast cancer cells in vitro and in vivo. Tyr23 phosphorylated Anxa2 binds and enhances the sensitivity of STAT3 activation in response to IL-6, thereby increasing the protein and mRNA expression levels of cyclin D1 and MMP2/9 which are STAT3 key target genes and serve pivotal regulatory functions in cell proliferation and invasion, respectively.. Our findings further confirmed the regulatory role of Anxa2 and revealed the direct relationship between Anxa2-Tyr23 phosphorylation and activation of STAT3. Moreover, this study provides novel insights into the function of Anxa2-Tyr23 phosphorylation in signal transduction for further understanding of the mechanism through which Anxa2 promotes the progression of breast cancer.

Topics: Animals; Annexin A2; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Female; Humans; Lung Neoplasms; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Mice; Neoplasm Invasiveness; Neoplasm Transplantation; Phosphorylation; STAT3 Transcription Factor

Ubiquitin specific protease 32 (USP32) is a highly conserved but uncharacterized gene, which has been reported to be associated with growth of breast cancer cells. However, the role of USP32 in human small cell lung cancer (SCLC) has not been uncovered. The aim of this study was to investigate and evaluate the clinical significance of USP32 in patients with SCLC.. Expression of USP32 was firstly investigated using public online data sets and then determined in SCLC tissues and cell lines using quantitative real-time PCR, Western blotting and immunohistochemical staining. SCLC cells were transfected with a small-interfering RNA targeting USP32 mRNA and analysed for cell viability, proliferation ability, cell cycle distribution, apoptosis and invasion.. USP32 was found to be overexpressed in SCLC tissues compared with normal tissues. High USP32 expression was significantly correlated with disease stage and invasion. In vitro experiments demonstrated that silencing of USP32 caused a significant decrease in the proliferation and migration rate of cells. Furthermore, USP32 silencing arrested cell cycle progression at G0/G1 phase via decreasing CDK4/Cyclin D1 complex and elevating p21. In addition, downregulation of USP32 significantly induced cell apoptosis by activating cleaved caspase-3 and cleaved PARP, as well as inhibiting cell invasiveness via altering epithelial mesenchymal transition expression.. Our results suggest for the first time that USP32 is important for SCLC progression and might be a potential target for molecular therapy of SCLC.

Topics: Aged; Caspase 3; Cell Movement; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase Inhibitor p21; Down-Regulation; Female; G1 Phase Cell Cycle Checkpoints; Humans; Immunohistochemistry; Lung Neoplasms; Male; Poly(ADP-ribose) Polymerases; RNA Interference; RNA, Messenger; RNA, Small Interfering; Small Cell Lung Carcinoma; Tumor Cells, Cultured; Ubiquitin Thiolesterase

Studies in several cancers have suggested that miR-218 has anti-tumor activities, but its function is yet to be elucidated. In this study, we investigated the regulation and function of miR-218 (miR-218-5p) in the cell cycle progression of gastric cancer (GC). We found that miR-218 could suppress proliferation of gastric cancer cells, induce cell cycle arrest at the G1 phase and inhibit tumor growth and metastasis in vivo. We also demonstrated that miR-218 specifically targeted the 3'-UTR regions of CDK6 and cyclin D1 and inhibited the expression of these molecules, which in turn repressed the pRb/E2F1 signaling pathway. Overexpression of CDK6 and Cyclin D1 reversed miR-218-mediated inhibition of pRB/E2F1 signaling and attenuated the miR-218-induced cell cycle arrest. More importantly, miR-218 expression was significantly reduced and inversely correlated with the levels of CDK6 and Cyclin D1 in gastric cancer tissues. Decreased miR-218 expression was also correlated with advanced clinical stage, lymph node metastasis, and poor prognosis in gastric cancer patients. Furthermore, we showed that miR-218 expression was directly activated by E2F1 through the transactivation of miR-218 host genes, SLIT2 and SLIT3, revealing a negative feedback regulation of miR-218 expression. Taken together, our results describe a regulatory loop miR-218-CDK6/CyclinD1-E2F1 whose disruption may contribute to cell cycle progression in gastric cancer and indicate the potential application of miR-218 in cancer therapy.

Topics: 3' Untranslated Regions; Animals; Binding Sites; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase 6; E2F1 Transcription Factor; Feedback, Physiological; G1 Phase Cell Cycle Checkpoints; Gene Expression Regulation, Neoplastic; Humans; Intercellular Signaling Peptides and Proteins; Lung Neoplasms; Lymphatic Metastasis; Membrane Proteins; Mice, Nude; MicroRNAs; Neoplasm Staging; Nerve Tissue Proteins; Protein Binding; Signal Transduction; Stomach Neoplasms; Time Factors; Transcription, Genetic; Transcriptional Activation; Transfection; Tumor Burden

The known oncogene

Topics: A549 Cells; Adenocarcinoma; Adenocarcinoma of Lung; Binding Sites; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cyclin D1; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Neoplasm Metastasis; Prognosis; Promoter Regions, Genetic; Survival Analysis; Thyroid Nuclear Factor 1

Circular RNAs (circRNAs) are associated with cancer progression and metastasis, although little is known about their role in lung adenocarcinoma (LAC). In the present study, microarrays were first used to screen for tumour-specific circRNA candidates in LAC tissue. Thirty-nine circRNAs were found to be up-regulated and 20 were down-regulated (fold change > 2.0). Among them, hsa_circ_0013958 was further confirmed to be up-regulated in all of the LAC tissues, cells and plasma. In addition, hsa_circ_0013958 levels were associated with TNM stage (P = 0.009) and lymphatic metastasis (P = 0.006). The area under the receiver operating characteristic curve was 0.815 (95% confidence interval = 0.727-0.903; P < 0.001). In addition, to further illustrate the bioactivities of hsa_circ_0013958 in LAC, siRNA-mediated inhibition of hsa_circ_0013958 was performed in vitro. The results showed that hsa_circ_0013958 promoted cell proliferation and invasion and inhibited cell apoptosis in LAC. Moreover, hsa_circ_0013958 was identified as a sponge of miR-134, and thus it up-regulated oncogenic cyclin D1, which plays a pivotal role in the development of non-small cell lung cancer. In conclusion, our results suggested that hsa_circ_0013958 could be used as a potential non-invasive biomarker for the early detection and screening of LAC.

Topics: Adenocarcinoma; Aged; Apoptosis; Biomarkers, Tumor; Cell Proliferation; Cyclin D1; Female; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Lymphatic Metastasis; Male; MicroRNAs; Middle Aged; Neoplasm Staging; RNA; RNA, Circular; Tumor Cells, Cultured

Accumulating evidence demonstrates that a series of differentially expressed lncRNAs is important in tumorigenesis. However, the function of many of the lncRNAs in lung cancer remains elusive. In the present study, we used microarray analysis to identify lncRNAs that are dysregulated in non-small-cell lung cancer (NSCLC) as compared with normal tissues. Among the dysregulated lncRNAs, we identified TFPI2AS1, an antisense transcript of the tumor suppressor TFPI2 (tissue factor pathway inhibitor 2). TFPI2AS1 was shown to be markedly upregulated in NSCLC patient tumors as compared to paired non-tumor samples. TFPI2AS1 knockdown increased NSCLC cell proliferation and migration, which was associated with enhanced G1/S transition and downregulation of cyclin D1 and cyclin-dependent kinases 2 (CDK2), while TFPI2AS1 overexpression had the opposite effect. Knockdown and overexpression experiments also suggested that TFPI2AS1 regulates NSCLC cell migration and AKT activation. Moreover, TFPI2AS1 is a positive regulator of TFPI2. Our findings bring new insights for understanding the role of TFPI2AS1 in mediating the proliferation and migration of NSCLC cells by regulating TFPI2 expression.

Topics: A549 Cells; Carcinoma, Non-Small-Cell Lung; Cell Movement; Cell Proliferation; Cyclin D1; Down-Regulation; G1 Phase Cell Cycle Checkpoints; Glycoproteins; Humans; Lung Neoplasms; Proto-Oncogene Proteins c-akt; RNA Interference; RNA, Long Noncoding; RNA, Small Interfering; Signal Transduction

Valid evidence has demonstrated that microRNAs have critical functions in cancer genesis and tumor progression. In the present study, aberrant expression of microRNA-149 (miR-149) was confirmed in non-small cell lung cancer (NSCLC) tissues. Low expression of miR-149 was associated with malignant clinical features and poor survival. Gain- and loss-of-function experiments demonstrated that miR-149 inhibited NSCLC tumor growth and metastasis in vitro and in vivo. Furthermore, oncogenic transcription factor FOXM1 was confirmed as a direct target of miR-149 in NSCLC. Cyclin D1 and MMP2 served as downstream targets of FOXM1 and were also inhibited by miR-149. In summary, the present study indicated that downregulation of miR-149 in NSCLC predicted poor clinical outcomes. miR-149 suppresses tumor growth and metastasis in NSCLC by inhibiting the FOXM1/cyclin D1/MMP2 signaling pathway.

Topics: A549 Cells; Adult; Aged; Animals; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Down-Regulation; Female; Forkhead Box Protein M1; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Male; Matrix Metalloproteinase 2; Mice; MicroRNAs; Middle Aged; Neoplasm Metastasis; Neoplasm Transplantation; Prognosis; Survival Analysis; Tumor Burden

Circular RNAs (circRNAs), a novel class of widespread and diverse endogenous RNAs, can regulate gene expression in mammals. CircRNAs have recently been identified as microRNA sponges and involved in the development of some human diseases. However, the role of circRNAs in the process of tumorigenesis and development of lung cancer remains vague. The purpose of this study is to investigate the role of circRNAs in the lung cancer. In this study, we chose hsa_circ_0000064 as a targeted circRNA to investigate its clinical significances in lung cancer patients. The result indicated that hsa_circ_0000064 was up-regulated in lung cancer tissues and lung cancer cell lines (A549 and H1229). Moreover, its aberrant expression was correlated with several clinical characteristics, including T stage, lymphatic metastasis, and TNM stage. Fluorescence in situ hybridization detected that hsa_circ_0000064 was mostly located in the cytoplasm in A549 and H1229 cells. In addition, knockdown of hsa_circ_0000064 with siRNA dramatically attenuated the proliferation, blocked cell cycle progression, and promoted cell apoptosis. Western blot analysis showed that the protein levels of caspase-3, caspase-9, bax, p21, CDK6 and cyclin D1 significantly restrained by si-hsa_circ_0000064, while the expression of bcl-2 notably increased in A549 and H1229 cells. Further, si-hsa_circ_0000064 also abated migration and invasion activities of A549 and H1229 cells, which may be associated with reduced expressions of MMP-2 and MMP-9. In general, our data suggest that hsa_circ_0000064 represents a novel potential biomarker and therapeutic target of lung cancer.

Topics: A549 Cells; Apoptosis; bcl-2-Associated X Protein; Biomarkers, Tumor; Carcinogenesis; Caspase 3; Caspase 9; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase 6; Female; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Lymphatic Metastasis; Male; MicroRNAs; Middle Aged; p21-Activated Kinases; RNA; RNA, Circular; Up-Regulation

Asbestos exposure causes malignant tumors such as lung cancer and malignant mesothelioma. Based on our hypothesis in which continuous exposure to asbestos of immune cells cause reduction of antitumor immunity, the decrease of natural killer cell killing activity with reduction of NKp46 activating receptor expression, inhibition of cytotoxic T cell clonal expansion, reduced CXCR3 chemokine receptor expression and production of interferon-γ production in CD4+ T cells were reported using cell line models, freshly isolated peripheral blood immune cells from health donors as well as asbestos exposed patients such as pleural plaque and mesothelioma. In addition to these findings, regulatory T cells (Treg) showed enhanced function through cell-cell contact and increased secretion of typical soluble factors, interleukin (IL)-10 and transforming growth factor (TGF)-β, in a cell line model using the MT-2 human polyclonal T cells and its sublines exposed continuously to asbestos fibers. Since these sublines showed a remarkable reduction of FoxO1 transcription factor, which regulates various cell cycle regulators in asbestos-exposed sublines, the cell cycle progression in these sublines was examined and compared with that of the original MT-2 cells. Results showed that cyclin D1 expression was markedly enhanced, and various cyclin-dependent kinase-inhibitors were reduced with increased S phases in the sublines. Furthermore, the increase of cyclin D1 expression was regulated by FoxO1. The overall findings indicate that antitumor immunity in asbestos-exposed individuals may be reduced in Treg through changes in the function and volume of Treg.

Topics: CD4-Positive T-Lymphocytes; Cell Cycle; Cyclin D1; Forkhead Box Protein O1; Gene Expression Regulation, Neoplastic; Humans; Interleukin-10; Killer Cells, Natural; Lung Neoplasms; Mesothelioma; Mesothelioma, Malignant; Natural Cytotoxicity Triggering Receptor 1; Receptors, CXCR3; T-Lymphocytes, Cytotoxic; T-Lymphocytes, Regulatory; Transforming Growth Factor beta

Sex-determining region Y-box 2 (SOX2) is an oncogene known to be amplified and overexpressed in various human malignancies, including lung squamous cell carcinoma (SCC). However, the role played by SOX2 in lung SCC development remains to be elucidated. We measured the levels of SOX2 and cyclin D1 mRNA and protein expression in lung SCC tissues and a lung SCC cell line, and found that both levels were dramatically upregulated in specimens of lung SCC tissue when compared with their expression levels in samples of adjacent nonneoplastic tissue. The lung SCC cell line also showed higher levels of SOX2 and cyclin D1 expression than a normal human bronchial epithelium cell line. After using RNA interference to knock down SOX2 expression in NCI-H520 lung SCC cells, their proliferation was reduced. Furthermore, overexpression of SOX2 promoted the proliferation of normal human bronchial epithelium cells. To further determine whether cyclin D1 was downstream target gene of SOX2, we measured the levels of cyclin D1 expression that occurred when SOX2 was knocked down or overexpressed. SOX2 knockdown significantly decreased the levels of cyclin D1 mRNA and protein expression, while SOX2 overexpression upregulated the levels of cyclin D1. We used bioinformatics data to identify potential cyclin D1 promoter binding sites for SOX2. Results of luciferase reporter assays, electrophoretic mobility shift assays, and chromatin immunoprecipitation assays confirmed that cyclin D1 was a direct target of transcription factor SOX2 in human lung SCC cells.

Topics: Bronchi; Carcinoma, Squamous Cell; Cell Line; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Epithelial Cells; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; SOXB1 Transcription Factors

Cisplatin and its analogues are widely used as anti-tumor drugs in lung cancer but many cisplatin-resistant lung cancer cases have been identified in recent years. Single-stranded DNA-binding protein 1 (SSDBP1) can effectively induce H69 cell resistance to cisplatin in our previous identification; thus, it is necessary to explore the mechanism underlying the effects of SSDBP1-induced resistance to cisplatin. First, SSDBP1-overexpressed or silent cell line was constructed and used to analyze the effects of SSDBP1 on chemoresistance of lung cancer cells to cisplatin. SSDBP1 expression was assayed by real-time PCR and Western blot. Next, the effects of SSDBP1 on cisplatin sensitivity, proliferation, and apoptosis of lung cancer cell lines were assayed by MTT and flow cytometry, respectively; ABC transporters, apoptosis-related genes, and cell cycle-related genes by real-time PCR, and DNA wound repair by comet assay. Low expression of SSDBP1 was observed in H69 cells, while increased expression in cisplatin-resistant H69 cells. Upregulated expression of SSDBP1 in H69AR cells was identified to promote proliferation and cisplatin resistance and inhibit apoptosis, while downregulation of SSDBP1 to inhibit cisplatin resistance and proliferation and promoted apoptosis. Moreover, SSDBP1 promoted the expression of P2gp, MRP1, Cyclin D1, and CDK4 and inhibited the expression of caspase 3 and caspase 9. Furthermore, SSDBP1 promoted the DNA wound repair. These results indicated that SSDBP1 may induce cell chemoresistance of cisplatin through promoting DNA repair, resistance-related gene expression, cell proliferation, and inhibiting apoptosis.

Topics: ATP Binding Cassette Transporter, Subfamily B; ATP-Binding Cassette Sub-Family B Member 4; Cell Line, Tumor; Cisplatin; Cyclin D1; DNA-Binding Proteins; Drug Resistance, Neoplasm; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Multidrug Resistance-Associated Proteins; Neoplasm Proteins; Ribonucleoproteins; Up-Regulation

Lung cancer is globally widespread and associated with high morbidity and mortality. DDA1 (DET1 and DDB1 associated 1) was first discovered and registered in the GenBank database by our colleagues. DDA1, an evolutionarily conserved gene, might have significant functions. Recent reports have demonstrated that DDA1 is linked to the ubiquitin-proteasome pathway and facilitates the degradation of target proteins. However, the function of DDA1 in lung cancer was previously unknown. This study aimed to investigate whether DDA1 contributes to tumorigenesis and progression of lung cancer. We found that the expression of DDA1 in normal lung cells and tissue was significantly lower than that in lung cancer and was associated with poor prognosis. DDA1 overexpression promoted proliferation of lung tumour cells and facilitated cell cycle progression in vitro and subcutaneous xenograft tumour progression in vivo. Mechanistically, this was associated with the regulation of S phase and cyclins including cyclin D1/D3/E1. These results indicate that DDA1 promotes lung cancer progression, potentially through promoting cyclins and cell cycle progression. Therefore, DDA1 may be a potential novel target for lung cancer treatment, and a biomarker for tumour prognosis.

Topics: Adenocarcinoma; Adenocarcinoma of Lung; Aged; Animals; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Proliferation; Cell Transformation, Neoplastic; Cyclin D1; Cyclin D3; Cyclin E; Disease Progression; DNA-Binding Proteins; Female; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Male; Mice; Mice, Nude; Middle Aged; Neoplasm Transplantation; Oncogene Proteins; Prognosis; S Phase; Signal Transduction; Survival Analysis; Tissue Array Analysis

Glioma-associated oncogene homolog 1 (Gli1) is involved in cancer stem cell (CSC) maintenance in various tumors; however, its expression and clinical significance in lung squamous cell carcinoma (LSCC) has not been reported. In this study, we aimed to reveal the clinical significance of Gli1 in LSCC and investigate the potential of Gli1 as a CSC marker by comparing its expression with that of other stemness-related genes in LSCC.. We assessed the expressions of Gli1, LSD1, CD44, Sox9 and Sox2 by immunohistochemistry in the tissue specimens obtained from 101 patients with LSCC. The relationship of Gli1 expression with clinicopathological parameters and cell-cycle regulating genes was investigated.. Gli1 expression was significantly correlated with T stage (P<0.001), lymph node metastasis (P=0.002), and clinical stage (P=0.005) of LSCC. The Kaplan-Meier survival analysis revealed that the expression of Gli1 in LSCC was all significantly associated with poor overall survival (OS: P=0.005). Cox regression analysis further confirmed that Gli1 is a prognostic marker of unfavorable clinical outcome of LSCC. Gli1 expression was significantly correlated with the expression of stemness-related genes such as LSD1 (P=0.009) and CD44 (P<0.001), but not with those of Sox2 and Sox9. However, Gli1 expression was associated with the expression of hypoxia-inducible factors1α (HIF1α; P<0.001) and Cyclin D1 (P=0.002), respectively. In additionally, microvessel density (MVD) was significantly higher in Gli1-positive LSCC than in the negative LSCC (P=0.026).. Our results suggest that Gli1 may be a potential LSCC stem cell marker and an independent indicator of poor prognosis for patients with LSCC.

Topics: Aged; Biomarkers, Tumor; Carcinoma, Non-Small-Cell Lung; Carcinoma, Squamous Cell; Cyclin D1; Female; Gene Expression Regulation, Neoplastic; Histone Demethylases; Humans; Hyaluronan Receptors; Immunohistochemistry; Kaplan-Meier Estimate; Lung Neoplasms; Male; Neoplastic Stem Cells; Prognosis; SOX9 Transcription Factor; SOXB1 Transcription Factors; Zinc Finger Protein GLI1

LRRC3B has emerged as a tumor suppressor in several human cancers. However, its expression pattern and biological roles in human non-small-cell lung cancer (NSCLC) have not been explored. In the present study, we investigated clinical significance of LRRC3B in 101 NSCLC specimens. We found that LRRC3B expression was downregulated in NSCLC tissues compared with normal bronchial epithelium and that its downregulation significantly correlated with tumor-node-metastasis (TNM) stage (p < 0.0001), nodal metastasis (p < 0.0001), and poor patient prognosis (p = 0.0016, log-rank test). We also checked LRRC3B levels in several lung cancer cell lines and found that its expression was downregulated in four of nine lung cancer cell lines compared with normal human bronchial epithelial (NHBE) cell line. We further explored the biological role of LRRC3B. LRRC3B plasmid transfection in H460 and A549 cell lines inhibited proliferation, colony formation ability, and invading ability. Furthermore, we identified that LRRC3B could inhibit cell cycle progression with downregulation of cyclin D1 and decreased MMP9 expression. In addition, LRRC3B depletion in HBE cells promoted proliferation and invasion. In conclusion, our data suggested that LRRC3B may serve as an important tumor suppressor in NSCLC.

Topics: A549 Cells; Aged; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Down-Regulation; Female; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Male; Matrix Metalloproteinase 9; Middle Aged; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasm Proteins; Real-Time Polymerase Chain Reaction; RNA, Small Interfering

ZNF322A encoding a classical Cys2His2 zinc finger transcription factor was previously revealed as a potential oncogene in lung cancer patients. However, the oncogenic role of ZNF322A and its underlying mechanism in lung tumorigenesis remain elusive. Here we show ZNF322A protein overexpression in 123 Asian and 74 Caucasian lung cancer patients. Multivariate Cox regression analysis indicated that ZNF322A was an independent risk factor for a poor outcome in lung cancer, corroborating the Kaplan-Meier results that patients with ZNF322A protein overexpression had significantly poorer overall survival than other patients. Overexpression of ZNF322A promoted cell proliferation and soft agar growth by prolonging cell cycle in S phase in multiple lung cell lines, including the immortalized lung cell BEAS-2B. In addition, ZNF322A overexpression enhanced cell migration and invasion, whereas knockdown of ZNF322A reduced cell growth, invasion and metastasis abilities in vitro and in vivo. Quantitative proteomic analysis revealed potential ZNF322A-regulated downstream targets, including alpha-adducin (ADD1), cyclin D1 (CCND1), and p53. Using luciferase promoter activity assay combined with site-directed mutagenesis and sequential chromatin immunoprecipitation-PCR assay, we found that ZNF322A could form a complex with c-Jun and cooperatively activate ADD1 and CCND1 but repress p53 gene transcription by recruiting differential chromatin modifiers, such as histone deacetylase 3, in an AP-1 element dependent manner. Reconstitution experiments indicated that CCND1 and p53 were important to ZNF322A-mediated promotion of cell proliferation, whereas ADD1 was necessary for ZNF322A-mediated cell migration and invasion. Our results provide compelling evidence that ZNF322A overexpression transcriptionally dysregulates genes involved in cell growth and motility therefore contributes to lung tumorigenesis and poor prognosis.

Topics: Aged; Calmodulin-Binding Proteins; Cell Line, Tumor; Cell Movement; Cell Proliferation; Chromatin; Cyclin D1; DNA-Binding Proteins; Female; Humans; JNK Mitogen-Activated Protein Kinases; Lung Neoplasms; Male; Middle Aged; Neoplasm Metastasis; Prognosis; Promoter Regions, Genetic; Transcription Factor AP-1; Transcription Factors; Transcription, Genetic; Tumor Suppressor Protein p53

Natural compounds such as curcumin have the ability to enhance the therapeutic effectiveness of common chemotherapy agents through cancer stem-like cell (CSC) sensitisation. In the present study, we showed that curcumin enhanced the sensitivity of the double-positive (CD166+/EpCAM+) CSC subpopulation in non-small cell lung cancer (NSCLC) cell lines (A549 and H2170) to cisplatin-induced apoptosis and inhibition of metastasis. Our results revealed that initial exposure of NSCLC cell lines to curcumin (10-40 µM) markedly reduced the percentage of viability to an average of ~51 and ~54% compared to treatment with low dose cisplatin (3 µM) with only 94 and 86% in both the A549 and H2170 cells. Moreover, sensitisation of NSCLC cell lines to curcumin through combined treatment enhanced the single effect induced by low dose cisplatin on the apoptosis of the double-positive CSC subpopulation by 18 and 20% in the A549 and H2170 cells, respectively. Furthermore, we found that curcumin enhanced the inhibitory effects of cisplatin on the highly migratory CD166+/EpCAM+ subpopulation, marked by a reduction in cell migration to 9 and 21% in the A549 and H2170 cells, respectively, indicating that curcumin may increase the sensitivity of CSCs to cisplatin-induced migratory inhibition. We also observed that the mRNA expression of cyclin D1 was downregulated, while a substantial increased in p21 expression was noted, followed by Apaf1 and caspase-9 activation in the double-positive (CD166+/EpCAM+) CSC subpopulation of A549 cells, suggested that the combined treatments induced cell cycle arrest, therefore triggering CSC growth inhibition via the intrinsic apoptotic pathway. In conclusion, we provided novel evidence of the previously unknown therapeutic effects of curcumin, either alone or in combination with cisplatin on the inhibition of the CD166+/EpCAM+ subpopulation of NSCLC cell lines. This finding demonstrated the potential therapeutic approach of using curcumin that may enhance the effects of cisplatin by targeting the CSC subpopulation in NSCLC.

Topics: Antineoplastic Agents; Carcinoma, Non-Small-Cell Lung; Cell Cycle; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cell Survival; Cisplatin; Curcumin; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Drug Synergism; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Neoplastic Stem Cells; Signal Transduction

The Forkhead box P3 (FOXP3) transcription factor is the key driver of the differentiation and immunosuppressive function of regulatory T cells (Tregs). Additionally, FOXP3 has been reported to be expressed in many solid tumor cell lines and tissues. However, its role in tumorigenesis and tumor progression is conflicting, both tumor suppressive and promoting functions have been described. In this study, we demonstrated that FOXP3 was expressed in both lung adenocarcinoma tissues and the lung adenocarcinoma cell line A549. FOXP3 inhibition decreased cell proliferation, migration, and invasion as well as the secretion of inhibitory cytokines (e.g., transforming growth factor beta 1 (TGF-β1), interleukin 35 (IL-35), and heme oxygenase-1 (HMOX1)), suggesting a positive role for FOXP3 in tumor development. Importantly, we found that FOXP3 could enhance lung adenocarcinoma cell proliferation via upregulating the levels of the cell cycle G1/S checkpoint gene CCND1. These data demonstrated that FOXP3 could be regarded as a novel therapeutic target for inhibiting lung adenocarcinoma progression.

Topics: A549 Cells; Adenocarcinoma; Carcinoma, Non-Small-Cell Lung; Cell Division; Cyclin D1; Cytokines; Forkhead Transcription Factors; G1 Phase Cell Cycle Checkpoints; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Neoplasm Invasiveness; Neoplasm Proteins; RNA Interference; RNA, Neoplasm; RNA, Small Interfering; Tumor Escape; Up-Regulation

It is largely recognized that PDCD4 is frequently lost in tumors of various origins, including lung cancer, and its loss contributes to tumor progression. However, its role and molecular mechanism remain largely unexplored in non-small cell lung cancer (NSCLC). In this study, downregulated PDCD4 mRNA expression was found in NSCLC tissues compared to their corresponding paracarcinoma tissues and distal paracarcinoma tissues. Induced expression of PDCD4 inhibited cell growth and proliferation and cell cycle transition in vitro. Conversely, knocking down PDCD4 expression promoted cell growth and proliferation. Mechanistically, PDCD4 inactivated PI3K/Akt signaling and its downstream cell cycle factors CCND1 and CDK4 to regulate cell growth in NSCLC. Additionally, PI3K-specific inhibitor Ly294002 suppressed the expression of pPI3K (Tyr458), pAkt (Ser473), CCND1, and CDK4 in PC9-shPDCD4 and A549-shPDCD4 cells. Furthermore, Akt-specific inhibitor MK2206 inhibited the expression of pAkt (Ser473), CCND1, and CDK4 in PC9-shPDCD4 and A549-shPDCD4 cells. Taken together, our study provides evidence that PDCD4 inhibits cell growth through PI3K/Akt signaling in NSCLC and may be a potential therapeutic target for NSCLC.

Topics: Apoptosis Regulatory Proteins; Carcinoma, Non-Small-Cell Lung; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase 4; Gene Expression; Gene Knockdown Techniques; Humans; Lung Neoplasms; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; RNA Interference; RNA-Binding Proteins; RNA, Small Interfering; Signal Transduction

Hsa-miRNA-326 (miR-326) has recently been discovered having anticancer efficacy in different organs. However, the role of miR-326 on non-small cell lung cancer (NSCLC) is still ambiguous. In this study, we investigated the role of miR-326 on the development of NSCLC. The results indicated that miR-326 was significantly down-regulated in primary tumor tissues and very low levels were found in NSCLC cell lines. Ectopic expression of miR-326 in NSCLC cell lines significantly suppressed cell growth as evidenced by cell viability assay, colony formation assay and BrdU staining, through inhibition of cyclin D1, cyclin D2, CDK4 and up-regulation of p57(Kip2) and p21(Waf1/Cip1). In addition, miR-326 induced apoptosis, as indicated by concomitantly with up-regulation of key apoptosis protein cleaved caspase-3, and down-regulation of anti-apoptosis protein Bcl2. Moreover, miR-326 inhibited cellular migration and invasiveness through inhibition of matrix metalloproteinases (MMP)-7 and MMP-9. Further, oncogene CCND1 was revealed to be a putative target of miR-326, which was inversely correlated with miR-326 expression in NSCLC. Taken together, our results demonstrated that miR-326 played a pivotal role on NSCLC through inhibiting cell proliferation, migration, invasion, and promoting apoptosis by targeting oncogenic CCND1.

Topics: Animals; Apoptosis; Blotting, Western; Carcinoma, Non-Small-Cell Lung; Cell Movement; Cell Proliferation; Cyclin D1; Fluorescent Antibody Technique; Follow-Up Studies; Gene Expression Regulation, Neoplastic; Humans; Immunoenzyme Techniques; Lung Neoplasms; Mice; Mice, Inbred BALB C; Mice, Nude; MicroRNAs; Neoplasm Staging; Prognosis; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Survival Rate; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

Radiation therapy is an important treatment choice for unresectable advanced human lung cancers, and a critical adjuvant treatment for surgery. However, radiation as a lung cancer treatment remains far from satisfactory due to problems associated with radiation resistance in cancer cells and severe cytotoxicity to non-cancer cells, which arise at doses typically administered to patients. We have recently identified a promising novel inhibitor of β-catenin/Tcf4 interaction, named BC-23 (C21H14ClN3O4S), which acts as a potent cell death enhancer when used in combination with radiation. Sequential exposure of human p53-null non-small cell lung cancer (NSCLC) H1299 cells to low doses of x-ray radiation, followed 1 hour later by administration of minimally cytotoxic concentrations of BC-23, resulted in a highly synergistic induction of clonogenic cell death (combination index <1.0). Co-treatment with BC-23 at low concentrations effectively inhibits Wnt/β-catenin signaling and down-regulates c-Myc and cyclin D1 expression. S phase arrest and ROS generation are also involved in the enhancement of radiation effectiveness mediated by BC-23. BC-23 therefore represents a promising new class of radiation enhancer.

Topics: Antineoplastic Agents; Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; beta Catenin; Binding, Competitive; Carcinoma, Non-Small-Cell Lung; Cell Cycle; Cell Line, Tumor; Cyclin D1; Down-Regulation; Gene Expression Regulation, Neoplastic; Humans; Luciferases; Lung Neoplasms; Neoplastic Stem Cells; Nimustine; Proto-Oncogene Proteins c-myc; Radiation Tolerance; Reactive Oxygen Species; Spectrometry, Fluorescence; Transcription Factor 4; Transcription Factors; X-Rays

Sulforaphane (SFN) is present in plants belonging to Cruciferae family and was first isolated from broccoli sprouts. Chemotherapeutic and anticarcinogenic properties of sulforaphane were demonstrated, however, the underlying mechanisms are not fully understood. In this study we evaluated the expression of cyclin D1 and p21 protein in SFN-treated A549 cells and correlated these results with the extent of cell death and/or cell cycle alterations, as well as determined a potential contribution of cyclin D1 to cell death. A549 cells were treated with increasing concentrations of SFN (30, 60 and 90 µM) for 24 h. Morphological and ultrastructural changes were observed using light, transmission electron microscope and videomicroscopy. Image-based cytometry was applied to evaluate the effect of SFN on apoptosis and the cell cycle. Cyclin D1 and p21 expression was determined by flow cytometry, RT-qPCR and immunofluorescence. siRNA was used to evaluate the role of cyclin D1 in the process of suforaphane-induced cell death. We found that the percentage of cyclin D1-positive cells decreased after the treatment with SFN, but at the same time mean fluorescence intensity reflecting cyclin D1 content was increased at 30 µM SFN and decreased at 60 and 90 µM SFN. Percentage of p21-positive cells increased following the treatment, with the highest increase at 60 µM SFN, at which concentration mean fluorescence intensity of this protein was also significantly increased. The 30-µM dose of SFN induced an increased G2/M phase population along with a decreased polyploid fraction of cells, which implies a functional G2/M arrest. The major mode of cell death induced by SFN was necrosis and, to a lower degree apoptosis. Transfection with cyclin D1-siRNA resulted in significantly compromised fraction of apoptotic and necrotic cells, which suggests that cyclin D1 is an important determinant of the therapeutic efficiency of SFN in the A549 cells.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Carcinoma, Non-Small-Cell Lung; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Gene Expression Regulation, Neoplastic; Humans; Isothiocyanates; Lung Neoplasms; Sulfoxides

Insulinoma associated-1 (INSM1) gene is expressed exclusively in early embryonic neuroendocrine tissues, but has been found highly re-activated in most of the neuroendocrine tumors including small cell lung carcinoma.. In order to elucidate the functional effects of INSM1 in normal lung development, we used a conditional lung-specific INSM1 transgenic mouse model. Transgenic (Tet-on system) CMV-INSM1 responder mice were bred with the lung-specific, club cell secretory protein (CCSP) promoter-rtTA activator mice to produce bi-transgenic progeny carrying both alleles, CCSP-rtTA and Tet-on-INSM1. Mice were fed with doxycycline containing food at the initial mating day to the postnatal day 21. Lung samples were collected at embryonic day 17.5, newborn, and postnatal day 21 for analyses.. Northern blot, RT-PCR, and immunohistochemical analyses revealed that doxycycline induced respiratory epithelium-specific INSM1 expression in bi-transgenic mice. Samples from postnatal day 21 mice revealed a larger lung size in the bi-transgenic mouse as compared to the single-transgenic or wild-type littermates. The histopathology results showed that the alveolar space in the bi-transgenic mice were 4 times larger than those in the single transgenic or wild-type littermates. In contrast, the size was not significantly different in the lungs collected at E17.5 or newborn among the bi-transgenic, single transgenic, or wild type mice. The respiratory epithelium with INSM1 ectopic expression suppressed cyclin D1 signal. Further in vitro studies revealed that the ectopic expression of INSM1 suppresses cyclin D1 expression and delays cell cycle progression.. The current study suggests that CCSP promoter-driven INSM1 ectopic expression impairs normal lung development especially in postnatal alveologenesis.

Topics: Animals; Blotting, Northern; Blotting, Western; Bronchi; Case-Control Studies; Cell Line; Cyclin D1; DNA-Binding Proteins; Ectopic Gene Expression; Epithelial Cells; Flow Cytometry; Gene Expression Regulation, Developmental; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Lung; Lung Neoplasms; Mice; Mice, Transgenic; Pulmonary Alveoli; Repressor Proteins; Respiratory Mucosa; Reverse Transcriptase Polymerase Chain Reaction; Small Cell Lung Carcinoma; Transcription Factors

LRRC3B expression is downregulated in many kinds of malignant tumors and it is regarded as tumor inhibition protein. However, the expression pattern and biological effect are still unclear in non-small cell lung cancer (NSCLC). Study of human cancer microarray showed LRRC3B was downregulated in breast cancer and colorectal cancer, which declares that LRRC3B takes part in tumor formation. This study is to investigate the expression of LRRC3B in lung cancer cell lines and the influence of LRRC3B during cell proliferation, invasion and cell cycle.. The expression of LRRC3B was detected through Western blot and Realtime RT-PCR. MTT assay, colony formation assay and matrigel invasion assay were performed to explore the effect of LRRC3B on cell proliferation, invasion and cell cycle. LRRC3B siRAN was transfected in lung cancer cell line H3255, and then the effect of LRRC3B on cell proliferation, invasion and cell cycle was analyzed.. During 4 of 9 lung cancer cell lines, the expression of LRRC3B is downregulated compared with normal epithelial cells. Cell proliferation and invasion were inhibited in A549 and H460 after LRRC3B transfected. LRRC3B suppressed the progress of cell cycle and downregulated the expression of cyclin D1 and MMP9. The activity of proliferation and invasion was promoted after LRRC3B was knocked out in H3255.. LRRC3B expression downreguated in lung cancer cell lines and LRRC3B could inhibit lung cancer cell proliferation, invasion and cell cycle progress. LRRC3B could be a new target for lung cancer therapy.
.. 背景与目的 已有的研究表明：在许多恶性肿瘤细胞中，LRRC3B表达显著下调，被视为肿瘤抑制蛋白。然而，在非小细胞肺癌中它的表达模式和生物学作用缺乏研究。人类癌症微阵列的研究显示LRRC3B在乳腺癌和结肠直肠癌表达下调，提示LRRC3B参与致癌作用。本研究的目的是研究LRRC3B在非小细胞肺癌中的必到状态及其与肺癌增殖、侵袭和细胞周期间的相关性，探讨LRRC3B在调控肺癌细胞增殖、侵袭及细胞周期中的作用。方法 应用Western blot和Realtime RT-PCR检测LRRC3B在几株肺癌细胞系中的mRNA和蛋白表达水平。应用MTT法检测对转染LRRC3B的A549和H460细胞系细胞增殖能力变化，应用集落形成实验以及细胞侵袭实验研究LRRC3B对细胞增殖和侵袭以及细胞周期进程的作用。肺癌细胞系H3255中转染LRRC3B siRAN验证LRRC3B对细胞的增殖以及侵袭能力和对细胞周期进程的影响。结果 与正常NHBE细胞系相比，NSCLC细胞系中LRRC3B蛋白表达量显著下调，特别是H460、H358、HCC827以及A549。A549和H460细胞系转染LRRC3B后，细胞增殖和侵袭能力受到抑制。LRRC3B抑制细胞周期进程，并下调cyclin D1和MMP9的表达。H3255细胞中敲除LRRC3B，细胞增殖和侵袭能力显著增强，同时与细胞周期及侵袭能力相关的蛋白cyclin D1和MMP9表达略微上调。结论 LRRC3B在肺癌细胞系中表达下调，而上调LRRC3B则能够抑制肺癌细胞增殖和侵袭能力，并抑制细胞周期进程，可能是未来肺癌治疗的一个新靶点。.

Topics: Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Down-Regulation; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Matrix Metalloproteinase 9; Neoplasm Invasiveness; Neoplasm Proteins

Hsa-miRNA-134 (miR-134) has recently been discovered to have anticancer efficacy in different organs. However, the role of miR-134 on non-small cell lung cancer (NSCLC) is still ambiguous. In this study, we investigated the role of miR-134 on the development of NSCLC. The results indicated that miR-134 was significantly down-regulated in primary tumor tissues and very low levels were found in NSCLC cell lines. Ectopic expression of miR-134 in NSCLC cell lines significantly suppressed cell growth as evidenced by cell viability assay, colony formation assay and BrdU staining, through inhibition of cyclin D1, cyclin D2, CDK4 and up-regulation of p57(Kip2) and p21(Waf1/Cip1). In addition, miR-134 induced apoptosis, as indicated by concomitantly with up-regulation of key apoptosis protein cleaved caspase-3, and down-regulation of anti-apoptosis protein Bcl2. Moreover, miR-134 inhibited cellular migration and invasiveness through inhibition of matrix metalloproteinases (MMP)-7 and MMP-9. Further, oncogene CCND1 was revealed to be a putative target of miR-134, which was inversely correlated with miR-134 expression in NSCLC. Taken together, our results demonstrated that miR-134 played a pivotal role on NSCLC through inhibiting cell proliferation, migration, invasion, and promoting apoptosis by targeting oncogenic CCND1.

Topics: A549 Cells; Aged; Animals; Apoptosis; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin D1; Down-Regulation; Female; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Male; Mice, Inbred BALB C; Mice, Nude; MicroRNAs; Middle Aged; Transplantation, Heterologous

Manumycin A (Manu A) is a natural product isolated from Streptomyces parvulus and has been reported to have anti-carcinogenic and anti-biotic properties. However, neither its molecular mechanism nor its molecular targets are well understood. Thus, the aim of the present study was to explore the possibility that Manu A has cancer preventive and chemotherapeutic effects on malignant pleural mesothelioma (MPM) through regulation of Sp1 and induction of mitochondrial cell death pathway. Manu A inhibited the cell viability of MSTO-211H and H28 cells in a concentration‑dependent manner as determined by MTS assay. IC50 values were calculated as 8.3 and 4.3 µM in the MSTO-311H and H28 cells following 48 h incubation, respectively. Manu A induced a significant increase in apoptotic indices as shown by DAPI staining, Annexin V assay, multi-caspase activity and mitochondrial membrane potential assay. The downregulation of Sp1 mRNA and protein expression by Manu A led to apoptosis by suppressing Sp1-regulated proteins (cyclin D1, Mcl-1 and survivin). Manu A decreased the protein levels of BID, Bcl-xL and PARP while it increased Bax levels. Manu A caused depolarization of the mitochondrial membrane with induction of CHOP, DR4 and DR5. Our results demonstrated that Manu A exerted anticancer effects by inducing apoptosis via inhibition of the Sp1-related signaling pathway in human MPM.

Topics: Annexin A5; Apoptosis; bcl-2-Associated X Protein; bcl-X Protein; BH3 Interacting Domain Death Agonist Protein; Cell Line, Tumor; Cell Membrane Permeability; Cell Proliferation; Cell Survival; Cyclin D1; Humans; Inhibitor of Apoptosis Proteins; Lung Neoplasms; Membrane Potential, Mitochondrial; Mesothelioma; Mesothelioma, Malignant; Mitochondria; Myeloid Cell Leukemia Sequence 1 Protein; Pleural Neoplasms; Poly(ADP-ribose) Polymerases; Polyenes; Polyunsaturated Alkamides; Receptors, TNF-Related Apoptosis-Inducing Ligand; Sp1 Transcription Factor; Survivin; Transcription Factor CHOP

Latcripin-13 domain, isolated from the transcriptome of Lentinula edodes C91-3, contains a regulator of chromosome condensation (RCC1) domain/β-lactamase-inhibitor protein II (BLIP-II) and a plant homeodomain (PHD). Latcripin-13 domain has been shown to have antitumor effects. However, the underlying molecular pharmacology is largely unknown. We report here that Latcripin-13 domain induced cell cycle arrest in the G1 phase and caused the apoptosis of human lung carcinoma A549 cells via the GSK3β-cyclin D1 and caspase-8/NF-κB signaling pathways. Western blot analysis showed that Latcripin-13 domain decreased cyclin D1 and cyclin-dependent kinase 4 (CDK4), while it increased the ratio of GSK3β/phosphorylated GSK3β. Importantly, Latcripin-13 domain induced nuclear fragmentation and chromatin condensation in the A549 cells. In addition, treatment of the A549 cells with Latcripin-13 domain resulted in the loss of mitochondrial membrane potential, accompanied by an increase in the Bax/Bcl-2 ratio and activation of caspase-3, -8, and -9. Intriguingly, western blot analysis revealed that NF-κB was significantly downregulated by Latcripin-13 domain. These results demonstrated that Latcripin-13 domain induced apoptosis and cell cycle arrest at G1 phase in the A549 cells, providing a mechanism for the antitumor effects of Latcripin-13 domain.

Topics: A549 Cells; Antineoplastic Agents; Apoptosis; Caspase 8; Cell Cycle Checkpoints; Cell Line, Tumor; Chromatin; Cyclin D1; DNA Fragmentation; Down-Regulation; Fungal Proteins; G1 Phase; Glycogen Synthase Kinase 3 beta; Humans; Lung Neoplasms; Membrane Potential, Mitochondrial; NF-kappa B; Signal Transduction

Though Farnesiferol c (FC) has been reported to have anti-angiogenic and antitumor activity, the underlying antitumor mechanism of FC still remains unclear. Thus, in the present study, we investigated the apoptotic mechanism of FC in human H1299 and H596 non-small lung cancer cells (NSCLCs). FC significantly showed cytotoxicity, increased sub-G1 accumulation, and attenuated the expression of Bcl-2, Bcl-xL, Survivin and procaspase 3 in H1299 and H596 cells. Furthermore, FC effectively suppressed the mRNA expression of G1 arrest related genes such as Cyclin D1, E2F1 transcription factor and CDC25A by RT-PCR. Interestingly, FC inhibited the expression of c-Myc, ribosomal protein L11 (L11) and nucleolin (NCL) in H1299 and H596 cells. Of note, silencing of L11 by siRNA transfection enhanced the expression of c-Myc through a negative feedback mechanism, while c-Myc knockdown downregulated L11 in H1299 cells. Additionally, combined treatment of FC and puromycin/doxorubicin promoted the activation of caspase 9/3, and attenuated the expression of c-Myc, Cyclin D1 and CDK4 in H1299 cells compared to single treatment. Taken together, our findings suggest that FC induces apoptosis and G1 arrest via regulation of ribosomal protein L11 and c-Myc and also enhances antitumor effect of puromycin or doxorubicin in NSCLCs.

Topics: Antineoplastic Agents; Apoptosis; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Survival; Coumarins; Cyclin D1; Cyclin-Dependent Kinase 4; Doxorubicin; Drug Therapy, Combination; G1 Phase Cell Cycle Checkpoints; Humans; Lung Neoplasms; Proto-Oncogene Proteins c-myc; Puromycin; Ribosomal Proteins

We explored the anti-cancer capacity of (-)-oleocanthal in human hepatocellular carcinoma (HCC). (-)-Oleocanthal inhibited proliferation and cell cycle progression and induced apoptosis in HCC cells in vitro and suppressed tumor growth in an orthotopic HCC model. (-)-Oleocanthal also inhibited HCC cell migration and invasion in vitro and impeded HCC metastasis in an in vivo lung metastasis model. ( )-Oleocanthal acted by inhibiting epithelial-mesenchymal transition (EMT) through downregulation Twist, which is a direct target of STAT3. (-)-Oleocanthal also reduced STAT3 nuclear translocation and DNA binding activity, ultimately downregulating its downstream effectors, including the cell cycle protein Cyclin D1, the anti-apoptotic proteins Bcl-2 and survivin, and the invasion-related protein MMP 2. Overexpression of constitutively active STAT3 partly reversed the anti cancer effects of (-)-oleocanthal, which inhibited STAT3 activation by decreasing the activities of JAK1 and JAK2 and increasing the activity of SHP-1. These data suggest that (-)-oleocanthal may be a promising candidate for HCC treatment.

Topics: Aldehydes; Animals; Antineoplastic Agents; Apoptosis; Carcinoma, Hepatocellular; Cell Cycle; Cell Line, Tumor; Cell Movement; Cell Nucleus; Cell Proliferation; Cyclin D1; Cyclopentane Monoterpenes; Down-Regulation; Epithelial-Mesenchymal Transition; Humans; Inhibitor of Apoptosis Proteins; Janus Kinase 1; Janus Kinase 2; Liver Neoplasms; Lung Neoplasms; Male; Mice, Inbred BALB C; Mice, Nude; Neoplasm Invasiveness; Nuclear Proteins; Olive Oil; Phenols; Protein Tyrosine Phosphatase, Non-Receptor Type 6; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-bcl-2; STAT3 Transcription Factor; Survivin; Twist-Related Protein 1; Xenograft Model Antitumor Assays

Aberrant activation of the Ras/MEK/ERK signaling pathway has been frequently observed in non-small-cell lung carcinoma (NSCLC) and its important role in cancer progression and malignant transformation has been documented. Hence, the ERK1/2 kinase cascade becomes a potential molecular target in cancer treatment. Xanthohumol (XN, a prenylated chalcone derived from hope cones) is known to possess a broad spectrum of chemopreventive and anticancer activities. In our studies, the MTT and BrdU assays revealed that XN demonstrated greater antiproliferative activity against A549 lung adenocarcinoma cells than against the lung adenocarcinoma H1563 cell line. We observed that XN was able to suppress the activities of ERK1/2 and p90RSK kinases, followed by inhibition of phosphorylation and activation of the CREB protein. Additionally, the XN treatment of the cancer cells caused upregulation of key cell cycle regulators p53 and p21 as well as downregulation of cyclin D1. As a result, the cytotoxic effect of XN was attributed to the cell cycle arrest at G1 phase and induction of apoptosis indicated by increased caspase-3 activity. Thus, XN might be a promising anticancer drug candidate against lung carcinomas.

Topics: A549 Cells; Adenocarcinoma; Adenocarcinoma of Lung; Antineoplastic Agents; Apoptosis; Caspase 3; Cell Line, Tumor; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Down-Regulation; Extracellular Signal-Regulated MAP Kinases; Flavonoids; G1 Phase Cell Cycle Checkpoints; Humans; Lung Neoplasms; MAP Kinase Signaling System; Propiophenones; Signal Transduction; Tumor Suppressor Protein p53; Up-Regulation

MicroRNAs are critical regulators in tumorigenesis. This study is aimed at investigating the function of miR-202 in the proliferation of human lung cancer cells.. Lung cancer tissues and paired adjacent normal tissues were collected from 20 patients; the expression of miR-202 was tested by Realtime PCR; cell proliferation and cell cycle distribution were determined by CCK-8 assay and flow cytometry, respectively; apoptosis was determined by TUNEL assay and Western blot analysis of cleaved-PARP; the relationship between miR-202 and cyclin D1 mRNA 3'UTR was determined by luciferase activity assay.. MiR-202 was significantly reduced in lung cancer tissues compared with the adjacent normal tissues. Overexpression of miR-202 inhibited cell proliferation and induced a G0/G1 cell cycle arrest and apoptosis. Luciferase activity assay and Western blot analysis together showed that miR-202 can bind to the 3'UTR of cyclin D1 mRNA directly and inhibits cyclin D1 expression at the protein level. In addition, restoration of cyclin D1 expression partially abolished cell cycle arrest and apoptosis induced by miR-202.. MiR-202 is constantly downregulated in lung cancer and functions as a tumor suppressive gene via targeting cyclin D1. Modulating the level of miR-202 may be a novel therapeutic method for lung cancer.

Topics: Apoptosis; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Humans; Lung Neoplasms; MicroRNAs

Lung cancer is still the leading cause of malignant deaths in the world. It is of great importance to find novel functional genes for the tumorigenesis of lung cancer. We demonstrated that Rac3 could promote cell proliferation and inhibit apoptosis in lung adenocarcinoma cell line A549 previously. The aim of this study was to investigate the function and mechanism of Rac3 in lung adenocarcinoma cell lines. Immunohistochemistry staining was performed in 107 lung adenocarcinoma tissues and matched non-tumor tissues. Multivariate analysis and Kaplan-Meier analysis were used to investigate the correlation between Rac3 expression and the clinical outcomes. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, colony formation assay, and flow cytometry analysis were employed to determine the proliferative ability, cell cycle distribution, and apoptosis in H1299 and H1975 cell lines. Gene expression microarray and pathway analysis between the Rac3-siRNA group and the control group in A549 cells were performed to investigate the pathways and mechanism of Rac3 regulation. Rac3 was shown to be positively expressed in lung adenocarcinoma tissues, and the expression of Rac3 associates with longer survival in lung adenocarcinoma patients. Silencing of Rac3 significantly induced cell growth inhibition, colony formation decrease, cell cycle arrest, and apoptosis of lung adenocarcinoma cell lines, which accompanied by obvious downregulation of CCND1, MYC, and TFDP1 of cell cycle pathway involving in the tumorigenesis of lung adenocarcinoma based on the gene expression microarray. In conclusion, these findings suggest that Rac3 has the potential of being a therapeutic target for lung adenocarcinoma.

Topics: Adenocarcinoma; Adenocarcinoma of Lung; Adult; Aged; Apoptosis; Cell Cycle Checkpoints; Cell Proliferation; Cyclin D1; Female; Humans; Lung Neoplasms; Male; Middle Aged; Prognosis; Proto-Oncogene Proteins c-myc; rac GTP-Binding Proteins; Transcription Factor DP1

Piwi-interacting RNAs (piRNAs or piRs) are a novel class of non-coding RNAs that participate in germline development by silencing transposable elements and regulating gene expression. To date, the association between piRNAs and non‑small cell lung carcinoma (NSCLC) has not yet been elucidated. In the present study, we have demonstrated that a significant increase in piR-651 expression occurs in NSCLC. Furthermore, the abnormal expression of piR-651 was associated with cancer progression in the patients with NSCLC. The upregulation of piR-651 in A549 cells caused a significant increase in cell viability and metastasis. The percentage of arrested cells in the G0/G1 phase was lower after piR-651 overexpression compared with the controls. We also examined the expression of oncogenes and cancer suppressor genes following piR-651 overexpression in NSCLC cells. Only the expression levels of cyclin D1 and CDK4 significantly correlated with piR-651 expression both in vivo and in vitro. Furthermore, by injecting nude mice with A549 cells transfected with piR-651 plasmids to establish a xenograft model, we demonstrated that there was a correlation between piR-651 overexpression and tumor growth, which was mediated by cyclin D1 and CDK4. These findings strongly support the notion that piR-651 induces NSCLC progression through the cyclin D1 and CDK4 pathway and it may have applications as a potential diagnostic indicator and therapeutic target in the management of NSCLC.

Topics: A549 Cells; Animals; Blotting, Western; Carcinoma, Non-Small-Cell Lung; Cell Survival; Cyclin D1; Cyclin-Dependent Kinase 4; Female; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Lung Neoplasms; Mice, Nude; Neoplasm Metastasis; Reverse Transcriptase Polymerase Chain Reaction; RNA, Small Interfering; Transplantation, Heterologous; Tumor Burden; Up-Regulation

Previous studies have indicated that miR-146a-5p acts as an oncogene in several types of cancer, yet a tumor suppressor gene in others. In non-small cell lung cancer (NSCLC), one report showed that it was downregulated and played the role of tumor suppressor. However, another study showed that miR-146a-5p was overexpressed in the serum of NSCLC patients compared to healthy controls. Therefore, it is obvious that further study of the function of miR-146a-5p in NSCLC is necessary to fully understand its importance. Herein, we have verified that miR- 146a- 5p acts as a tumor suppressor in NSCLC. Our data revealed that the expression level of miR-146a-5p was significantly decreased in several human NSCLC cell lines, and also less abundant in human NSCLC tissues, when compared with controls. Moreover, we observed that miR-146a-5p could suppress cell proliferation, both in vitro and in vivo. Our results also showed that miR-146a-5p directly targeted the 3'-UTR of CCND1 and CCND2 mRNAs as well as decreased their expression at both mRNA and protein levels, causing cell cycle arrest at the G0/G1 phase. Furthermore, siRNA-mediated downregulation of CCND1 or CCND2 yielded the same effects on proliferation and cell cycle arrest as miR-146a-5p upregulation did in the NSCLC cell lines. We confirmed that the expression of miR-146a-5p had negative relationship with CCND1 or CCND2. Besides, we also found that miR-146a-5p could inhibit tumor growth in xengroft mouse models, and CCND1 and CCND2 were downregulated in miR-146a-5p overexpressed xengroft tumor tissues. In summary, our results demonstrated that miR-146a-5p could suppress the proliferation and cell cycle progression in NSCLC cells by inhibiting the expression of CCND1 and CCND2.

Topics: 3' Untranslated Regions; Animals; Carcinoma, Non-Small-Cell Lung; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin D2; Female; Humans; Lung Neoplasms; Mice; Mice, Nude; MicroRNAs; RNA, Small Interfering; Xenograft Model Antitumor Assays

Low-dose radiation (LDR) induces hormesis and adaptive response in normal cells but not in cancer cells, suggesting its potential protection of normal tissue against damage induced by conventional radiotherapy. However, the underlying mechanisms are not well established. We addressed this in the present study by examining the role of the ataxia telangiectasia mutated (ATM) signaling pathway in response to LDR using A549 human lung adenocarcinoma cells and HBE135-E6E7 (HBE) normal lung epithelial cells. We found that LDR-activated ATM was the initiating event in hormesis and adaptive response to LDR in HBE cells. ATM activation increased the expression of CDK4/CDK6/cyclin D1 by activating the AKT/glycogen synthase kinase (GSK)-3β signaling pathway, which stimulated HBE cell proliferation. Activation of ATM/AKT/GSK-3β signaling also increased nuclear accumulation of nuclear factor erythroid 2-related factor 2, leading to increased expression of antioxidants, which mitigated cellular damage from excessive reactive oxygen species production induced by high-dose radiation. However, these effects were not observed in A549 cells. Thus, the failure to activate these pathways in A549 cells likely explains the difference between normal and cancer cells in terms of hormesis and adaptive response to LDR.

Topics: Ataxia Telangiectasia Mutated Proteins; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Glycogen Synthase Kinase 3 beta; Humans; Lung; Lung Neoplasms; NF-E2-Related Factor 2; Proto-Oncogene Proteins c-akt; Reactive Oxygen Species; Signal Transduction

Multiple nodes in the one-carbon metabolism pathway play important regulatory roles in cancer cell growth and tumorigenesis. The specific biological functions of metabolic enzymes in regulating the signaling pathways that are associated with tumor cell growth and survival, however, remain unclear. Our current study found that phosphoserine aminotransferase 1 (PSAT1), an enzyme catalyzing serine biosynthesis, was significantly up-regulated in non-small cell lung cancer (NSCLC) and was involved in the regulation of E2F activity. Loss- and gain-of-function experiments demonstrated that PSAT1 promoted cell cycle progression, cell proliferation and tumorigenesis. Mechanistic study suggested that elevated PSAT1 led to inhibition of cyclin D1 degradation and subsequently an alteration in Rb-E2F pathway activity, which in turn enhanced G1 progression and proliferation of NSCLC cells. Moreover, phosphorylation of cyclin D1 at threonine 286 by GSK-3β was required for PSAT1-induced blockage of cyclin D1 degradation. We also found that the activity of p70S6K mediated the effects of PSAT1 on GSK-3β phosphorylation and cyclin D1 degradation. We further identified that PSAT1 was over-expressed in NSCLC and predicted poor clinical outcome of patients with the disease. Correlation analysis showed that PSAT1 expression positively correlated with the levels of phosphorylated GSK-3β, cyclin D1 and phosphorylated Rb in NSCLC primary tumors. These findings uncover a mechanism for constitutive activation of E2F via which unrestrained cell cycle progression occurs in NSCLC and may represent a prognostic biomarker and therapeutic target.

Topics: Animals; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Proliferation; Cyclin D1; E2F Transcription Factors; G1 Phase Cell Cycle Checkpoints; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Humans; Lung Neoplasms; Mice, Inbred BALB C; Mice, Nude; Phosphorylation; Prognosis; Protein Processing, Post-Translational; Proteolysis; Signal Transduction; Transaminases; Transcriptome; Tumor Burden

To better understand the complete genomic architecture of lung adenocarcinoma.. We used array experiments to determine copy number variations and sequenced the complete exomes of the 247 lung adenocarcinoma tumor samples along with matched normal cells obtained from the same patients. Fully annotated clinical data were also available, providing an unprecedented opportunity to assess the impact of genomic alterations on clinical outcomes.. We discovered that genomic alternations in the RB pathway are associated with significantly shorter disease-free survival in early-stage lung adenocarcinoma patients. This association was also observed in our independent validation cohort. The current treatment guidelines for early-stage lung adenocarcinoma patients recommend follow-up without adjuvant therapy after complete resection, except for high-risk patients. However, our findings raise the interesting possibility that additional clinical interventions might provide medical benefits to early-stage lung adenocarcinoma patients with genomic alterations in the RB pathway. When examining the association between genomic mutation and histologic subtype, we uncovered the characteristic genomic signatures of various histologic subtypes. Notably, the solid and the micropapillary subtypes demonstrated great diversity in the mutated genes, while the mucinous subtype exhibited the most unique landscape. This suggests that a more tailored therapeutic approach should be used to treat patients with lung adenocarcinoma.. Our analysis of the genomic and clinical data for 247 lung adenocarcinomas should help provide a more comprehensive genomic portrait of lung adenocarcinoma, define molecular signatures of lung adenocarcinoma subtypes, and lead to the discovery of useful prognostic markers that could be used in personalized treatments for early-stage lung adenocarcinoma patients.

Topics: Adenocarcinoma; Adenocarcinoma of Lung; Aged; Cyclin D1; Disease-Free Survival; DNA Copy Number Variations; Female; Genome, Human; Genomics; Humans; Lung Neoplasms; Male; Middle Aged; Mutation; Neoplasm Staging; Prognosis; Retinoblastoma Protein

SCIN (scinderin) is a calcium-dependent actin severing and capping protein. Homologue in zebrafish has been found to be related with cell death. In the present study, we found that SCIN is highly expressed in human lung cancer specimens. However, the role of SCIN in lung cancer has not yet been determined. To investigate the function of SCIN in lung carcinoma cells, we took advantage of lentivirus-mediated RNA interference (RNAi) to knockdown SCIN expression in two lung carcinoma cell lines A549 and H1299. Silencing of SCIN significantly inhibited the proliferation and colony formation ability of both cell lines in vitro. Moreover, flow cytometry analysis showed that knockdown of SCIN led to G0/G1 phase cell cycle arrest as well as an excess accumulation of cells in the sub-G1 phase. Furthermore, depletion of SCIN resulted in a significant increase in Cyclin B1, p21 and PARP expression, and a little decrease in Cyclin D1 expression. These results suggest that SCIN plays an important role in lung carcinoma cell proliferation, and lentivirus-mediated silencing of SCIN might be a potential therapeutic approach for the treatment of lung cancer.

Topics: Apoptosis; Carcinoma; Cell Line, Tumor; Cell Proliferation; Cyclin B1; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Gelsolin; Humans; Immunohistochemistry; Lentivirus; Lung; Lung Neoplasms; Poly(ADP-ribose) Polymerases; RNA Interference

Lung cancer is the leading cause of cancer-related mortality worldwide. microRNAs (miRNAs) are small post-transcriptional regulatory non-coding RNAs that function as oncogenes or tumor suppressors in human cancers. Emerging evidence reveals that deregulation of miRNAs contributes to the progression of human lung cancer, which is the leading cause of cancer-related deaths worldwide. In the present study, we found that upregulation of the miR-212/132 cluster significantly suppressed the growth and focus formation of A549 and H1299 cells. Moreover, forced expression of this cluster conferred radiosensitivity and inhibited the migration of lung cancer cells, whereas downregulation of miR-212/132 reversed the above effects. Furthermore, miR-212/132 overexpression induced cell cycle arrest at the G1/S phase transition of the lung cancer cells, and inhibition of miR-132 and miR-212 abrogated this arrest. In addition, miR-212/132 overexpression increased the percentage of cells undergoing apoptosis. Cells transfected with the miR-212/132 cluster exhibited upregulated p21 expression and reduced cyclin D1 expression. Conversely, cells transfected with the miR-212/132 inhibitor showed reduced expression of p21 and upregulated expression of cyclin D1, suggesting that miR-212/132 may mediate proliferation and cell cycle arrest through p21 and cyclin D1. Our study provides insight into the biological function of the miR-212/132 cluster in lung cancer. The present study may provide a potential therapeutic target for the treatment of lung cancer.

Topics: Apoptosis; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Movement; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; MicroRNAs

Ubiquitin carboxyl terminal hydrolase 1 (UCHL1), a member of the UCH class of DUBs, has been reported as either an oncogene or a tumor suppressor. However, the molecular mechanism underlying the biological function of UCHL1 in osteosarcoma is still unclear. This study was aimed at elucidating the roles of UCHL1 in regulating the biological behavior of osteosarcoma cells. In this study, we found that UCHL1 was elevated in osteosarcoma compared with normal bone tissue. Moreover, UCHL1 expression level was correlated with tumor maximum diameter, high rate of lung metastases and short survival time. Then, we found that knockdown of UCHL1 in osteosarcoma cell MG63 inhibited cell proliferation and significantly increased cell population in the G1 phase. Several cyclins promoting G1/S phase transition were reduced after UCHL1 knockdown, including cell cycle regulator cyclin D1, cyclin E1 and CDK6. Moreover, inhibition of UCHL1 in MG63 cells dramatically induced cell apoptosis. We also found that down-regulation of UCHL1 in MG63 significantly inhibited cell invasion. Then, we found that there was a positive correlation between UCHL1 expression level and the Akt and ERK phosphorylation status. Finally, in vivo data showed that knockdown of UCHL1 inhibited osteosarcoma growth in nude mice. These results indicate that UCHL1 could work as an oncogene and may serve as a promising therapeutic strategy for osteosarcoma.

Topics: Adolescent; Animals; Apoptosis; Bone Neoplasms; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin E; Cyclin-Dependent Kinase 6; Extracellular Signal-Regulated MAP Kinases; G1 Phase Cell Cycle Checkpoints; Humans; Kaplan-Meier Estimate; Lung Neoplasms; Male; Mice, Nude; Neoplasm Invasiveness; Oncogene Proteins; Osteosarcoma; Phosphorylation; Proto-Oncogene Proteins c-akt; RNA Interference; Signal Transduction; Time Factors; Transfection; Tumor Burden; Ubiquitin Thiolesterase; Xenograft Model Antitumor Assays

The anticancer effects and mechanism of all-trans retinoic acid (ATRA), C-phycocyanin (C-PC) or ATRA+C-PC on the growth of A549 cells were studied in in vitro and in vivo experiments. The effects of C-PC and ATRA on the growth of A549 cells were determined. The expression of CDK-4 and caspase-3, and the cellular apoptosis levels were detected. The tumor model was established by subcutaneous injection of A549 cells to the left axilla of the NU/NU mice. The weights of tumor and the spleen were tested. The viabilities of T-cells and spleen cells, TNF levels, the expression of Bcl-2 protein and Cyclin D1 gene were examined. Results showed both C-PC and ATRA could inhibit the growth of tumor cells in vivo and in vitro. ATRA+C-PC cooperatively showed a higher antitumor activity. The dosage of ATRA was reduced when it was administered with C-PC together, and the toxicity was reduced as well. ATRA+C-PC could decrease CDK-4 but increase caspase-3 protein expression level and induce cell apoptosis. ATRA alone could lower the activities of T lymphocytes and spleen weights, but the combination with C-PC could effectively promote viability of T cells and spleen. C-PC+ATRA could up-regulate TNF, and down-regulate Bcl-2 and Cyclin D1 gene. The combination might inhibit tumor growth by inhibiting the progress of cell cycle, inducing cell apoptosis and enhancing the body immunity.

Topics: Animals; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Caspase 3; Cell Line, Tumor; Cell Survival; Cyclin D1; Cyclin-Dependent Kinase 4; Drug Synergism; Female; Humans; Lung Neoplasms; Male; Mice, Nude; Phycocyanin; Proto-Oncogene Proteins c-bcl-2; Spleen; T-Lymphocytes; Tretinoin; Tumor Burden; Tumor Necrosis Factor-alpha

We have previously reported that decreased expression of PTEN in lung cancer PC9 cells harboring an EGFR-activating mutation (del E746-A750) results in acquisition of resistance to EGFR-TKIs, gefitinib and erlotinib, accompanied by enhanced phosphorylation of Akt and decreased nuclear translocation of a transcription factor EGR-1 [8]. In the present study, PTEN promoter methylation accounted for the decreased expression of PTEN in our gefitinib-resistant mutant.. DNA methylation status of the PTEN promoter in PC9 and gefitinib-resistant cells were examined using methylation-specific PCR. The effect of DNA methylation on PTEN expression was evaluated by treatment of lung cancer cell lines with 5-aza-2'-deoxycytidine (5AZA-CdR).. We observed the characteristics of two gefitinib-resistant sublines, GEF1-1 and GEF2-1, derived from PC9 as follows. (1) PTEN overexpression suppressed AKT phosphorylation and restored the sensitivity to gefitinib and erlotinib in GEF1-1 cells. (2) EGR-1 siRNA mediated knockdown suppressed the expression of cyclin D1 and ICAM-1 genes but not of PTEN gene in PC9 cells. (3) Transfection of EGR-1 cDNA into a drug-resistant subline induced the expression of cyclin D1 and ICAM-1 but not of PTEN. (4) Treatment with 5AZA-CdR induced the expression of PTEN in resistant sublines but not in the parental line PC9. (5) A CpG site near the translational start point of the 5'-regulatory region was methylated in GEF1-1 and GEF2-1 but not in PC9.. Our results strongly suggest that CpG hypermethylation of the PTEN gene contributes to the decreased expression of PTEN during acquired resistance to gefitinib or erlotinib.

Topics: Antineoplastic Agents; Cell Line, Tumor; CpG Islands; Cyclin D1; DNA Methylation; Drug Resistance, Neoplasm; Early Growth Response Protein 1; Gefitinib; Gene Expression; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Humans; Intercellular Adhesion Molecule-1; Lung Neoplasms; Phosphorylation; Promoter Regions, Genetic; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; PTEN Phosphohydrolase; Quinazolines

Human non-small cell lung carcinoma (NSCLC) is one of the most common cancer worldwide. In previous studies, lovastatin, acting as an inhibitor of 3-hydroxy-3-methylglutaryl Co A (HMG-CoA) reductase, exhibited significant antitumor activity during tumorigenesis. However, whether or not this effect is mediated through changes in minichromosome maintenance (MCM) 2 expression remains unclear. The present study investigated whether lovastatin inhibits proliferation due to MCM2 in NSCLCs. We first assessed the effects of lovastatin on cell anti-proliferation, cell cycle progression and apoptosis in NSCLC cells. We found, by quantitative RT-PCR and western blot analysis, that lovastatin treatment markedly and consistently inhibited the expression of MCM2. Then, to further explore the anticancer mechanism of lovastatin involving MCM2, we silenced MCM2 by siRNA in two cell lines (A549 and GLC-82). Silencing of MCM2 triggered G1/S arrest. Following further examination of cell cycle-related factors, MCM2 knockdown inhibited protein retinoblastoma (Rb), cyclin D1 and CDK4 expression, but increased p21 and p53 expression, suggesting that siMCM2 indeed triggered cell cycle arrest. In addition, siMCM2 induced apoptosis. Finally, lovastatin treatment increased p-JNK, which is involved in the downregulation of MCM2. In conclusion, our data suggest that MCM2 may be a novel therapeutic target of lovastatin treatment in NSCLCs.

Topics: Apoptosis; Carcinoma, Non-Small-Cell Lung; Cell Cycle Checkpoints; Cell Cycle Proteins; Cell Line, Tumor; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase Inhibitor p21; G1 Phase Cell Cycle Checkpoints; Humans; Lovastatin; Lung Neoplasms; MAP Kinase Signaling System; Minichromosome Maintenance Complex Component 2; Retinoblastoma Protein; Tumor Suppressor Protein p53

The aim of the present study was to investigate the effect of short hairpin (sh)RNA targeting AKT1 and phosphatidylinositol 3-kinase (PI3K)/p85 on the proliferation and self-renewal of lung cancer stem cells (LCSCs). The recombinant adenovirus expression vector, which contained shRNA targeting open reading frames of AKT1 and PI3K/p85, was transfected into LCSCs. It was found that AKT1 and PI3K/p85 expression was upregulated in LCSCs compared with that in the primary lung cancer cells. Recombinant adenovirus vector rAd5-siAKT1-siPI3K/p85 significantly downregulated AKT1 and PI3K/p85 mRNA and protein expression in LCSCs. The downstream factors, proliferating cell nuclear antigen (PCNA) and cyclin D1 were also downregulated, while p53 was upregulated. Following silencing of AKT1 and PI3K/p85, cell proliferation, tumor sphere formation and tumor formation in NOD/SCID mice were also reduced. According to the present results, it was hypothesized that the PI3K/Akt signaling pathway is important in the self‑renewal and proliferation of LCSCs, and that targeting the PI3K/Akt signaling pathway decreases the rate of tumor formation in vivo.

Topics: Animals; Cell Line, Tumor; Cell Proliferation; Class Ia Phosphatidylinositol 3-Kinase; Cyclin D1; Down-Regulation; G1 Phase Cell Cycle Checkpoints; Humans; Lung Neoplasms; Mice; Mice, Inbred NOD; Mice, SCID; Neoplastic Stem Cells; Phosphoinositide-3 Kinase Inhibitors; Proliferating Cell Nuclear Antigen; Proto-Oncogene Proteins c-akt; RNA Interference; RNA, Messenger; RNA, Small Interfering; Transplantation, Heterologous; Tumor Suppressor Protein p53

We sought to elucidate the role of Rho guanine nucleotide exchange factor 5 (ARHGEF5) in tumorigenesis of lung adenocarcinoma cells. ARHGEF5 protein levels were assessed in 91 human lung adenocarcinoma specimens, and A549 and NCI-H1650 cells, by IHC and Western blotting. In addition, ARHGEF5 mRNA expression was evaluated by quantitative reverse transcriptase-PCR. Furthermore, ARHGEF5 long and short isoform coexpression was detected by immunofluorescence. Finally, flow cytometry; CCK8 and wound-healing assays; cell invasion, migration and adhesion; and xenografts were used to evaluate the biologic significance of ARHGEF5. ARHGEF5 was significantly increased in lung adenocarcinoma tissues and cell lines. Interestingly, ARHGEF5 levels were significantly associated with tumor grade and pathologic stage, but not age, gender, T stage, or lymph node metastasis status. ARHGEF5 knockdown by RNAi resulted in dramatically reduced proliferation, adhesion, invasion, and migratory capability of A549 and NCI-H1650 cells. Likewise, protein levels of p-Src, p-Akt, and NF-κB were significantly decreased after ARHGEF5 knockdown. In parallel, increased S-phase population and MMP-2/cyclin D1 expression were observed in the cancer cells, which were not apoptotic. In addition, ARHGEF5 knockdown A549 and NCI-H1650 cells injected s.c. and i.v. into nude mice exhibited decreased xenograft volume and overtly reduced metastasis. Conversely, ARHGEF5 overexpression in A549 and NCI-H1650 cells increased their tumorigenicity in vitro. ARHGEF5 acts as a proto-oncogene in human lung adenocarcinoma cell tumorigenesis.

Topics: Adenocarcinoma; Adult; Animals; Carcinogenesis; Cell Line, Tumor; Cell Movement; Cell Survival; Cell Transformation, Neoplastic; Cyclin D1; Female; Humans; Immunoblotting; Lung Neoplasms; Male; Matrix Metalloproteinase 2; Mice, Nude; Microscopy, Confocal; Middle Aged; Proto-Oncogene Mas; Reverse Transcriptase Polymerase Chain Reaction; Rho Guanine Nucleotide Exchange Factors; RNA Interference; Transplantation, Heterologous; Up-Regulation

Increased expression of pituitary tumor-transforming gene 1 (PTTG1) is expressed in many tumors and regulates tumor growth and progression. However, the precise function of PTTG1 in the tumorigenesis of lung adenocarcinoma (LAC) is not defined yet. Here, we examined the expression of PTTG1 in human LAC tissues by immunohistochemical assay using a tissue microarray procedure. A loss-of-function experiment was carried out to investigate the effects of lentiviral vector-mediated PTTG1 shRNA (shPTTG1) on cell growth and invasive potential in LAC cell lines (A549 and LETPα-2), assessed by MTT and Transwell assays. As a consequence, we found that the expression of PTTG1 protein was markedly upregulated in LAC tissues compared with the adjacent non-cancerous tissues (ANCT) (54.0% vs. 28.0%, P = 0.008), and was positively associated with the lymphatic invasion of the tumor (P = 0.01). Moreover, knockdown of PTTG1 expression inhibited tumor proliferation and invasion of LAC cells, companied by the decreased expression of CyclinD1 and MMP-2 and increased expression of p-TGFβ1 and p-SMAD3. Collectively, our findings indicate that high expression of PTTG1 is correlated with the tumor metastasis of LAC patients, and knockdown of PTTG1 suppresses the growth and invasion of LAC cells through upregulation of the TGFβ1/SMAD3 signaling, suggesting that PTTG1 may be a potential target for developing an effective immunotherapeutic strategy for LAC.

Topics: A549 Cells; Adenocarcinoma; Adenocarcinoma of Lung; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Lymphatic Metastasis; Matrix Metalloproteinase 2; Neoplasm Invasiveness; Securin; Signal Transduction; Smad3 Protein; Transforming Growth Factor beta1; Up-Regulation

Metastasis is a leading cause of mortality for osteosarcoma patients. The molecular pathological mechanism remains to be elucidated. In the previously study, we established two osteosarcoma cell lines with different metastatic potentials. Differential expressed genes and proteins regarding metastatic ability have been identified. MicroRNAs are important regulators in tumorigenesis and tumor progression. In this study, microRNA microarray was used to assess the differential expressed miRNAs level between these two cell lines. One of the top ranked miRNAs-miR-195 was identified highly expressing in lowly metastatic cells. It was showed that over-expression of miR-195 substantially inhibits migration and invasion of osteosarcoma cells in vitro and pulmonary metastasis formation in vivo. Meanwhile, CCND1 was identified as the target gene of miR-195 and further studied. More importantly, using real-time PCR, we evaluated the expression of miR-195 and CCND1 in osteosarcoma samples from 107 frozen biopsy tissues and 99 formalin- or paraformalin-fixed, paraffin-embedded (FFPE) tissues. Results indicated lowly expressed miR-195 or highly CCND1 correlated with positive overall survival and their expression inversely related to each other. In summary, our study suggests miR-195 functions as a tumor metastasis suppressor gene by down-regulating CCND1 and can be used as a potential target in the treatment of osteosarcoma.

Topics: Adolescent; Adult; Animals; Bone Neoplasms; Cell Line, Tumor; Cell Movement; Cyclin D1; Female; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Heterografts; Humans; Lung Neoplasms; Male; Mice; Mice, Inbred BALB C; Mice, Nude; MicroRNAs; Neoplasm Invasiveness; Osteosarcoma; Proportional Hazards Models; Recombinant Fusion Proteins; RNA, Neoplasm; Tissue Array Analysis; Young Adult

We investigated the role of macrophages in promoting benzopyrene (BaP)-induced malignant transformation of human bronchial epithelial cells using a BaP-induced tumor transformation model with a bionic airway chip in vitro and in animal models. The bionic airway chip culture data showed that macrophages promoted BaP-induced malignant transformation of human bronchial epithelial cells, which was mediated by nuclear factor (NF)-κB and STAT3 pathways to induce cell proliferation, colony formation in chip culture, and tumorigenicity in nude mice. Blockage of interleukin (IL)-6 or tumor necrosis factor (TNF)-α signaling or inhibition of NF-κB, STAT3, or cyclinD1 expression abrogated the effect of macrophages on malignant transformation in the bionic airway chip culture. In vivo, macrophages promoted lung tumorigenesis in a carcinogen-induced animal model. Similarly, blockage of NF-κB, STAT3, or cyclinD1 using siRNA transfection decreased the carcinogen-induced tumorigenesis in rats. We demonstrated that macrophages are critical in promoting lung tumorigenesis and that the macrophage-initiated TNF-α/NF-κB/cyclinD1 and IL-6/STAT3/cyclinD1 pathways are primarily responsible for promoting lung tumorigenesis.

Topics: Adenocarcinoma; Aged; Animals; Benzo(a)pyrene; Bronchi; Carcinoma, Squamous Cell; Cell Count; Cell Transformation, Neoplastic; Cyclin D1; Epithelial Cells; Female; Humans; Interleukin-6; Lab-On-A-Chip Devices; Lung Neoplasms; Macrophages; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Middle Aged; Neoplasm Proteins; NF-kappa B; Rats; Rats, Wistar; STAT3 Transcription Factor; Tobacco Smoke Pollution; Tumor Necrosis Factor-alpha

Isorhamnetin (Iso), a novel and essential monomer derived from total flavones of Hippophae rhamnoides that has long been used as a traditional Chinese medicine for angina pectoris and acute myocardial infarction, has also shown a spectrum of antitumor activity. However, little is known about the mechanisms of action Iso on cancer cells.. To investigate the effects of Iso on A549 lung cancer cells and underlying mechanisms.. A549 cells were treated with 10~320 μg/ml Iso. Their morphological and cellular characteristics were assessed by light and electronic microscopy. Growth inhibition was analyzed by MTT, clonogenic and growth curve assays. Apoptotic characteristics of cells were determined by flow cytometry (FCM), DNA fragmentation, single cell gel electrophoresis (comet) assay, immunocytochemistry and terminal deoxynucleotidyl transferase nick end labeling (TUNEL) . Tumor models were setup by transplanting Lewis lung carcinoma cells into C57BL/6 mice, and the weights and sizes of tumors were measured.. Iso markedly inhibited the growth of A549 cells with induction of apoptotic changes. Iso at 20 μg/ml, could induce A549 cell apoptosis, up-regulate the expression of apoptosis genes Bax, Caspase-3 and P53, and down-regulate the expression of Bcl-2, cyclinD1 and PCNA protein. The tumors in tumor-bearing mice treated with Iso were significantly smaller than in the control group. The results of apoptosis-related genes, PCNA, cyclinD1 and other protein expression levels of transplanted Lewis cells were the same as those of A549 cells in vitro.. Iso, a natural single compound isolated from total flavones, has antiproliferative activity against lung cancer in vitro and in vivo. Its mechanisms of action may involve apoptosis of cells induced by down-regulation of oncogenes and up-regulation of apoptotic genes.

Topics: Animals; Apoptosis; bcl-2-Associated X Protein; Carcinoma, Lewis Lung; Caspase 3; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Down-Regulation; Humans; Lung Neoplasms; Mice; Mice, Inbred C57BL; Proliferating Cell Nuclear Antigen; Quercetin; Tumor Suppressor Protein p53; Up-Regulation

Long noncoding RNAs (lncRNAs) have emerged as key regulators of tumor development and progression. The lncRNA HNF1A-antisense 1 (HNF1A-AS1) is a 2455-bp transcript on chromosome 12 with a potential oncogenic role in esophageal adenocarcinoma. Nevertheless, current understanding of the involvement of HNF1A-AS1 in lung adenocarcinoma tumorigenesis remains limited. In this study, we analyzed the roles of HNF1A-AS1 in 40 lung adenocarcinoma tissues and five lung cancer cell lines. Our results showed that HNF1A-AS1 was significantly up-regulated in lung adenocarcinoma tissues compared with corresponding non-tumor tissues, and its expression level was significantly correlated with TNM stage, tumor size, and lymph node metastasis. The UCSC Cancer Genomics Browser's Kaplan-Meier plot suggested that patients in the high HNF1A-AS1 expression subgroup experienced worse overall survival compared to the low expression subgroup. Moreover, HNF1A-AS1 was determined to promote tumor proliferation and metastasis, both in vitro and in vivo, by regulating cyclin D1, E-cadherin, N-cadherin and β-catenin expression. In addition, the binding of HNF1A-AS1 to DNMT1 may explain its regulation of E-cadherin. In conclusions, we demonstrated that increased HNF1A-AS1 expression could regulate cell proliferation and metastasis and identified it as a poor prognostic biomarker in lung adenocarcinoma.

Topics: Adenocarcinoma; Adenocarcinoma of Lung; Aged; Animals; Apoptosis; beta Catenin; Biomarkers, Tumor; Cadherins; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cell Transformation, Neoplastic; Cyclin D1; Disease Progression; DNA (Cytosine-5-)-Methyltransferase 1; DNA (Cytosine-5-)-Methyltransferases; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation, Neoplastic; Hepatocyte Nuclear Factor 1-alpha; Humans; Lung Neoplasms; Lymphatic Metastasis; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Invasiveness; Neoplasm Staging; Neoplasm Transplantation; Protein Binding; RNA Interference; RNA, Long Noncoding; RNA, Small Interfering; Transplantation, Heterologous; Up-Regulation

Chronic inflammation is an important risk factor for lung cancer. Therefore, identification of chemopreventive agents that suppress inflammation-driven lung cancer is indispensable. We studied the efficacy of combinations of indole-3-carbinol (I3C) and silibinin (Sil), 20 µmol/g diet each, against mouse lung tumors induced by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and driven by lipopolysaccharide (LPS), a potent inflammatory agent and constituent of tobacco smoke. Mice treated with NNK + LPS developed 14.7±4.1 lung tumors/mouse, whereas mice treated with NNK + LPS and given combinations of I3C and Sil had 7.1±4.5 lung tumors/mouse, corresponding to a significant reduction of 52%. Moreover, the number of largest tumors (>1.0mm) was significantly reduced from 6.3±2.9 lung tumors/mouse in the control group to 1.0±1.3 and 1.6±1.8 lung tumors/mouse in mice given I3C + Sil and I3C alone, respectively. These results were paralleled by significant reductions in the level of proinflammatory and procarcinogenic proteins (pSTAT3, pIκBα and COX-2) and proteins that regulate cell proliferation (pAkt, cyclin D1, CDKs 2, 4, 6 and pRB). Further studies in premalignant bronchial cells showed that the antiproliferative effects of I3C + Sil were higher than the individual compounds and these effects were mediated by targeting cyclin D1, CDKs 2, 4 and 6 and pRB. I3C + Sil suppressed cyclin D1 by reducing its messenger RNA level and by enhancing its proteasomal degradation. Our results showed the potential lung cancer chemopreventive effects of I3C + Sil in smokers/former smokers with chronic pulmonary inflammatory conditions.

Topics: Animals; Anticarcinogenic Agents; Cell Cycle Proteins; Cell Line, Tumor; Cell Proliferation; Cell Transformation, Neoplastic; Chemoprevention; Cyclin D1; Cyclin-Dependent Kinases; Cyclooxygenase 2; Drug Combinations; Female; Humans; I-kappa B Proteins; Indoles; Inflammation; Interleukin-6; Lipopolysaccharides; Lung; Lung Neoplasms; Mice; Mice, Inbred A; NF-KappaB Inhibitor alpha; Nitrosamines; Proto-Oncogene Proteins c-akt; Random Allocation; Retinoblastoma Protein; Silybin; Silymarin; Smoke; STAT3 Transcription Factor; Tumor Necrosis Factor-alpha

Lung cancer as an aggressive type tumor is rapidly growing and has become the leading cause of cancer-related death worldwide. γ-Glutamylcyclotransferase (GGCT) has been shown as a diagnostic marker in various cancers. To reveal whether there is a correlation between GGCT and lung cancer, GGCT expression in human lung cancer cell lines was first determined by real-time quantitative PCR and western blot. GGCT is expressed in all tested lung cancer cell lines, A549, H1299, and H460. Then, a lentivirus-based system was applied to knock down GGCT in A549 cells, which were thus divided into Lv-shGGCT, Lv-shCon, and Con (noninfected) groups. Methylthiazol tetrazolium assay showed that the cell proliferation was decreased by over 50% in the Lv-shGGCT group compared with controls. The size and number of colonies were dramatically reduced in the GGCT knockdown group, as measured by colony formation assay. Moreover, A549 cells infected with Lv-shGGCT were arrested in the G0/G1 phase as assayed by flow cytometry. Furthermore, the expression levels of CDK4, CDK6, and cyclin D1 were decreased and the cleaved level of PARP was increased in GGCT knockdown cells. In conclusion, GGCT plays a critical role in lung cancer cell proliferation and may be a potential cancer therapeutic target.

Topics: Cell Line, Tumor; Cell Proliferation; Colony-Forming Units Assay; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase 6; G1 Phase Cell Cycle Checkpoints; gamma-Glutamylcyclotransferase; Gene Expression; Genetic Vectors; Humans; Lentivirus; Lung Neoplasms; Poly(ADP-ribose) Polymerases; RNA Interference

SET oncoprotein is an endogenous inhibitor of protein phosphatase 2A (PP2A), and SET-mediated PP2A inhibition is an important regulatory mechanism for promoting cancer initiation and progression of several types of human leukemia disease. However, its potential relevance in solid tumors as non-small cell lung cancer (NSCLC) remains mostly unknown. In this study, we showed that SET was evidently overexpressed in human NSCLC cell lines and NSCLC tissues. Clinicopathologic analysis showed that SET expression was significantly correlated with clinical stage (p < 0.001), and lymph node metastasis (p < 0.05). Kaplan-Meier analysis revealed that patients with high SET expression had poorer overall survival rates than those with low SET expression. Moreover, knockdown of SET in NSCLC cells resulted in attenuated proliferative and invasive abilities. The biological effect of SET on proliferation and invasion was mediated by the inhibition of the PP2A, which in turn, activation of AKT and ERK, increased the expression of cyclin D1 and MMP9, and decreased the expression of p27. Furthermore, we observed that restoration of PP2A using SET antagonist FTY720 impaired proliferative and invasive potential in vitro, as well as inhibited tumor growth in vivo of NSCLC cells. Taken together, SET oncoprotein plays an important role in NSCLC progression, which could serve as a potential prognosis marker and a novel therapeutic target for NSCLC patients.

Topics: Animals; Blotting, Western; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p27; Disease Progression; DNA-Binding Proteins; Extracellular Signal-Regulated MAP Kinases; Fingolimod Hydrochloride; Histone Chaperones; Humans; Kaplan-Meier Estimate; Lung Neoplasms; Lymphatic Metastasis; Matrix Metalloproteinase 9; Mice; Neoplasm Staging; Prognosis; Protein Phosphatase 2; Proto-Oncogene Proteins c-akt; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; Transcription Factors; Transplantation, Heterologous

Despite great efforts to improve survival rates, the prognosis of lung cancer patients is still very poor, mainly due to high invasiveness. We developed brain metastatic PC14PE6/LvBr4 cells through intracardiac injection of lung adenocarcinoma PC14PE6 cells. Western blot and RT-qPCR analyses revealed that PC14PE6/LvBr4 cells had mesenchymal characteristics and higher invasiveness than PC14PE6 cells. We found that cyclin D1 was upregulated, miR-95-3p was inversely downregulated, and pri-miR-95 and its host gene, ABLIM2, were consistently decreased in PC14PE6/LvBr4 cells. MiR-95-3p suppressed cyclin D1 expression through direct binding to the 3' UTR of cyclin D1 mRNA and suppressed invasiveness, proliferation, and clonogenicity of PC14PE6/LvBr4 cells. Ectopic cyclin D1 reversed miR-95-3p-mediated inhibition of invasiveness and clonogenicity, demonstrating cyclin D1 downregulation is involved in function of miR-95-3p. Using bioluminescence imaging, we found that miR-95-3p suppressed orthotopic tumorigenicity and brain metastasis in vivo and increased overall survival and brain metastasis-free survival. Consistent with in vitro metastatic cells, the levels of miR-95-3p, pri-miR-95, and ABLIM2 mRNA were decreased in brain metastatic tissues compared with lung cancer tissues and higher cyclin D1 expression was involved in poor prognosis. Taken together, our results demonstrate that miR-95-3p is a potential therapeutic target for brain metastasis of lung adenocarcinoma cells.

Topics: Adenocarcinoma; Adenocarcinoma of Lung; Animals; Brain Neoplasms; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Down-Regulation; Female; Heterografts; Humans; Lung Neoplasms; Mice; Mice, Inbred BALB C; Mice, Nude; MicroRNAs; Neoplasm Metastasis; Prognosis; Transfection

Unlike p53, which is mutated at a high rate in human cancers, its homologue p73 is not mutated but is often overexpressed, suggesting a possible context-dependent role in growth promotion. Previously, we have shown that co-expression of TAp73 with the proto-oncogene c-Jun can augment cellular growth and potentiate transactivation of activator protein (AP)-1 target genes such as cyclin D1. Here, we provide further mechanistic insights into the cooperative activity between these two transcription factors. Our data show that TAp73-mediated AP-1 target gene transactivation relies on c-Jun dimerization and requires the canonical AP-1 sites on target gene promoters. Interestingly, only selected members of the Fos family of proteins such as c-Fos and Fra1 were found to cooperate with TAp73 in a c-Jun-dependent manner to transactivate AP-1 target promoters. Inducible expression of TAp73 led to the recruitment of these Fos family members to the AP-1 target promoters on which TAp73 was found to be bound near the AP-1 site. Consistent with the binding of TAp73 and AP-1 members on the target promoters in a c-Jun-dependent manner, TAp73 was observed to physically interact with c-Jun specifically at the chromatin via its carboxyl-terminal region. Furthermore, co-expression of c-Fos or Fra1 was able to cooperate with TAp73 in potentiating cellular growth, similarly to c-Jun. These data together suggest that TAp73 plays a vital role in activation of AP-1 target genes via direct binding to c-Jun at the target promoters, leading to enhanced loading of other AP-1 family members, thereby leading to cellular growth.

Topics: Cell Line, Tumor; Cell Proliferation; Cyclin D1; DNA-Binding Proteins; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Nuclear Proteins; Promoter Regions, Genetic; Proto-Oncogene Mas; Proto-Oncogene Proteins c-fos; Proto-Oncogene Proteins c-jun; Transcription Factor AP-1; Transcriptional Activation; Tumor Protein p73; Tumor Suppressor Proteins

Recent studies suggest that high expression of the proinflammatory cytokine IL6 is associated with poor survival of lung cancer patients. Accordingly, IL6 has been a target of great interest for lung cancer therapy. However, the role of IL6 in lung cancer has not been determined yet. Here, we demonstrate that IL6 plays opposite roles in the initiation and growth of lung cancer in a mouse model of lung cancer induced by the K-Ras oncogene. We find that compared with wild-type mice, IL6-deficient mice developed much more lung tumors after an activating mutant of K-Ras was induced in the lungs. However, lung tumors developed in IL6-deficient mice were significantly smaller. Notably, both the lung tumor-suppressing and -promoting functions of IL6 involve its ability in activating the transcription factor STAT3. IL6/STAT3 signaling suppressed lung cancer initiation through maintaining lung homeostasis, regulating lung macrophages, and activating cytotoxic CD8 T cells under K-Ras oncogenic stress, whereas it promoted lung cancer cell growth through inducing the cell proliferation regulator cyclin D1. These studies reveal a previously unexplored role of IL6/STAT3 signaling in maintaining lung homeostasis and suppressing lung cancer induction. These studies also significantly improve our understanding of lung cancer and provide a molecular basis for designing IL6/STAT3-targeted therapies for this deadliest human cancer.

Topics: Animals; CD8-Positive T-Lymphocytes; Cell Proliferation; Chemokines; Cyclin D1; Disease Progression; Gene Expression; Homeostasis; Immunohistochemistry; Interleukin-10; Interleukin-6; Lung; Lung Neoplasms; Macrophages; Mice, Knockout; Mutation; Nitric Oxide Synthase Type II; Proto-Oncogene Proteins; Proto-Oncogene Proteins p21(ras); ras Proteins; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; STAT3 Transcription Factor

We have sequenced the genomes of 110 small cell lung cancers (SCLC), one of the deadliest human cancers. In nearly all the tumours analysed we found bi-allelic inactivation of TP53 and RB1, sometimes by complex genomic rearrangements. Two tumours with wild-type RB1 had evidence of chromothripsis leading to overexpression of cyclin D1 (encoded by the CCND1 gene), revealing an alternative mechanism of Rb1 deregulation. Thus, loss of the tumour suppressors TP53 and RB1 is obligatory in SCLC. We discovered somatic genomic rearrangements of TP73 that create an oncogenic version of this gene, TP73Δex2/3. In rare cases, SCLC tumours exhibited kinase gene mutations, providing a possible therapeutic opportunity for individual patients. Finally, we observed inactivating mutations in NOTCH family genes in 25% of human SCLC. Accordingly, activation of Notch signalling in a pre-clinical SCLC mouse model strikingly reduced the number of tumours and extended the survival of the mutant mice. Furthermore, neuroendocrine gene expression was abrogated by Notch activity in SCLC cells. This first comprehensive study of somatic genome alterations in SCLC uncovers several key biological processes and identifies candidate therapeutic targets in this highly lethal form of cancer.

Topics: Alleles; Animals; Cell Line, Tumor; Chromosome Breakpoints; Cyclin D1; Disease Models, Animal; DNA-Binding Proteins; Female; Gene Expression Profiling; Genome, Human; Genomics; Humans; Lung Neoplasms; Male; Mice; Mutation; Neurosecretory Systems; Nuclear Proteins; Receptors, Notch; Retinoblastoma Protein; Signal Transduction; Small Cell Lung Carcinoma; Tumor Protein p73; Tumor Suppressor Protein p53; Tumor Suppressor Proteins

The DEAD-box-protein DDX5 is an ATP-dependent RNA helicase that is frequently overexpressed in various cancers and acts as a transcriptional co-activator of several transcription factors, including β-catenin. DDX5 is reported to be involved in cancer progression by promoting cell proliferation and epithelial-mesenchymal transition. However, the clinical significance and biological role of DDX5 in non-small-cell lung cancer (NSCLC) remain largely unknown. In this study, we examined the expression of DDX5 in clinical NSCLC samples, investigated its role in regulating NSCLC cell proliferation and tumorigenesis, and explored the possible molecular mechanism. We found that DDX5 was significantly overexpressed in NSCLC tissues as compared with the matched normal adjacent tissues. In addition, overexpression of DDX5 was associated with advanced clinical stage, higher Ki67 index, and shorter overall survival in NSCLC patients. Upregulation of DDX5 promoted proliferation of NSCLC cells in vitro and growth of NSCLC xenografts in vivo, whereas downregulation of DDX5 showed the opposite effects. Furthermore, DDX5 directly interacted with β-catenin, promoted its nuclear translocation, and co-activated the expression of cyclin D1 and c-Myc. β-catenin silencing significantly abrogated DDX5-induced cyclin D1 and c-Myc expression and proliferation in NSCLC cells. Interestingly, DDX5 and cyclin D1 expression followed positive correlation in the same set of NSCLC samples. These findings indicated that DDX5 played an important role in the proliferation and tumorigenesis of NSCLC cells by activating the β-catenin signaling pathway. Therefore, DDX5 may serve as a novel prognostic marker and potential therapeutic target in the treatment of NSCLC.

Topics: Active Transport, Cell Nucleus; Animals; beta Catenin; Biomarkers, Tumor; Carcinogenesis; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Proliferation; Cyclin D1; DEAD-box RNA Helicases; Enzyme Activation; Gene Expression Regulation, Neoplastic; Humans; Ki-67 Antigen; Lung Neoplasms; Male; Mice; Mice, Nude; Prognosis; Proto-Oncogene Proteins c-myc; RNA Interference; RNA, Small Interfering; Signal Transduction

Lung cancer is one of the leading causes of cancer-related deaths worldwide. Early diagnosis is the best defense against this threat and is therefore of vital importance. In this study, we investigated the role of long non-coding RNA HOTTIP in the tumor growth of lung cancer. Initially, we found that expression of HOTTIP was significantly elevated in 20 cases of lung cancer. HOTTIP was also differentially expressed in a consecutive of lung cancer cell lines. Furthermore, specific shRNA against HOTTIP was employed to deplete expression of HOTTIP in A549 cells and NCI-H446 cells. After successfully depletion of HOTTIP, cell proliferation and colony formation were significantly inhibited in vitro. Tumor growth in vivo was also suppressed after depletion of HOTTIP in a mouse model of lung cancer. Moreover, depletion of HOTTIP caused cell cycle arrest in G0/G1 phase and induced significant cell apoptosis. Cell cycle regulators Cdc25C, Cyclin B1 and Cyclin D1 were decreased upon depletion of HOTTIP. Pro-apoptotic factor Bad was up-regulated, whereas anti-apoptotic factors Bcl-2 and Bcl-xL were down-regulated after HOTTIP ablation. These data suggest that lncRNA HOTTIP contributes to tumor growth in vivo and in vitro and inhibits cell apoptosis in lung cancer.

Topics: Animals; Apoptosis; cdc25 Phosphatases; Cell Line, Tumor; Cell Proliferation; Cyclin B1; Cyclin D1; G1 Phase Cell Cycle Checkpoints; Humans; Lung Neoplasms; Male; Mice; Mice, Inbred BALB C; Neoplasm Transplantation; RNA Interference; RNA, Long Noncoding; RNA, Small Interfering; Transplantation, Heterologous

Hsa-miRNA-139-5p (miR-139-5p) has recently been discovered having anticancer efficacy in different organs. However, the role of miR-139-5p on lung cancer is still ambiguous. In this study, we investigated the role of miR-139-5p on development of lung cancer. Results indicated miR-139-5p was significantly down-regulated in primary tumor tissues and very low levels were found in a non-small cell lung cancer (NSCLC) cell lines. Ectopic expression of miR-139-5p in NSCLC cell lines significantly suppressed cell growth through inhibition of cyclin D1 and up-regulation of p57(Kip2). In addition, miR-139-5p induced apoptosis, as indicated by up-regulation of key apoptosis gene cleaved caspase-3, and down-regulation of anti-apoptosis gene Bcl2. Moreover, miR-139-5p inhibited cellular metastasis through inhibition of matrix metalloproteinases (MMP)-7 and MMP-9. Further, oncogene c-Met was revealed to be a putative target of miR-139-5p, which was inversely correlated with miR-139-5p expression. Taken together, our results demonstrated that miR-139-5p plays a pivotal role in lung cancer through inhibiting cell proliferation, metastasis, and promoting apoptosis by targeting oncogenic c-Met.

Topics: Animals; Apoptosis; Blotting, Western; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Down-Regulation; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Mice, Inbred BALB C; Mice, Nude; MicroRNAs; Microscopy, Fluorescence; Proto-Oncogene Proteins c-met; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; Survival Analysis; Tumor Burden; Xenograft Model Antitumor Assays

This study was aimed at identifying prognostic biomarkers for stage II-IIIA non-small cell lung cancer (NSCLC) according to histology and at investigating the effect of vorinostat on the expression of these biomarkers.. Expression levels of cyclin D1, cyclin A2, cyclin E, and p16 proteins that are involved in the G1-to-S phase progression of cell cycle were analyzed using immunohistochemistry in formalin-fixed paraffin-embedded tissues from 372 samples of stage II-IIIA NSCLC. The effect of vorinostat on the expression of these proteins, impacts on cell cycle, and histone modification was explored in lung cancer cells.. Abnormal expression of cyclin A2, cyclin D1, cyclin E, and p16 was found in 66, 47, 34, and 51 % of 372 cases, respectively. Amongst the four proteins, only cyclin D1 overexpression was significantly associated with poor recurrence-free survival (adjusted hazard ratio = 1.87; 95 % confidence interval = 1.12 - 2.69, P = 0.02) in adenocarcinoma but not in squamous cell carcinoma (P = 0.44). Vorinostat inhibited cell cycle progression to the S-phase and induced down-regulation of cyclin D1 in vitro. The down-regulation of cyclin D1 by vorinostat was comparable to a siRNA-mediated knockdown of cyclin D1 in A549 cells, but vorinostat in the presence of benzo[a]pyrene showed a differential effect in different lung cancer cell lines. Cyclin D1 down-regulation by vorinostat was associated with the accumulation of dimethyl-H3K9 at the promoter of the gene.. The present study suggests that cyclin D1 may be an independent prognostic factor for recurrence-free survival in stage II-IIIA adenocarcinoma of lung and its expression may be modulated by vorinostat.

Topics: Aged; Antineoplastic Agents; Carcinoma, Non-Small-Cell Lung; Cell Cycle; Cell Line, Tumor; Cyclin D1; Disease-Free Survival; DNA Methylation; Female; Gene Expression Regulation, Neoplastic; Humans; Hydroxamic Acids; In Vitro Techniques; Lung Neoplasms; Male; Middle Aged; Prognosis; Up-Regulation; Vorinostat

To examine the expression pattern of CCND1 and analyze the correlation of its nuclear expression with clinicopathologic features and prognosis in lung adenocarcinoma.. CCND1 mRNA and protein levels in lung adenocarcinoma tissues were examined. The relationship between nuclear CCND1 protein expression and clinical features including survival prognosis was analyzed.. CCND1 mRNA levels were markedly increased in lung adenocarcinoma (P=0.0019). Western blot analysis confirmed increased nuclear CCND1 protein expression in lung adenocarcinoma specimens. Immunohistochemistry analysis confirmed that CCND1 protein was predominantly nuclear localized in lung adenocarcinoma cells and significantly elevated relative to normal lung tissues (P<0.001). Furthermore, high levels of nuclear CCND1 were positively correlated with clinical stage (P=0.026). Patients with nuclear CCND1 expression had a significantly shorter overall survival time than did patients with low expression. Interestingly, nuclear CCND1 expression in clinical stage I+II, but not clinical stage III, was shown associated with poor prognosis and shorter overall survival time for lung adenocarcinoma patients by strata analysis. Finally, nuclear CCND1 expression tended to be an independent prognostic indicator (P=0.087) for lung adenocarcinoma patient survival.. Increased nuclear CCND1 is a potential unfavorable prognostic factor for lung adenocarcinoma patients, especially those with clinical early stage (stage I+II).

Topics: Adenocarcinoma; Adenocarcinoma of Lung; Biomarkers, Tumor; Blotting, Western; Cell Nucleus; Cyclin D1; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Kaplan-Meier Estimate; Lung Neoplasms; Neoplasm Staging; Predictive Value of Tests; Proportional Hazards Models; Real-Time Polymerase Chain Reaction; Risk Factors; RNA, Messenger; Time Factors; Tissue Array Analysis; Up-Regulation

Furanodiene (FUR) is a natural terpenoid isolated from Rhizoma Curcumae, a well-known Chinese medicinal herb that presents anti-proliferative activities in several cancer cell lines. Recently, we found that the combined treatment of FUR with paclitaxel (TAX) showed synergetic anti-proliferative activities in 95-D lung cancer cells. Herein, we showed that FUR reduced the cell numbers distributed in mitosis phase induced by TAX while increased those in G1 phase. The protein levels of cyclin D1, cyclin B1, CDK6 and c-Myc were all down-regulated in the group of combined treatment. The dramatically down-regulated expression of integrin β4, focal adhesion kinase and paxillin might partially contribute to the synergic effect. Though FUR alone obviously induced endoplasmic reticulum stress, this signaling pathway may not contribute to the synergetic anti-proliferative effect as the protein expression of CHOP and BIP was similar in FUR alone and combined treatment group.

Topics: Cell Cycle; Cell Line, Tumor; Curcuma; Cyclin B1; Cyclin D1; Cyclin-Dependent Kinase 6; Drug Synergism; Drugs, Chinese Herbal; Endoplasmic Reticulum Stress; Focal Adhesion Kinase 1; Furans; Heterocyclic Compounds, 2-Ring; Humans; Integrin beta4; Lung Neoplasms; Paclitaxel; Paxillin; Proto-Oncogene Proteins c-myc; Signal Transduction

Recent population studies provide clues that the use of curcumin may be associated with reduced incidence and improved prognosis of certain cancers. In the present study, we demonstrated that curcumin acted as a growth inhibitor for lung cancer cells. Our results found that curcumin inhibited cell proliferation, which was associated with upregulation of the cyclin-dependent kinase inhibitors, p27 and p21, and downregulation of cyclin D1. In addition, we showed that curcumin induced the expression of forkhead box protein O1 (FOXO1) through activation of extracellular signal-regulated kinase 1/2 signaling. These findings provide evidence for a mechanism that may contribute to the antineoplastic effects of curcumin and justify further work to explore potential roles for activators of FOXO1 in the prevention and treatment of lung cancer.

Topics: Antineoplastic Agents; Cell Line, Tumor; Cell Proliferation; Curcumin; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase Inhibitor p27; Disease Progression; Dose-Response Relationship, Drug; Forkhead Box Protein O1; Forkhead Transcription Factors; Humans; Lung Neoplasms; MAP Kinase Signaling System; Neoplasm Metastasis

N-cadherin and HER2/neu were found to be co-expressed in invasive breast carcinomas. To test the contribution of N-cadherin and HER2 in mammary tumor metastasis, we targeted N-cadherin expression in the mammary epithelium of the MMTV-Neu mouse. In the context of ErbB2/Neu, N-cadherin stimulated carcinoma cell invasion, proliferation and metastasis. N-cadherin caused fibroblast growth factor receptor (FGFR) upmodulation, resulting in epithelial-to-mesenchymal transition (EMT) and stem/progenitor like properties, involving Snail and Slug upregulation, mammosphere formation and aldehyde dehydrogenase activity. N-cadherin potentiation of the FGFR stimulated extracellular signal regulated kinase (ERK) and protein kinase B (AKT) phosphorylation resulting in differential effects on metastasis. Although ERK inhibition suppressed cyclin D1 expression, cell proliferation and stem/progenitor cell properties, it did not affect invasion or EMT. Conversely, AKT inhibition suppressed invasion through Akt 2 attenuation, and EMT through Snail inhibition, but had no effect on cyclin D1 expression, cell proliferation or mammosphere formation. These findings suggest N-cadherin/FGFR has a pivotal role in promoting metastasis through differential regulation of ERK and AKT, and underscore the potential for targeting the FGFR in advanced ErbB2-amplified breast tumors.

Topics: Aldehyde Dehydrogenase; Animals; Benzamides; Breast Neoplasms; Cadherins; Cell Movement; Cell Proliferation; Cyclin D1; Diphenylamine; Epithelial-Mesenchymal Transition; Extracellular Signal-Regulated MAP Kinases; Female; Humans; Lung Neoplasms; MAP Kinase Kinase 1; Mice; Mice, Transgenic; Neoplasm Invasiveness; Neoplasm Metastasis; Phosphorylation; Proto-Oncogene Proteins c-akt; Pyrimidines; Receptor, ErbB-2; Receptors, Fibroblast Growth Factor; RNA Interference; RNA, Small Interfering; Signal Transduction; Snail Family Transcription Factors; Spheroids, Cellular; Stem Cells; Transcription Factors; Tumor Cells, Cultured

MicroRNA-7 (miR-7) has been reported to be a tumour suppressor gene. However, whether it has a role in the growth of non-small-cell lung cancer (NSCLC) and what is its target involved in the tumour growth is still under investigation.. NSCLC tissue sample, NSCLC cell lines and tissue microarray were investigated in this study. Total RNA, miRNA and protein were used for RT-PCR and western blot analysis. Immunohistochemistry was performed in tissues microarray. Cell culture and intervention experiments were performed in vitro and in vivo. Bioinformatics prediction, western blot and luciferase assay were identified the target of miR-7.. In this study, we found that the expression of miR-7 was significantly downregulated not only in NSCLC cell lines, but also in human NSCLC tissues compared with the matched adjacent tissues. Restoration of its expression through miR-7 mimics in A549 and H1299 NSCLC cells inhibited cell proliferation, colony formation, and cell-cycle progression in vitro. More importantly, the tumorigenicity in nude mice was reduced after administration of miR-7 in vivo. In advance, through bioinformatic analysis, luciferase assay and western blot, we identified a novel target of miR-7, PA28gamma (a proteasome activator) to be enrolled in the regulation with tumour. PA28gamma mRNA and protein levels are markedly upregulated in NSCLC cell lines and tumour samples, exhibiting a strong inverse relation with that of miR-7. In addition, knockdown of PA28gamma induced similar effects as overexpression of miR-7 in NSCLC cells. Furthermore, miR-7 overexpression or silencing of PA28gamma reduced the cyclinD1 expression at mRNA and protein level in NSCLC cell lines.. All these findings strongly imply that the overexpression of PA28gamma resulted from miR-7 downexpression in NSCLC has an important role in promoting cancer cell progress and consequently results in NSCLC growth. Thus, strategies targeting PA28gamma and/or miR-7 may become promising molecular therapies in NSCLC treatment.

Topics: Animals; Autoantigens; Carcinogenesis; Carcinoma, Non-Small-Cell Lung; Cell Cycle; Cell Line; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Down-Regulation; Female; Gene Expression Regulation, Neoplastic; Genes, Tumor Suppressor; HEK293 Cells; Heterografts; Humans; Lung Neoplasms; Mice; Mice, Nude; MicroRNAs; Molecular Targeted Therapy; Proteasome Endopeptidase Complex; Up-Regulation

Epigallocatechin gallate (EGCG), the major biologically active compound in green tea, is a well-known chemoprevention agent. Although several reports have shown that EGCG exerts its anticancer activity by targeting specific cell signaling pathways, the underlying molecular mechanism(s) are only partially understood. In the present study, we report that EGCG had a profound antiproliferative effect on human lung cancer cells. EGCG inhibited anchorage-independent growth and induced cell cycle G0/G1 phase arrest. The mechanism underlying EGCG antitumor potency was mainly dependent on suppression of the EGFR signaling pathway. Short-term EGCG exposure substantially decreased EGF-induced EGFR, AKT and ERK1/2 activation. Moreover, long-term EGCG treatment not only inhibited total and membranous EGFR expression, but also markedly attenuated EGFR nuclear localization and expression of the downstream target gene cyclin D1, indicating that EGCG treatment suppressed EGFR transactivation. Additionally, knockdown of EGFR in lung cancer cells decreased their sensitivity to EGCG. Thus, inhibition of the EGFR signaling pathway may partly contribute to the anticancer activity of EGCG.

Topics: Antineoplastic Agents; Catechin; Cell Adhesion; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Drug Resistance, Neoplasm; Drug Screening Assays, Antitumor; ErbB Receptors; G1 Phase Cell Cycle Checkpoints; Gene Expression; Gene Knockdown Techniques; Humans; Lung Neoplasms; Signal Transduction

The aim of this study was to investigate the role of apelin in the cell proliferation and autophagy of lung adenocarcinoma. The over-expression of APJ in lung adenocarcinoma was detected by immunohistochemistry, while plasma apelin level in lung cancer patients was measured by enzyme-linked immunosorbent assay. Our findings revealed that apelin-13 significantly increased the phosphorylation of ERK1/2, the expression of cyclin D1, microtubule-associated protein 1 light chain 3A/B (LC3A/B), and beclin1, and confirmed that apelin-13 promoted A549 cell proliferation and induced A549 cell autophagy via ERK1/2 signaling. Moreover, there are pores on the surface of human lung adenocarcinoma cell line A549 and apelin-13 causes cell surface smooth and glossy as observed under atomic force microscopy. These results suggested that ERK1/2 signaling pathway mediates apelin-13-induced lung adenocarcinoma cell proliferation and autophagy. Under our experimental condition, autophagy associated with 3-methyladenine was not involved in cell proliferation.

Topics: Adenocarcinoma; Adenocarcinoma of Lung; Apoptosis Regulatory Proteins; Autophagy; Beclin-1; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Extracellular Signal-Regulated MAP Kinases; Flavonoids; Humans; Intercellular Signaling Peptides and Proteins; Lung Neoplasms; Membrane Proteins; Microtubule-Associated Proteins; Phosphorylation; Signal Transduction

MicroRNAs (miRNAs) are small non-coding RNAs that negatively regulate the translation of messenger RNAs by binding their 3'-untranslated region (3'UTR). In this study, we found that miR-490-3p is significantly down-regulated in A549 lung cancer cells compared with the normal bronchial epithelial cell line. To better characterize the role of miR-490-3p in A549 cells, we performed a gain-of-function analysis by transfecting the A549 cells with chemically synthesized miR-490-3P mimics. Overexpression of miR-490-3P evidently inhibits cell proliferation via G1-phase arrest. We also found that forced expression of miR-490-3P decreased both mRNA and protein levels of CCND1, which plays a key role in G1/S phase transition. In addition, the dual-luciferase reporter assays indicated that miR-490-3P directly targets CCND1 through binding its 3'UTR. These findings indicated miR-490-3P could be a potential suppressor of cellular proliferation.

Topics: 3' Untranslated Regions; Base Sequence; Bronchi; Cell Line; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Down-Regulation; G1 Phase Cell Cycle Checkpoints; Humans; Lung Neoplasms; MicroRNAs; Molecular Mimicry; RNA, Messenger; RNA, Small Interfering; Tumor Stem Cell Assay

Non-small cell lung cancer (NSCLC) is a lethal disease due to the absence of effective diagnostic biomarkers and therapeutic targets. Therefore, novel molecular targets are critically needed to formulate new approaches for this devastating disease. In the present study, using quantitive real-time PCR and immunohistochemistry. we initially found that expression of the ribosome assembly factor NIN/RPN12 binding protein (NOB1) was elevated in the majority of NSCLC tissues when compared to that in the normal lung tissue counterparts, and its expression level was correlated with key pathological characteristics including tumor differentiation, stage and metastasis. Then, the recombinant lentiviral shRNA expression vector carrying NOB1 was constructed and infected into the human NSCLC A549 cell line. Cell proliferation, cell apoptosis, cell cycle distribution and colony formation ability in A549 cells were assessed following downregulation of NOB1 by siRNA. In addition, tumor growth ability in nude mice was evaluated to define the function of NOB1 in cell transformation and tumorigenesis. It was found that downregulation of NOB1 expression using the RNA silencing approach in A549 tumor cells significantly suppressed the proliferation and colony formation ability, and induced tumor apoptosis in vitro. Tumor growth was also suppressed in vivo. These data suggest that NOB1 is an important regulator of the tumorigenic properties of human NSCLC and may be used as a new promising diagnostic biomarker and a potential anticancer therapeutic target for NSCLC.

Topics: Animals; Apoptosis; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin D3; Cyclin-Dependent Kinase Inhibitor p21; Female; Gene Knockdown Techniques; Humans; Lung Neoplasms; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Middle Aged; Neoplasm Transplantation; Nuclear Proteins; RNA-Binding Proteins; RNA, Small Interfering

Studies have suggested a possible correlation between the newly identified E3 ubiquitin ligase ring finger protein 146 (RNF146) and tumor development. However, until now, studies on RNF146 have been restricted to poly(ADP-ribosyl)ation and ubiquitin ligation, whereas the role of RNF146 in tumor biology has rarely been reported. In the present study, the role of RNF146 in non-small cell lung cancer (NSCLC) was investigated. The results showed that the expression of RNF146 was increased in clinical lung cancer samples and cell lines. RNF146 expression correlated with tumor size, differentiation level, lymphatic metastasis, pTNM staging, and prognosis of patients in stage I. RNF146 expression was negatively correlated with Axin expression but positively correlated with the nuclear expression of β-catenin in NSCLC tissues. RNF146 downregulated the expression of Axin in lung cancer cell lines and induced the expression and nuclear distribution of β-catenin. Overexpression of RNF146 in NSCLC cell lines increased the levels of cyclinD1, cyclinE, and CDK4, promoted cell cycle G0/G1-S transitions, and regulated cell proliferation. Overexpression of RNF146 led to upregulated levels of matrix metalloproteinases 2 and 7 and enhanced lung cancer cell invasiveness, events that were mediated by the classical Wnt/β-catenin signaling pathway. In summary, the data in the present study indicate that RNF146 regulated the development and progression of NSCLC by enhancing cell growth, invasion, and survival, suggesting that RNF146 may be a potential treatment target in NSCLC.

Topics: Active Transport, Cell Nucleus; Axin Protein; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Nucleus; Cell Proliferation; Cyclin D1; Female; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Male; Matrix Metalloproteinase 7; Middle Aged; Neoplasm Invasiveness; Ubiquitin-Protein Ligases; Wnt Signaling Pathway

The transcription factor PAX6 is primarily expressed in embryos. PAX6 is also expressed in several tumors and plays an oncogenic role. However, little is known about the role of PAX6 in lung cancer.. The function of PAX6 in lung cancer cells was evaluated by small interfering RNA-mediated depletion of the protein followed by analyses of cell proliferation, anchorage-independent growth, and cell cycle arrest. The changes of cyclin D1, pRB, ERK1/2, p38 expression caused by PAX6 inhibition were detected using western-blotting. The PAX6 mRNA level in 52 pairs of tumors and corresponding matched adjacent normal tissues from non-small cell lung cancer patients and lung cancer cell lines was detected by real-time PCR.. Suppression of PAX6 expression inhibited cell growth and colony formation in A549 and H1299 cells. The percentage of cells in G1-phase increased when PAX6 expression was inhibited. The cyclin D1 protein level, as well as the pRB phosphorylation level, decreased as a result of PAX6 down-regulation. The activity of ERK1/2 and p38 was also suppressed in PAX6 knock-down cells. The PAX6 mRNA was highly expressed in lung cancer tissue and lung cancer cell lines. In most patients (about 65%), the relative ratio of PAX6 mRNA in primary NSCLC versus adjacent tissues exceeded 100.. Our data implicated that PAX6 accelerates cell cycle progression by activating MAPK signal pathway. PAX6 mRNA levels were significantly elevated in primary lung cancer tissues compared to their matched adjacent tissues.

Topics: Carcinoma, Non-Small-Cell Lung; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Eye Proteins; Female; Gene Expression; Gene Knockdown Techniques; Homeodomain Proteins; Humans; Lung Neoplasms; Male; MAP Kinase Signaling System; Middle Aged; Paired Box Transcription Factors; PAX6 Transcription Factor; Phosphorylation; Protein Processing, Post-Translational; Repressor Proteins; Retinoblastoma Protein; RNA, Messenger; RNA, Small Interfering; S Phase

B lymphoma Mo-MLV insertion region 1 (Bmi-1) is highly expressed in a variety of cancers and has been shown to regulate cell proliferation. The INK4a/ARF tumor suppressor gene locus is one of the major targets of Bmi-1. In the present study, we chose two lung adenocarcinoma cell lines, A549 cells (without INK4a locus) and SPC-A1 cells (with INK4a locus), to investigate if the small hairpin RNA-mediated knockdown of Bmi-1 could inhibit the proliferation of lung adenocarcinoma cells, and to delineate the possible mechanism underlying Bmi-1 modulation of cell proliferation. We also investigated the potential pathway underlying Bmi-1 regulation of lung adenocarcinoma cell proliferation in different genetic backgrounds. To this end, we used shRNA to knockdown Bmi-1 expression in lung adenocarcinoma cells, which led to inhibition of cell growth, colony formation in vitro, and tumorigenesis in vivo. In addition, phosphorylated Akt and cyclin D1 expression were downregulated, p21 and p27 levels were upregulated, and p16 expression remained unchanged in SPC-A1 cells. These data indicate that Bmi-1 might modulate the growth of lung adenocarcinoma cells in an INK4a-p16 independent pathway.

Topics: Adenocarcinoma; Adenocarcinoma of Lung; Animals; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p16; Cyclin-Dependent Kinase Inhibitor p27; G1 Phase Cell Cycle Checkpoints; Gene Expression Regulation, Neoplastic; Genetic Loci; Humans; Lung Neoplasms; Mice; Mice, Nude; Organ Specificity; p21-Activated Kinases; Polycomb Repressive Complex 1; Proto-Oncogene Proteins; RNA, Small Interfering; Signal Transduction

Oncogenic mutation or misregulation of small GTPases in the Ras and Rho families can promote unregulated cell cycle progression in cancer. Post-translational modification by prenylation of these GTPases allows them to signal at the cell membrane. Splice variants of SmgGDS, named SmgGDS-607 and SmgGDS-558, promote the prenylation and membrane trafficking of multiple Ras and Rho family members, which makes SmgGDS a potentially important regulator of the cell cycle. Surprisingly little is known about how SmgGDS-607 and SmgGDS-558 affect cell cycle-regulatory proteins in cancer, even though SmgGDS is overexpressed in multiple types of cancer. To examine the roles of SmgGDS splice variants in the cell cycle, we compared the effects of the RNAi-mediated depletion of SmgGDS-558 vs. SmgGDS-607 on cell cycle progression and the expression of cyclin D1, p27, and p21 in pancreatic, lung, and breast cancer cell lines. We show for the first time that SmgGDS promotes proliferation of pancreatic cancer cells, and we demonstrate that SmgGDS-558 plays a greater role than SmgGDS-607 in cell cycle progression as well as promoting cyclin D1 and suppressing p27 expression in multiple types of cancer. Silencing both splice variants of SmgGDS in the cancer cell lines produces an alternative signaling profile compared with silencing SmgGDS-558 alone. We also show that loss of both SmgGDS-607 and SmgGDS-558 simultaneously decreases tumorigenesis of NCI-H1703 non-small cell lung carcinoma (NSCLC) xenografts in mice. These findings indicate that SmgGDS promotes cell cycle progression in multiple types of cancer, making SmgGDS a valuable target for cancer therapeutics.

Topics: Animals; Breast Neoplasms; Carcinoma, Non-Small-Cell Lung; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Female; Guanine Nucleotide Exchange Factors; Heterografts; Humans; Lung Neoplasms; Mice; Mice, SCID; Pancreatic Neoplasms; Proliferating Cell Nuclear Antigen; Protein Isoforms; rho GTP-Binding Proteins

ARID1A (AT-rich interactive domain 1A) is a key member of the SWI/SNF chromatin-modeling complex, and the gene has emerged as a tumor suppressor in various human cancers. In the present study, we investigated the expression and clinical significance of ARID1A in non-small cell lung cancer (NSCLC). We found that ARID1A expression was decreased in NSCLC tissues compared with normal bronchial epithelium and was significantly correlated with nodal metastasis, tumor, node, metastasis (TNM) stage, and poor differentiation. ARID1A expression was lower in lung cancer cell lines than normal bronchial epithelial HBE cell line. We also explored the involvement of ARID1A in biological behavior of lung cancer cell lines. ARID1A depletion by small interfering RNA (siRNA) in H460 and H1299 cell lines promoted proliferation, colony formation ability, and inhibited paclitaxel-induced apoptosis. Furthermore, we identified that ARID1A regulated several cell cycle and apoptosis-related targets such as cyclin D1 and Bcl-2. In addition, the activity of Akt phosphorylation was also enhanced after ARID1A depletion. In conclusion, our data suggested that ARID1A may serve as an important tumor suppressor in NSCLC.

Topics: Adult; Aged; Apoptosis; Carcinoma, Non-Small-Cell Lung; Cell Proliferation; Cyclin D1; DNA-Binding Proteins; Down-Regulation; Female; Humans; Lung Neoplasms; Male; Middle Aged; Nuclear Proteins; Phosphorylation; Proto-Oncogene Proteins c-akt; Transcription Factors; Tumor Suppressor Proteins

The present study aims to investigate expression pattern and biological roles of TRIM31 in human non-small cell lung cancer (NSCLC). We examined TRIM31 expression in 116 NSCLC tissues and 20 corresponding normal lung tissues by immumohistochemistry. We found TRIM31 downregulation in 47 out of 116 (40.5 %) cancer samples, which correlated with tumor status (p=0.0132), advanced p-TNM stage (p=0.001), and nodal metastasis (p=0.0382). TRIM31 expression was lower in lung cancer cell lines than normal bronchial cell line HBE. Transfection of TRIM31 plasmid was performed in H157 and H1299 cells. TRIM31 overexpression inhibited cell growth rate and colony formation ability in both cell lines. In addition, expression of cell cycle regulator cyclin D1 and cyclin E were decreased after TRIM31 transfection. In conclusion, TRIM31 might serve as a tumor suppressor in non-small cell lung cancer.

Topics: Adult; Aged; Carcinoma, Non-Small-Cell Lung; Cell Proliferation; Cyclin D1; Cyclin E; Down-Regulation; Female; Humans; Lung Neoplasms; Male; Middle Aged; Neoplasm Invasiveness; Tripartite Motif Proteins; Tumor Suppressor Proteins; Ubiquitin-Protein Ligases

Cancer heterogeneity is a big hurdle in achieving complete cancer treatment, which has led to the emergence of combinational therapy. In this study, we investigated the potential use of nuclear receptor (NR) ligands for combinational therapy with other anti-cancer drugs. We first profiled all 48 NRs and 48 biological anti-cancer targets in four pairs of lung cell lines, where each pair was obtained from the same patient. Two sets of cell lines were normal and the corresponding tumor cell lines while the other two sets consisted of primary versus metastatic tumor cell lines. Analysis of the expression profile revealed 11 NRs and 15 cancer targets from the two pairs of normal versus tumor cell lines, and 9 NRs and 9 cancer targets from the primary versus metastatic tumor cell lines had distinct expression patterns in each category. Finally, the evaluation of nuclear receptor ligand T0901317 for liver X receptor (LXR) demonstrated its combined therapeutic potential with tyrosine kinase inhibitors. The combined treatment of cMET inhibitor PHA665752 or EGFR inhibitor gefitinib with T0901317 showed additive growth inhibition in both H2073 and H1993 cells. Mechanistically, the combined treatment suppressed cell cycle progression by inhibiting cyclinD1 and cyclinB expression. Taken together, this study provides insight into the potential use of NR ligands in combined therapeutics with other biological anti-cancer drugs.

Topics: Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Cell Line, Tumor; Cyclin B; Cyclin D1; ErbB Receptors; Gefitinib; Humans; Hydrocarbons, Fluorinated; Indoles; Liver X Receptors; Lung Neoplasms; Orphan Nuclear Receptors; Protein Kinase Inhibitors; Quinazolines; Receptors, Cytoplasmic and Nuclear; Sulfonamides; Sulfones

Cyclin D1, an oncogenic G1 cyclin, and YB-1, a transcription factor involved in cell growth, are both over-expressed in several human cancers. In human lung cancer, the functional association between YB-1 and cyclin D1 has never been elucidated. In this study, we show YB-1 is involved in the transcription of cyclin D1 in human lung cancer. Depletion of endogenous YB-1 by siRNA inhibited progression of G1 phase and down-regulated both the protein and mRNA levels of cyclin D1 in human lung cancer cells. Forced over-expression of YB-1 with a cyclin D1 reporter plasmid increased luciferase activity, and ChIP assay results showed YB-1 bound to the cyclin D1 promoter. Moreover, the amount of YB-1 mRNA positively correlated with cyclin D1 mRNA levels in clinical non-small-cell lung cancer (NSCLC) specimens. Immunohistochemical analysis also indicated YB-1 expression correlated with cyclin D1 expression in NSCLC specimens. In addition, most of the cases expressing both cyclin D1 and CDC6, another molecule controlled by YB-1, had co-existing YB-1 over-expression. Together, our results suggest that aberrant expression of both cyclin D1 and CDC6 by YB-1 over-expression may collaboratively participate in lung carcinogenesis.

Topics: Adenocarcinoma; Adult; Aged; Aged, 80 and over; Carcinoma, Large Cell; Carcinoma, Non-Small-Cell Lung; Carcinoma, Squamous Cell; Cell Cycle Proteins; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Female; Gene Knockdown Techniques; Humans; Lung Neoplasms; Male; Middle Aged; Nuclear Proteins; Promoter Regions, Genetic; RNA, Messenger; Y-Box-Binding Protein 1

The current treatment for advanced nonsmall cell lung cancer (NSCLC) remains unsatisfactory due to resistance to chemotherapy and ionizing radiation. The ubiquitin-proteasome system (UPS) regulates multiple cellular processes that are crucial for the proliferation and survival of all kinds of cells. Carbobenzoxyl-leucinyl-leucinyl-leucinal-H (MG132), a specific and selective reversible inhibitor of the 26S proteasome, represents a novel approach for cancer therapy. However, whether MG132 can potentiate the effect of radiation against the growth and metastasis of NSCLC is not clear. We found that MG132 inhibited the proliferation of human NSCLC cell lines (A549 and H1299) in a dose- and time-dependent manner by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Then MG132 at a nontoxic dose (100 nM) was selected for following studies. Pretreatment of A549 and H1299 cells with 100 nM MG132 before ionizing radiation (IR) potentiated the anticancer effect of IR. Moreover, pretreatment with 100 nM MG132 before IR-enhanced radiation induced cell cycle arrest by decreased CyclinD1 but increased Wee1 expression in A549 and H1299 cells. In addition, pretreatment of MG132 combined with IR significantly suppressed cell migration and invasion abilities in NSCLC cell lines, which was accompanied by decreased expression of matrix metalloproteinase (MMP)-2 and MMP-9 in NSCLC cell lines. Taken together, our results demonstrate that MG132 enhances the antigrowth and antimetastatic effects of irradiation in NSCLC cells by modulating expression of cell cycle and invasion- related genes.

Topics: Antineoplastic Agents; Carcinoma, Non-Small-Cell Lung; Cell Cycle; Cell Cycle Proteins; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin D1; Humans; Leupeptins; Lung Neoplasms; Neoplasm Metastasis; NF-kappa B; Nuclear Proteins; Proteasome Inhibitors; Protein-Tyrosine Kinases; Radiation Tolerance

The aims of this study were to investigate the influence of ubiquitin- conjugating enzyme E2C (UBE2C) on biological behavior of lung cancer cells. Using MTT, flow cytometry and invasion assays, we detected UBE2C expression and evaluated its biological properties in these cells, including effects on proliferation, the cell cycle profile and invasive capability. Compared with control cells, the UBE2C transfected cells demonstrated increased cellular proliferation (p<0.05). UBE2C transfected cells also had a lower percentage in G1 phase and a higher percentage in S phase (p<0.05). Importantly, the UBE2C transfected cells had a notable enhancement of cell numbers penetrating the basement membrane compared with the control group (p<0.05). Ectopic up- regulation UBE2C promoted the growth of lung cancer cells in vivo. Furthermore, we found UBE2C increased the expression of cyclin D1 and MMP-2. These results show UBE2C may represent a potential therapeutic target for lung cancer.

Topics: Biomarkers, Tumor; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Humans; Lung Neoplasms; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Neoplasm Invasiveness; S Phase; Ubiquitin-Conjugating Enzymes; Up-Regulation

Crebanine is an alkaloid known to exhibit anticancer, but its mechanism is not well understood. Besides, the nuclear factor-kappa B (NF-κB) transcription factor has been correlated with inflammation, carcinogenesis, tumor cell survival, invasion, and angiogenesis. In this study, we investigated the effects of crebanine on tumor necrosis factor alpha (TNF-α)-induced NF-κB activation and the expression of NF-κB-regulated gene products. We found that crebanine reduced the cell proliferation of lung, ovarian, and breast cancer cells. Crebanine also potentiated TNF-α-induced apoptosis which correlated with the suppression of the gene products linked to cell survival, B cell lymphoma-extra large, and proliferation, cyclin D1. In addition, crebanine affected TNF-α-induced activation of caspase-8, caspase-3, and poly(ADP-ribose) polymerase cleavage, indicating that the apoptotic effects of TNF-α were enhanced by crebanine. Moreover, crebanine reduced TNF-α-induced A549 cell invasion and migration. Furthermore, crebanine suppressed the TNF-α-mediated expression of proteins that involved cancer cell invasion (matrix metalloproteinase 9 urokinase-type plasminogen activator, urokinase-type plasminogen activator receptor and intercellular adhesion molecule 1) and angiogenesis (COX-2 and VEGF), all of which are known to be regulated by NF-κB. We also demonstrated that TNF-α induced NF-κB DNA-binding activity, which was inhibited by crebanine. Moreover, crebanine suppressed the TNF-α-induced degradation of inhibitor of NF-κB alpha (IκBa), which led to reduced NF-κB translocation to the nucleus. Taken together, our results demonstrated that crebanine reduced TNF-α-induced cancer cell proliferation, invasion, and survival by suppressing NF-κB activity and expression profile of its downstream genes.

Topics: Adenocarcinoma; Animals; Apoptosis; Aporphines; bcl-X Protein; Blotting, Western; Caspase 3; Caspase 8; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cell Survival; Cyclin D1; Dose-Response Relationship, Drug; Drug Synergism; Humans; I-kappa B Proteins; Lung Neoplasms; MCF-7 Cells; Mice; Neoplasm Invasiveness; NF-kappa B; NIH 3T3 Cells; Poly(ADP-ribose) Polymerases; Tumor Necrosis Factor-alpha

ARMC8 proteins are novel armadillo repeat containing proteins, which are well conserved in eukaryotes and are involved in a variety of processes such as cell migration, proliferation, tissue maintenance, signal transduction, and tumorigenesis. Armadillo repeat proteins include well-known proteins such as β-catenin and p120ctn. Our current knowledge of ARMC8, especially its role in cancer, is limited. In this study, we quantified ARMC8 expression in 112 non-small cell lung cancer (NSCLC) tissues and adjacent non-cancerous tissues, and seven lung cancer cell lines using immunohistochemistry staining and Western blotting. ARMC8 level was significantly higher in NSCLC tissues than in the adjacent normal tissues (67.9 % versus 5.4 %, p < 0.05) and was significantly associated with TNM stage (p = 0.022), lymph node metastasis (p = 0.001), and poor prognosis (p < 0.001) in NSCLC patients. Cox regression analysis demonstrated that ARMC8 was an independent prognostic factor for NSCLC. Consistent with this, ARMC8α downregulation by siRNA knockdown inhibited growth, colony formation, and invasion in A549 lung cancer cells, while ARMC8α overexpression promoted growth, colony formation, and invasion in H1299 lung cancer cells. In addition, ARMC8α knockdown downregulated canonical Wnt-signaling pathway activity and cyclin D1 and matrix metalloproteinase (MMP)-7 expression. Consistent with this, ARMC8α overexpression upregulated canonical Wnt-signaling pathway activity and cyclin D1 and MMP-7 expression. These results indicate that ARMC8α upregulates cyclin D1 and MMP7 expression by activating the canonical Wnt-signaling pathway and thereby promoting lung cancer cell proliferation and invasion. Therefore, ARMC8 might serve as a novel therapeutic target in NSCLC.

Topics: Adult; Aged; Aged, 80 and over; Armadillo Domain Proteins; Blotting, Western; Carcinoma, Non-Small-Cell Lung; Cell Proliferation; Cyclin D1; Female; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Kaplan-Meier Estimate; Lung Neoplasms; Lymphatic Metastasis; Male; Matrix Metalloproteinase 7; Middle Aged; Neoplasm Invasiveness; Outcome Assessment, Health Care; Prognosis; Proportional Hazards Models; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; Wnt Signaling Pathway

Claudin-2 is expressed in human lung adenocarcinoma tissue and cell lines, although it is absent in normal lung tissue. However, the role of claudin-2 in cell proliferation and the regulatory mechanism of intracellular distribution remain undefined. Proliferation of human adenocarcinoma A549 cells was decreased by claudin-2 knockdown together with a decrease in the percentage of S phase cells. This knockdown decreased the expression levels of ZONAB and cell cycle regulators. Claudin-2 was distributed in the nucleus in human adenocarcinoma tissues and proliferating A549 cells. The nuclear distribution of ZONAB and percentage of S phase cells were higher in cells exogenously expressing claudin-2 with a nuclear localization signal than in cells expressing claudin-2 with a nuclear export signal. Nuclear claudin-2 formed a complex with ZO-1, ZONAB, and cyclin D1. Nuclear distribution of S208A mutant, a dephosphorylated form of claudin-2, was higher than that of wild type. We suggest that nuclear distribution of claudin-2 is up-regulated by dephosphorylation and claudin-2 serves to retain ZONAB and cyclin D1 in the nucleus, resulting in the enhancement of cell proliferation in lung adenocarcinoma cells.

Topics: Adenocarcinoma; Adenocarcinoma of Lung; CCAAT-Enhancer-Binding Proteins; Cell Cycle Proteins; Cell Line, Tumor; Cell Nucleus; Cell Proliferation; Claudins; Colforsin; Cyclin D1; G1 Phase; Gene Knockdown Techniques; Heat-Shock Proteins; Humans; Lung Neoplasms; Mutant Proteins; Nuclear Export Signals; Nuclear Localization Signals; Phosphorylation; S Phase; Time Factors; Zonula Occludens-1 Protein

Prolyl hydroxylase-1 (PHD1), a member of the hypoxia inducible factor (HIF)-PHD family, plays an important role in regulating the stability of HIFs. The nuclear factor-κB (NF-κB) pathway consists of a family of transcription factors that play critical roles in inflammation, immunity, cell proliferation, differentiation, and survival. In this study, we demonstrate that PHD1 can inhibit NF-κB activity and its target genes in lung cancer cells based on both over-expression and RNA interference-mediated knockdown of PHD1 in human A549 lung cancer cells and HEK293 T cells. Of medical importance, PHD1 could induce cell cycle arrest in lung cancer cells, resulting in the suppression of cell proliferation. Xenograft tumor growth assays indicate that PHD1 plays a critical role in suppressing lung cancer growth. These findings reveal a new role of PHD1 in lung cancer and provide new treatment perspectives for cancer therapy by characterizing PHD1 as a potential target.

Topics: Amino Acids, Dicarboxylic; Animals; Cell Line, Tumor; Cell Proliferation; Cyclin D1; HEK293 Cells; Humans; Hypoxia-Inducible Factor-Proline Dioxygenases; Interleukin-1beta; Interleukin-8; Lung Neoplasms; Male; Mice; Mice, Inbred BALB C; Mice, Nude; RNA Interference; RNA, Messenger; S Phase Cell Cycle Checkpoints; Transcription Factor RelA; Tumor Necrosis Factor-alpha; Xenograft Model Antitumor Assays

It has been proven that Cyclin D1 and Cyclin E are the important positive regulators of cell cycle, they are closely related to the tumor proliferation. The aim of this study is to explore the relationship between Brucine and the proliferation in human lung cancer cell line PC-9, and the effect of it on the expression of Cyclin D1 and Cyclin E.. PC-9 cells were divided to 4 groups: the normal control group, the DMSO control group (2‰), the 150 μM Brucine group, and the 300 μM Brucine group. The proliferation rate of PC-9 cells was determined by The CellTiter-Glo Luminescent Cell Viability Assay and Colony Formation assay. The change of cell cycle was detected by Flow cytome try. Expressions of cell cycle regulators Cyclin D1, Cyclin E mRNA were determined by qRT-PCR. Protein expression of cell cycle regulators Cyclin D1, Cyclin E were determined by Western blot.. Compared with the control, Brucine remarkably inhibited the proliferation of PC-9 cells in a dose- and time-dependent manner (P<0.01); Flow cytome try showed that Brucine blocked the cell cycle of PC-9 cells at G0/G1, and the differences were statistically significant (P<0.01); qRT-PCR showed that the expression of Cyclin D1, Cyclin E mRNA were down-regulated; Western blot showed that the protein expression of Cyclin D1, Cyclin E were down-regulated.. Brucine can inhibit the proliferation of human lung cancer cell line PC-9 mainly by blocking the cell cycle at G0/G1 via down-regulating the expression of Cyclin D1, Cyclin E.. 背景与目的 已有的研究表明：Cyclin D1和Cyclin E是细胞周期中重要的正性调控因子，其高表达与肿瘤的增殖密切相关。本研究旨在探讨马钱子碱（Brucine）对人肺癌细胞株PC-9增殖的影响，及其与Cyclin D1和Cyclin E表达的影响。方法 将PC-9细胞分为4组：空白对照组、DMSO对照组（2‰）、150 μM Brucine组、300 μM Brucine组。CellTiter-Glo发光法、平板克隆形成实验观察该药对PC-9细胞增殖的影响，流式细胞仪检测细胞周期，qRT-PCR检测细胞周期相关基因Cyclin D1、Cyclin E mRNA的表达，Western blot检测细胞周期相关基因Cyclin D1、Cyclin E蛋白的表达。结果 与对照组比较，CellTiter-Glo发光法、平板克隆形成实验结果显示：Brucine可以抑制人肺癌细胞株PC-9的增殖，并呈时间-剂量依赖性（P<0.01）；流式结果显示对细胞周期的影响主要是阻滞PC-9细胞于G0/G1期；qRT-PCR结果显示Cyclin D1、Cyclin E mRNA的表达下调；Western blot结果显示Brucine使Cyclin D1、Cyclin E的表达降低。结论 Brucine能明显抑制人肺癌细胞株PC-9的增殖，机制主要与其通过下调Cyclin D1、Cyclin E表达，进而阻滞细胞周期有关。

Topics: Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cyclin D1; Cyclin E; Down-Regulation; Drugs, Chinese Herbal; Gene Expression Regulation, Neoplastic; Growth Inhibitors; Humans; Lung Neoplasms; Strychnine

The epidermal growth factor receptor (EGFR), a ubiquitously expressed receptor tyrosine kinase, is recognized as a key mediator of tumorigenesis in many human epithelial tumors. Erlotinib is tyrosine kinase inhibitor approved by FDA for use in oncology. It inhibits the intracellular phosphorylation of tyrosine kinase associated with the EGFR to restrain the development of the tumor. To investigate the antitumor effect of erlotinib at different dosing times and the underlying molecular mechanism via the PI3K/AKT pathway, we established a mouse model of Lewis lung cancer xenografts. The tumor-bearing mice were housed four or five per cage under standardized light-dark cycle conditions (light on at 7:00 AM, 500 Lux, off at 7:00 PM, 0 Lux) with food and water provided ad libitum. The mice were observed for quality of life, their body weight and tumor volume measured, and the tumor growth curves drawn. After being bled, the mice were sacrificed by cervical dislocation. The tumor masses were removed at different time points and weighed. The mRNA expression of EGFR, AKT, Cyclin D1 and CDK-4 were assayed by quantitative real-time PCR (qRT-PCR). Protein expression levels of AKT, P-AKT and Cyclin D1 were determined by Western blot analysis. The results suggest that erlotinib has a significant antitumor effect on xenografts of non-small cell lung cancer in mice, and its efficacy and toxicity is dependent on the time of day of administration. Its molecular mechanism of action might be related to the EGFR-AKT-Cyclin D1-CDK-4 pathway which plays a crucial role in the development of pathology. Therefore, our findings suggest that the time of day of administration of Erlotinib may be a clinically important variable.

Topics: Animals; Antineoplastic Agents; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Dose-Response Relationship, Drug; Erlotinib Hydrochloride; Female; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Mice; Phosphoproteins; Proto-Oncogene Proteins c-akt; Quinazolines; Time Factors; Xenograft Model Antitumor Assays

Aberrant epidermal growth factor (EGF)-dependent signaling plays a key role in the progression of human carcinomas. We found that TIP30, a tumor suppressor protein, translocated into the nucleus of human lung adenocarcinoma cells following EGF treatment, and the selective inhibitors of EGFR signaling pathways blocked this effect. Chromatin immunoprecipitation assays revealed that TIP30 negatively regulated EGF-dependent transcriptional activation of CCND1 through a HDAC1-dependent mechanism. In lung adenocarcinoma patients, the level of nuclear TIP30 was inversely correlated with that of EGFR and cyclin D1. These findings suggest that nuclear TIP30-induced downregulation of cyclin D1 transcription antagonizes EGFR signaling and suppresses tumorigenesis.

Topics: Acetyltransferases; Active Transport, Cell Nucleus; Adenocarcinoma; Animals; Cell Line, Tumor; Cell Nucleus; Cyclin D1; Epidermal Growth Factor; ErbB Receptors; Gene Expression Regulation, Neoplastic; HEK293 Cells; Humans; Lung Neoplasms; Mice; Protein Binding; Signal Transduction; Transcription Factors; Transcription, Genetic

Cyclin D1b, a splice variant of the cell cycle regulator cyclin D1, holds oncogenic functions in human cancer. However, the mechanisms underlying cyclin D1b function remain poorly understood. Here we introduced wild-type cyclin D1a or cyclin D1b variant into non-metastatic MCF-7 cells. Our results show that ectopic expression of cyclin D1b promotes invasiveness of the cancer cells in a cyclin D1a independent manner. Specifically, cyclin D1b is found to modulate the expression of αvβ3, which characterizes the metastatic phenotype, and enhance tumor cell invasive potential in cooperating with HoxD3. Notably, cyclin D1b promotes αvβ3-mediated adhesion and invasive migration, which are associated with invasive potential of breast cancer cells. Further exploration indicates that cyclin D1b makes breast cancer cells more sensitive to toll-like receptor 4 ligand released from damaged tumor cells. These findings reveal a role of cyclin D1b as a possible mediator of αvβ3 transcription to promote tumor metastasis.

Topics: Animals; Breast Neoplasms; Cell Adhesion; Cell Movement; Cyclin D1; Female; Homeodomain Proteins; Humans; Integrin alphaVbeta3; Integrin beta3; Ligands; Lung Neoplasms; Lymphatic Metastasis; MCF-7 Cells; Mice, Inbred BALB C; Mice, Nude; Neoplasm Invasiveness; Phenotype; Protein Isoforms; Time Factors; Toll-Like Receptor 4; Transcription Factors; Transfection

Human cells release nano-sized vesicles called exosomes, containing mRNA, miRNA and specific proteins. Exosomes from one cell can be taken up by another cell, which is a recently discovered cell-to-cell communication mechanism. Also, exosomes can be taken up by different types of cancer cells, but the potential functional effects of mast cell exosomes on tumor cells remain unknown.. Exosomes were isolated from the human mast cell line, HMC-1, and uptake of PKH67-labelled exosomes by the lung epithelial cell line, A549, was examined using flow cytometry and fluorescence microscopy. The RNA cargo of the exosomes was analyzed with a Bioanalyzer and absence or presence of the c-KIT mRNA was determined by RT-PCR. The cell proliferation was determined in a BrdU incorporation assay, and proteins in the KIT-SCF signaling pathway were detected by Western blot. Our result demonstrates that exosomes from mast cells can be taken up by lung cancer cells. Furthermore, HMC-1 exosomes contain and transfer KIT protein, but not the c-KIT mRNA to A549 cells and subsequently activate KIT-SCF signal transduction, which increase cyclin D1 expression and accelerate the proliferation in the human lung adenocarcinoma cells.. Our results indicate that exosomes can transfer KIT as a protein to tumor cells, which can affect recipient cell signaling events through receptor-ligand interactions.

Topics: Adenocarcinoma; Adenocarcinoma of Lung; Animals; Cell Line; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Exosomes; Humans; Lung Neoplasms; Mast Cells; Mice; NIH 3T3 Cells; Phosphatidylinositol 3-Kinases; Protein Transport; Proto-Oncogene Proteins c-kit; RNA, Messenger; Signal Transduction

Promyelocytic leukemia protein (PML) is emerging as an important tumor suppressor. Its expression is lost during the progression of several types of cancer, including lung cancer. The EGF receptor (EGFR), a membrane-bound receptor tyrosine kinase, transduces intracellular signals responsible for cell proliferation, differentiation and migration. EGFR activity is frequently abnormally upregulated in lung adenocarcinoma (LAC) and thus is considered to be a driving oncogene for LAC. EGFR translocates into the nucleus and transcriptionally activates genes, such as CCND1, that promote cell growth. Recently, we demonstrated that PML interacted with nuclear EGFR (nEGFR) and suppressed the nEGFR-mediated transcriptional activation of CCND1 in lung cancer cells, thereby restraining cell growth. When we further investigated the interplay between PML and EGFR in lung cancer metastasis, we found that the matrix metalloprotease-2 gene (MMP2) was a novel nEGFR target gene and was repressed by PML. We provide evidence that nEGFR bound to the AT-rich sequence (ATRS) in the MMP2 promoter and enhanced its transcriptional activity. In addition, we demonstrated that PML repressed nEGFR-induced MMP2 transcription and reduced cell invasion. PML was recruited by nEGFR to the MMP2 promoter where it reduced histone acetylation, leading to the transcriptional repression of MMP2. Finally, we demonstrated that PML upregulation by interferon-β (IFNβ) in lung cancer cells decreased MMP2 expression and cell invasion. Together, our results suggested that IFNβ induced PML to inhibit lung cancer metastasis by repressing the nEGFR-mediated transcriptional activation of MMP2.

Topics: Adenocarcinoma; Animals; Base Sequence; Binding Sites; Cell Line, Tumor; Cell Nucleus; Cyclin D1; Epidermal Growth Factor; ErbB Receptors; HEK293 Cells; Histones; Humans; Interferon-beta; Lung Neoplasms; Male; Matrix Metalloproteinase 2; Mice; Mice, Inbred NOD; Mice, SCID; Neoplasm Metastasis; Nuclear Proteins; Promoter Regions, Genetic; Promyelocytic Leukemia Protein; RNA, Small Interfering; STAT3 Transcription Factor; Transcription Factors; Transcriptional Activation; Tumor Suppressor Proteins; Up-Regulation

Lung cancer is the most common cause of cancer-related death in the world. The main types of lung cancer are small cell lung carcinoma (SCLC) and non-small-cell lung carcinoma (NSCLC); non small cell lung carcinoma (NSCLC) includes squamous cell carcinoma (SCC), adenocarcinoma and large cell carcinoma, Non small cell lung carcinoma accounts for about 80% of the total lung cancer cases. Dihydroartemisinin (DHA) inhibits cell proliferation and induces apoptosis in several cancer cell lines. The effects of DHA on cell growth and proliferation in lung cancer cells remain to be elucidated. Here, we demonstrate that DHA inhibited cell proliferation in the A549 lung cancer cell line through suppression of the AKT/Gsk-3β/cyclin D1 signaling pathway. DHA significantly inhibited cell proliferation of A549 cells in a concentration and time dependent manner as determined by MTS assay. Flow cytometry analysis demonstrated that DHA treatment of A549 cells resulted in cell cycle arrest at the G1 phase, which correlated with apparent downregulation of both mRNA and protein levels of both PCNA and cyclin D1. These results suggest that DHA is a potential natural product for the treatment of lung cancer.

Topics: Antineoplastic Agents; Apoptosis; Artemisinins; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Flow Cytometry; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Humans; Immunohistochemistry; Lung Neoplasms; Proto-Oncogene Proteins c-akt; Real-Time Polymerase Chain Reaction; Signal Transduction

The objective of the current study was to investigate the expression pattern and clinicopathological significance of differentiated embryo-chondrocyte-expressed gene 1 (DEC1) in patients with non-small-cell lung cancer (NSCLC). In 118 archived NSCLC tissues, the positive rate of DEC1 was reduced in human lung cancer samples (36/118, 30.5 %) compared with adjacent normal lung tissues (106/118, 89.8 %), as measured by immunohistochemical staining. Loss of DEC1 was correlated with poor differentiation (p=0.005) and high p-TNM stage (p=0.002). Consistently, downregulation of DEC1 by siRNA knockdown promoted the growth and colony formation in the A549 lung cancer cell line, and overexpression of DEC1 inhibited the growth and colony formation in the BE1 lung cancer cell line. In addition, a significant negative correlation was found between DEC1 and cyclin D1 (p=0.014) in 118 cases of NSCLC. Knockdown of DEC1 resulted in the upregulation of cyclin D1, and overexpression of DEC1 led to the downregulation of cyclin D1. Together with the observation that DEC1 bound directly to the promoter region of cyclin D1 in A549 cells, these results indicate that loss of DEC1 may promote tumor progression in NSCLC through upregulation of cyclin D1, and DEC1 might serve as a novel therapeutic target of NSCLC.

Topics: Adenocarcinoma; Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Carcinoma, Non-Small-Cell Lung; Carcinoma, Squamous Cell; Case-Control Studies; Cell Differentiation; Cyclin D1; Female; Follow-Up Studies; Humans; Lung; Lung Neoplasms; Male; Middle Aged; Neoplasm Staging; Prognosis; RNA, Small Interfering; Survival Rate; Tumor Cells, Cultured; Tumor Suppressor Proteins

Renal cell carcinoma (RCC) is the most common type of kidney cancer, and represents the third most common urological malignancy. Despite the advent of targeted therapies for RCC and the improvement of the lifespan of patients, its cost-effectiveness restricted the therapeutic efficacy. In a recent report, we showed that synthetic phosphoethanolamine (Pho-s) has a broad antitumor activity on a variety of tumor cells and showed potent inhibitor effects on tumor progress in vivo.. We show that murine renal carcinoma (Renca) is more sensitive to Pho-s when compared to normal immortalized rat proximal tubule cells (IRPTC) and human umbilical vein endothelial cells (HUVEC). In vitro anti-angiogenic activity assays show that Pho-s inhibits endothelial cell proliferation, migration and tube formation. In addition, Pho-s has anti-proliferative effects on HUVEC by inducing a cell cycle arrest at the G2/M phase. It causes a decrease in cyclin D1 mRNA, VEGFR1 gene transcription and VEGFR1 receptor expression. Pho-s also induces nuclear fragmentation and affects the organization of the cytoskeleton through the disruption of actin filaments. Additionally, Pho-s induces apoptosis through the mitochondrial pathway. The putative therapeutic potential of Pho-s was validated in a renal carcinoma model, on which our remarkable in vivo results show that Pho-s potentially inhibits lung metastasis in nude mice, with a superior efficacy when compared to Sunitinib.. Taken together, our findings provide evidence that Pho-s is a compound that potently inhibits lung metastasis, suggesting that it is a promising novel candidate drug for future developments.

Topics: Angiogenesis Inhibitors; Animals; Antineoplastic Agents; Apoptosis; Carcinoma, Renal Cell; Caspase 3; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin D1; Cytoskeleton; Ethanolamines; Gene Expression Regulation, Neoplastic; Human Umbilical Vein Endothelial Cells; Humans; Indoles; Kidney Tubules, Proximal; Lung Neoplasms; Male; Mice; Mice, Inbred BALB C; Mitochondria; Neoplasm Metastasis; Neovascularization, Pathologic; Oxidation-Reduction; Pyrroles; Rats; Sunitinib; Transcription, Genetic; Vascular Endothelial Growth Factor Receptor-1; Wound Healing

The aim of the present study was to explore mechanisms by which let-7c suppresses NSCLC cell proliferation.. The expression level of let-7c was quantified by qRT-PCR. A549 and H1299 cells were transfected with let-7c mimics to restore the expression of let-7c. The effects of let-7c were then assessed by cell proliferation, colony formation and cell cycle assay. Mouse experiments were used to confirm the effect of let-7c on tumorigenicity in vivo. Luciferase reporter assays and Western blotting were performed to identify target genes for let-7c.. HOXA1 was identified as a novel target of let-7c. MTS, colony formation and flow cytometry assays demonstrated that forced expression of let-7c inhibited NSCLC cell proliferation by inducing G1 arrest in vitro, consistent with inhibitory effects induced by knockdown of HOXA1. Mouse experiments demonstrated that let-7c expression suppressed tumorigenesis. Furthermore, we found that let-7c could regulate the expression of HOXA1 downstream effectors CCND1, CDC25A and CDK2.. Collectively, these results demonstrate let-7c inhibits NSCLC cell proliferation and tumorigenesis by partial direct targeting of the HOXA1 pathway, which suggests that restoration of let-7c expression may thus offer a potential therapeutic intervention strategy for NSCLC.

Topics: 3' Untranslated Regions; Animals; Carcinoma, Non-Small-Cell Lung; cdc25 Phosphatases; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cell Transformation, Neoplastic; Cyclin D1; Cyclin-Dependent Kinase 2; Down-Regulation; Flow Cytometry; G1 Phase Cell Cycle Checkpoints; Gene Expression Regulation; HEK293 Cells; Homeodomain Proteins; Humans; Lung Neoplasms; Male; Mice; Mice, Inbred BALB C; MicroRNAs; RNA, Messenger; Transcription Factors; Transfection; Tumor Stem Cell Assay

The expression of polypyrimidine tract-binding protein (PTB) is up-regulated in many types of cancer. Here, we studied the role of PTB in the growth of non small cell lung cancer cells. Data showed that PTB overexpression inhibited the growth of H1299 cells at least by inhibiting DNA synthesis. Quantitative real-time PCR and Western blot analyses showed that PTB overexpression in H1299 cells specifically induced the expression of p19(Ink4d), an inhibitor of cyclin-dependent kinase 4. Repression of p19(Ink4d) expression partially rescued PTB-caused proliferation inhibition. PTB overexpression also inhibited the growth and induced the expression of p19(Ink4d) mRNA in A549 cells. However, Western blot analyses failed to detect the presence of p19(Ink4d) protein in A549 cells. To address how PTB induced p19(Ink4d) in H1299 cells, we showed that PTB might up-regulate the activity of p19(Ink4d) gene (CDKN2D) promoter. Besides, PTB lacking the RNA recognition motif 3 (RRM3) was less effective in growth inhibition and p19(Ink4d) induction, suggesting that RNA-binding activity of PTB plays an important role in p19(Ink4d) induction. However, immunoprecipitation of ribonuclearprotein complexes plus quantitative real-time PCR analyses showed that PTB might not bind p19(Ink4d) mRNA, suggesting that PTB overexpression might trigger the other RNA-binding protein(s) to bind p19(Ink4d) mRNA. Subsequently, RNA electrophoretic mobility-shift assays revealed a 300-base segment (designated as B2) within the 3'UTR of p19(Ink4d) mRNA, with which the cytoplasmic lysates of PTB-overexpressing cells formed more prominent complexes than did control cell lysates. Insertion of B2 into a reporter construct increased the expression of the chimeric luciferase transcripts in transfected PTB-overexpressing cells but not in control cells; conversely, overexpression of B2-containing reporter construct in PTB-overexpressing cells abolished the induction of p19(Ink4d) mRNA. In sum, we have shown that PTB plays as a negative regulator in H1299 cell proliferation at least by inducing p19(Ink4d) expression at transcriptional and post-transcriptional levels.

Topics: 3' Untranslated Regions; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p19; Gene Expression; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Humans; Lung Neoplasms; Polypyrimidine Tract-Binding Protein; Promoter Regions, Genetic; Protein Binding; Transcriptional Activation

The lysine acetyltransferases play crucial but complex roles in cancer development. GCN5 is a lysine acetyltransferase that generally regulates gene expression, but its role in cancer development remains largely unknown. In this study, we report that GCN5 is highly expressed in non-small cell lung cancer tissues and that its expression correlates with tumor size. We found that the expression of GCN5 promotes cell growth and the G1/S phase transition in multiple lung cancer cell lines. Further study revealed that GCN5 regulates the expression of E2F1, cyclin D1, and cyclin E1. Our reporter assays indicated that the expression of GCN5 enhances the activities of the E2F1, cyclin D1, and cyclin E1 promoters. ChIP experiments suggested that GCN5 binds directly to these promoters and increases the extent of histone acetylation within these regions. Mechanistic studies suggested that GCN5 interacts with E2F1 and is recruited by E2F1 to the E2F1, cyclin D1, and cyclin E1 promoters. The function of GCN5 in lung cancer cells is abrogated by the knockdown of E2F1. Finally, we confirmed that GCN5 regulates the expression of E2F1, cyclin D1, and cyclin E1 and potentiates lung cancer cell growth in a mouse tumor model. Taken together, our results demonstrate that GCN5 specifically potentiates lung cancer growth by directly promoting the expression of E2F1, cyclin D1, and cyclin E1 in an E2F1-dependent manner. Our study identifies a specific and novel function of GCN5 in lung cancer development and suggests that the GCN5-E2F1 interaction represents a potential target for lung cancer treatment.

Topics: Adult; Aged; Aged, 80 and over; Animals; Carcinoma, Non-Small-Cell Lung; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin E; E2F1 Transcription Factor; Female; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Lysine; Male; Mice; Mice, Nude; Middle Aged; Neoplasm Transplantation; Oligonucleotide Array Sequence Analysis; Oncogene Proteins; p300-CBP Transcription Factors

To investigate the antitumor activity and action mechanism of fengycin using the human lung cancer cell line 95D. The antitumor activity of fengycin was tested in vitro and in vivo. Reactive oxygen species production, Ca(2+) uptake, and mitochondrial membrane potential loss induced by fengycin in 95D cells were measured by flow cytometry and a laser confocal microscope. Lactate dehydrogenase release and caspase activity in fengycin-treated 95D cells were assayed using cytotoxicity detection kits. Apoptosis triggered by fengycin was identified by 4,6-diamidino-2-phenylindole (DAPI) staining and flow cytometry. The effects of fengycin on cell-cycle and apoptosis-related proteins were evaluated by quantitative reverse-transcription PCR and western blot. Treatment with fengycin not only significantly decreased cell proliferation in various cancer cell lines including 95D but inhibited the growth of xenografted 95D cells in nude mice. Fengycin also induced reactive oxygen species production and Ca(2+) uptake, as well as lactate dehydrogenase release and mitochondrial membrane potential loss. Further experiments showed that fengycin could trigger apoptosis in 95D cells and cause cell-cycle arrest at the G0/G1 stage by downregulating cyclin D1 and cyclin-dependent kinase 4 (CDK4). While investigating caspase activity and the expression of apoptosis-related proteins, fengycin was found to induce apoptosis in 95D cells through the mitochondrial pathway, evidenced by increased caspase activity, Bax expression, and cytochrome c release into the cytoplasm, as well as decreased Bcl-2 levels. Fengycin can inhibit the growth of the cancer cell line 95D by regulating the cell cycle and promoting apoptosis, suggesting that it may have potential as an anticancer treatment.

Topics: Antineoplastic Agents; Apoptosis; Calcium; Caspases; Cell Cycle; Cell Line, Tumor; Cyclin D1; Cyclin-Dependent Kinase 4; Flow Cytometry; Gene Expression Regulation, Neoplastic; Genes, Neoplasm; Humans; L-Lactate Dehydrogenase; Lipopeptides; Lung Neoplasms; Microscopy, Confocal; Mitochondrial Membranes; Reactive Oxygen Species

Epidermal growth factor receptor (EGFR) is a membrane-bound receptor tyrosine kinase, which can transduce intracellular signals responsible for cell proliferation. It is frequently overexpressed and/or constitutively activated in non-small cell lung cancer and thus is considered as a major cause of this disease. Recently, EGFR has been found in the nucleus where the nuclear EGFR (nEGFR) can function as a transcription factor activating the transcription of genes such as cyclin D1 gene (CCND1), which is essential for cell proliferation. Nevertheless, how nEGFR's transcriptional activity is regulated remains unclear. Promyelocytic leukemia protein (PML) is a tumor suppressor, which is lost in various cancers including lung cancer. However, the role of PML in the suppression of lung cancer growth is still unclear. When we investigated the role of PML in the regulation of lung cancer cell growth, we found that PML isoform IV (PMLIV) preferentially represses the growth of lung cancer cells bearing constitutively active EGFR. Besides, when growing in the EGFR activating conditions, the growth of EGFR wild-type bearing A549 cells has been repressed by PMLIV overexpression. We also found that PMLIV can interact physically with nEGFR and represses the transcription of nEGFR target genes. We showed that PMLIV is recruited by nEGFR to the target promoters and reduces the promoter histone acetylation level via HDAC1. Together, our results suggest that PMLIV interacts with nEGFR upon EGFR activation and represses the transcription of nEGFR target genes such as CCND1 and thus leading to inhibition of the lung cancer cell growth.

Topics: Acetylation; Animals; Cell Growth Processes; Cell Line; Cell Line, Tumor; Cyclin D1; ErbB Receptors; Female; HEK293 Cells; Histone Deacetylase 1; Histones; Humans; Lung Neoplasms; Mice; Mice, Inbred BALB C; Mice, Nude; Nuclear Proteins; Promoter Regions, Genetic; Promyelocytic Leukemia Protein; Protein Isoforms; Transcription Factors; Transcription, Genetic; Tumor Suppressor Proteins

Lung cancer is the leading cause of cancer-related mortality. Therapies against non-small cell lung cancer (NSCLC) are particularly needed, as this type of cancer is relatively insensitive to chemotherapy and radiation therapy. We recently identified GGTI compounds that are designed to block geranylgeranylation and membrane association of signaling proteins including the Rho family G-proteins. One of the GGTIs is P61A6 which inhibits proliferation of human cancer cells, causes cell cycle effects with G1 accumulation and exhibits tumor-suppressing effects with human pancreatic cancer xenografts. In this paper, we investigated effects of P61A6 on non-small cell lung cancer (NSCLC) cells in vitro and in vivo.. Three non-small cell lung cancer cell lines were used to test the ability of P61A6 to inhibit cell proliferation. Further characterization involved analyses of geranylgeranylation, membrane association and activation of RhoA, and anchorage-dependent and -independent growth, as well as cell cycle effects and examination of cell cycle regulators. We also generated stable cells expressing RhoA-F, which bypasses the geranylgeranylation requirement of wild type RhoA, and examined whether the proliferation inhibition by P61A6 is suppressed in these cells. Tumor xenografts of NSCLC cells growing in nude mice were also used to test P61A6's tumor-suppressing ability.. P61A6 was shown to inhibit proliferation of NSCLC lines H358, H23 and H1507. Detailed analysis of P61A6 effects on H358 cells showed that P61A6 inhibited geranylgeranylation, membrane association of RhoA and caused G1 accumulation associated with decreased cyclin D1/2. The effects of P61A6 to inhibit proliferation could mainly be ascribed to RhoA, as expression of the RhoA-F geranylgeranylation bypass mutant rendered the cells resistant to inhibition by P61A6. We also found that P61A6 treatment of H358 tumor xenografts growing in nude mice reduced their growth as well as the membrane association of RhoA in the tumors.. Thus, P61A6 inhibits proliferation of NSCLC cells and causes G1 accumulation associated with decreased cyclin D1/2. The result with the RhoA-F mutant suggests that the effect of P61A6 to inhibit proliferation is mainly through the inhibition of RhoA. P61A6 also shows efficacy to inhibit growth of xenograft tumor.

Topics: Alkyl and Aryl Transferases; Animals; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin D2; Enzyme Inhibitors; Female; G1 Phase Cell Cycle Checkpoints; Humans; Lung Neoplasms; Mice; Mice, Inbred BALB C; Mice, Nude; Phenylalanine; Prenylation; rhoA GTP-Binding Protein; Sulfonamides

Numblike (Numbl), a conserved homolog of Drosophila Numb, has been proved to be implicated in early development of the nervous system. A recent study also showed that Numbl played an important role in tumorigenesis and invasion by suppressing NF-κB activation. However, the biological role of Numbl remains unknown in lung cancer up to now. To address the expression of Numbl in the lung cancer cell, four lung cancer cell lines (metastatic cell lines NCI-H292, 95-D, and non-metastatic cell lines A549, HCC827) and non-cancerous human bronchial epithelial cells were used to detect the protein expression of Numbl by western blotting. The results in this study indicated that the expression of Numbl was downregulated in human lung cancer cell lines, especially in metastatic cell lines. To investigate the role of Numbl in lung cancer cell proliferation, apoptosis, and invasion, we generated human lung cancer 95-D cell lines in which Numbl was either overexpressed or depleted. Subsequently, the effects of Numbl on the cell viability, cycle, apoptosis, and invasion properties in 95-D cells were determined with MTT [3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide] assay, flow cytometry analysis, and Transwell invasion assays. The results indicated that Numbl could decrease cell viability, suppress cell proliferation and invasion, and promote cell apoptosis. In addition, we investigated the effects of Numbl on the expression of the following proteins: TRAF6 (tumor necrosis factor receptor-associated factor 6), p-p65 (phosphor-NF-κB), cyclin D1, caspase-3, and matrix metalloproteinase 9 (MMP9). Results showed that Numbl could decrease the expression of TRAF6, p-p65, cyclin D1, and MMP9 and increase the expression of caspase-3. All these results suggested that Numbl might be involved in the inhibition of growth, proliferation, and invasion of 95-D cells, as well as the potentiation of apoptosis of 95-D cells by abrogating TRAF6-induced activation of NF-κB.

Topics: Apoptosis; Caspase 3; Cell Cycle; Cell Movement; Cell Proliferation; Cell Survival; Cyclin D1; Down-Regulation; Gene Expression; Humans; Intracellular Signaling Peptides and Proteins; Lung Neoplasms; Matrix Metalloproteinase 9; Neoplasm Invasiveness; TNF Receptor-Associated Factor 6; Transcription Factor RelA

Usnic acid (UA) is a secondary metabolite abundantly found in lichens. Some studies have shown the anticancer potential of UA; however, its efficacy and associated mechanisms are yet to be fully explored. Herein, we assessed the anticancer potency and associated molecular alterations by UA in human lung carcinoma A549 cells. UA treatment (25-100 μM) for 24 and 48 h decreased total cell number by 39-67% (P < 0.01) and 68-89% (P < 0.001), respectively, and enhanced cell death by up to twofold and eightfold (P < 0.001), respectively. UA (1-10 μM) also significantly (P < 0.001) suppressed colony formation of A549 cells. The cell growth inhibition was associated with cell cycle arrest at G0/ G1 phase. UA decreased the expression of cyclin-dependent kinase (CDK)4, CDK6, and cyclin D1 and increased the expression of CDK inhibitor (CDKI) p21/cip1 protein. While examining the cell death associated molecular changes, we observed that UA induces mitochondrial membrane depolarization and led to more than twofold increase (P < 0.01) in apoptotic cells. The apoptotic effect of UA was accompanied by enhanced poly(ADP-ribose) polymerase cleavage. This study shows that UA inhibits cell growth involving G0/G1 phase cell cycle arrest and induces cell death via mitochondrial membrane depolarization and induction of apoptosis in human lung carcinoma cells.

Topics: Antineoplastic Agents; Apoptosis; Benzofurans; Cell Cycle Checkpoints; Cell Line, Tumor; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase 6; Cyclin-Dependent Kinase Inhibitor p21; G1 Phase; Gene Expression Regulation; Humans; Lichens; Lung Neoplasms; Poly(ADP-ribose) Polymerases

Histone deacetylase inhibitor (HDACi; vorinostat) responses were studied in murine and human lung cancer cell lines and genetically engineered mouse lung cancer models. Findings were compared with a window of opportunity trial in aerodigestive tract cancers. In human (HOP62, H522, and H23) and murine transgenic (ED-1, ED-2, LKR-13, and 393P, driven, respectively, by cyclin E, degradation-resistant cyclin E, KRAS, or KRAS/p53) lung cancer cell lines, vorinostat reduced growth, cyclin D1, and cyclin E levels, but induced p27, histone acetylation, and apoptosis. Other biomarkers also changed. Findings from transgenic murine lung cancer models were integrated with those from a window of opportunity trial that measured vorinostat pharmacodynamic responses in pre- versus posttreatment tumor biopsies. Vorinostat repressed cyclin D1 and cyclin E expression in murine transgenic lung cancers and significantly reduced lung cancers in syngeneic mice. Vorinostat also reduced cyclin D1 and cyclin E expression, but increased p27 levels in post- versus pretreatment human lung cancer biopsies. Notably, necrotic and inflammatory responses appeared in posttreatment biopsies. These depended on intratumoral HDACi levels. Therefore, HDACi treatments of murine genetically engineered lung cancer models exert similar responses (growth inhibition and changes in gene expression) as observed in lung cancer cell lines. Moreover, enhanced pharmacodynamic responses occurred in the window of opportunity trial, providing additional markers of response that can be evaluated in subsequent HDACi trials. Thus, combining murine and human HDACi trials is a strategy to translate preclinical HDACi treatment outcomes into the clinic. This study uncovered clinically tractable mechanisms to engage in future HDACi trials.

Topics: Aged; Animals; Antineoplastic Agents; Apoptosis; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin E; Disease Models, Animal; Dose-Response Relationship, Drug; Female; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Lung Neoplasms; Male; Mice; Mice, Transgenic; Middle Aged; Neoplasm Staging; Oncogene Proteins; Vorinostat

(-)-Epigallocatechin gallate (EGCG) and curcumin are two naturally derived agents that have been widely investigated worldwide. They exhibit their anti-tumor effects in many types of cancers. In the current study, the effect of the combination of the two agents on non-small cell lung cancer (NSCLC) cells was investigated. The results revealed that at low concentrations, the combination of the EGCG and curcumin strongly enhanced cell cycle arrest. Flow cytometry analysis showed that the cells were arrested at G1 and S/G2 phases. Two main cell cycle related proteins cyclin D1 and cyclin B1 were significantly inhibited at the present of EGCG and curcumin. EdU (5-ethynyl-2'-deoxyuridine) fluorescence staining showed that the DNA replication was significantly blocked. A clonal growth assay also confirmed a marked repression of cell growth. In a lung cancer xenograft node mice model, combination of EGCG and curcumin exhibited protective effect against weight loss due to tumor burden. Tumor growth was strongly repressed by the combination of the two agents, without causing any serious side-effect. Overall, these results strongly suggest that EGCG in combination with curcumin could be a candidate for chemoprevention agent of NSCLC.

Topics: Animals; Anticarcinogenic Agents; Antineoplastic Combined Chemotherapy Protocols; Carcinoma, Non-Small-Cell Lung; Catechin; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Clone Cells; Curcumin; Cyclin B1; Cyclin D1; DNA Replication; Female; Lung Neoplasms; Mice, Inbred BALB C; Mice, Nude

Lung cancer is the most common cancer worldwide. Every year, as many people die of lung cancer as of breast, colon and rectum cancers combined. Because most patients are being diagnosed in advanced, not resectable stages and therefore have a poor prognosis, there is an urgent need for alternative therapies. Since it has been demonstrated that a high number of tumor- and stromal-infiltrating cytotoxic T cells (CTLs) is associated with an increased disease-specific survival in lung cancer patients, it can be assumed that immunotherapy, e.g. peptide vaccines that are able to induce a CTL response against the tumor, might be a promising approach.. We analyzed surgically resected lung cancer tissues with respect to HLA class I- and II-presented peptides and gene expression profiles, aiming at the identification of (novel) tumor antigens. In addition, we tested the ability of HLA ligands derived from such antigens to generate a CTL response in healthy donors.. Among 170 HLA ligands characterized, we were able to identify several potential targets for specific CTL recognition and to generate CD8+ T cells which were specific for peptides derived from cyclin D1 or protein-kinase, DNA-activated, catalytic polypeptide and lysed tumor cells loaded with peptide.. This is the first molecular analysis of HLA class I and II ligands ex vivo from human lung cancer tissues which reveals known and novel tumor antigens able to elicit a CTL response.

Topics: Amino Acid Sequence; Antigen Presentation; CD8-Positive T-Lymphocytes; Cyclin D1; Dendritic Cells; DNA-Activated Protein Kinase; Epitopes, T-Lymphocyte; Gene Expression; HLA Antigens; Humans; Immunohistochemistry; Immunotherapy; Ligands; Lung Neoplasms; Molecular Sequence Data; Nuclear Proteins; Peptides; T-Lymphocytes, Cytotoxic

This study investigates the expression of micro-ribonucleic acid-21 (miRNA-21) and B cell translocation gene 2 (BTG2) in lung cancer cells. We examined the impact of miRNA-21 on biological characteristics of lung cancer cells, such as growth, proliferation, apoptosis, and invasion. The expression of miRNA-21 and BTG2 protein in lung cancer cell lines (A549, HCC827, NCI-H292, and 95-D) was examined using quantitative reverse transcription-polymerase chain reaction and Western blot analysis, respectively. Subsequently, the regulatory role of miRNA-21 on BTG2 was explored by inhibiting miRNA-21 expression in 95-D cells using miRNA-21-antisense oligonucleotides (miRNA-21 ASO). The impact of miRNA-21 on the biological characteristics of 95-D cells was further studied using methylthiazol tetrazolium assays, flow cytometry, and Transwell invasion chamber assays. The impact of miRNA-21 on the expression of cyclin D1, caspase-3, and matrix metalloprotease-9 (MMP9) was also studied. miRNA-21 expression was significantly higher in lung cancer cell lines (A549, HCC827, NCI-H282, and 95-D) than that in normal human bronchial epithelial cells (HBE; p < 0.05). The pattern of BTG2 protein expression was exactly the opposite of miRNA-21 expression in lung cancer cells. BTG2 was highly expressed in HBE cells and was expressed at very low levels in lung cancer cell lines (A549, HCC827, NCI-H292, and 95-D). High miRNA-21 expression may inhibit BTG2 protein expression, whereas the inhibition of miRNA-21 expression may promote BTG2 protein expression in 95-D cells. Cell viability and invasion of 95-D cells were significantly lower in the miRNA-21 ASO-transfected group than that in the control ASO-transfected group and untransfected group (p < 0.05). The number of apoptotic cells was significantly higher in the miRNA-21 ASO-transfected group than that in the control ASO-transfected and untransfected groups (p < 0.05). The expression level of cyclin D1 and MMP9 in 95-D cells was significantly lower in the miRNA-21 ASO-transfected group than in the control ASO-transfected and untransfected groups (p < 0.05). Meanwhile, caspase-3 expression was significantly higher in the miRNA-21 ASO-transfected group than that in the control ASO-transfected and untransfected groups (p < 0.05). miRNA-21 overexpression may inhibit the BTG2 gene in lung cancer cells. miRNA-21 may promote cell proliferation and invasion and inhibit cell apoptosis in 95-D cells.

Topics: Apoptosis; Blotting, Western; Caspase 3; Cell Line; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Gene Expression Regulation, Neoplastic; Humans; Immediate-Early Proteins; Lung Neoplasms; Matrix Metalloproteinase 9; MicroRNAs; Oligonucleotides, Antisense; Reverse Transcriptase Polymerase Chain Reaction; Tumor Suppressor Proteins

The roles of many genes in the pathophysiology of lung cancer have been investigated in different studies. Cyclin D1 (CCND1) gene plays a significant role in the transition from G1 to S phase of the cell cycle and in the phosphorylation of retinoblastoma tumor suppressor protein. In this study, we aimed to identify the relationship between CCND1 A870G gene polymorphism with lung cancer. CCND1 A870G genotypes were determined in 75 patients with lung cancer and in 65 control subjects. DNA was isolated from blood samples and then CCND1 A870G gene polymorphism was identified using PCR and RFLP assay. The distribution of CCND1 A870G polymorphism did not show any significant differences in all lung cancer patients and controls. There was no correlation between CCND1 A870G polymorphism and histopathological findings. However, the AA + AG genotype was significantly higher in metastatic patients, when compared with non-metastatic patients. Thus, the results show that CCND1 gene polymorphism may be a predictor for detecting patients with poor survival who having metastatic disease.

Topics: Cyclin D1; Female; Genetic Predisposition to Disease; Humans; Lung Neoplasms; Male; Middle Aged; Polymorphism, Single Nucleotide; Prevalence; Risk Factors; Turkey

The Wilms' tumor suppressor gene (WT1) has been identified as an oncogene in many malignant diseases such as leukaemia, breast cancer, mesothelioma and lung cancer. However, the role of WT1 in non-small-cell lung cancer (NSCLC) carcinogenesis remains unclear. In this study, we compared WT1 mRNA levels in NSCLC tissues with paired corresponding adjacent tissues and identified significantly higher expression in NSCLC specimens. Cell proliferation of three NSCLC cell lines positively correlated with WT1 expression; moreover, these associations were identified in both cell lines and a xenograft mouse model. Furthermore, we demonstrated that up-regulation of Cyclin D1 and the phosphorylated retinoblastoma protein (p-pRb) was mechanistically related to WT1 accelerating cells to S-phase. In conclusion, our findings demonstrated that WT1 is an oncogene and promotes NSCLC cell proliferation by up-regulating Cyclin D1 and p-pRb expression.

Topics: Animals; Carcinoma, Non-Small-Cell Lung; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cell Transformation, Neoplastic; Cyclin D1; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Mice; Phosphoproteins; Retinoblastoma Protein; RNA, Messenger; STAT3 Transcription Factor; Transcriptional Activation; Up-Regulation; WT1 Proteins

To investigate the effect of the selective PI3K inhibitor and MEK inhibitor on KRAS and PTEN co-mutated non-small cell lung cancer cell line NCI-H157 and the relevant mechanisms.. NCI-H157 was cultured routinely and treated with different concentrations of the two inhibitors. Cell proliferation was detected by MTT cell cycle assay. Based on the MTT results the cells were divided into four groups: the control group, PI3K inhibitor group (GDC-0941, 0.5 and 5.0 µmol/L), combination group I (0.5 µmol/L AZD6244 + 0.5 µmol/L GDC-0941) and combination group II (5.0 µmol/L AZD6244 + 5.0 µmol/L GDC-0941). Colony formation assay was performed to detect colony formation efficiency. The cell cycle and apoptosis were analyzed by flow cytometry. The expression of protein related to apoptosis was tested with Western blot.. Cell growth was inhibited by the two inhibitors. Combination groups led to stronger cell proliferation inhibition: combination group Ishowed synergistic effect of their actions and combination group II showed an additive effect; in both groups, there were decreased colony number [(77.2 ± 1.54)/well vs (61.50 ± 2.12)/well, P < 0.01] and [(51.00 ± 4.00)/ well vs (22.50 ± 3.53)/well, P < 0.01]; and enhanced apoptotic ratios [(18.30 ± 0.82)% vs (21.32 ± 0.56)%, P < 0.01] and [(27.14 ± 1.58)% vs (42.45 ± 4.42)%, P < 0.01]. In addition, compared to the PI3K inhibitor alone group, the NCI-H157 cells in the combination groups showed increased G0/G1 phase and decreased S phase (P < 0.01). Western blotting showed that the combination groups demonstrated significantly decreased expression of cyclin D1 and cyclin B1, increased p21 and cleaved PARP and decreased bcl-2/bax ratio, compared to the PI3K inhibitor only group.. The combined inhibition of PI3K (AZD6244) and MEK (GDC-0941) has synergistic effects on the proliferation of NCI-H157 cells, but such effects appear to be in a dose-dependent manner.

Topics: Apoptosis; bcl-2-Associated X Protein; Benzimidazoles; Carcinoma, Non-Small-Cell Lung; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cyclin B1; Cyclin D1; Dose-Response Relationship, Drug; Drug Synergism; Humans; Indazoles; Lung Neoplasms; Mitogen-Activated Protein Kinase Kinases; Mutation; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Poly(ADP-ribose) Polymerases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Proto-Oncogene Proteins p21(ras); PTEN Phosphohydrolase; ras Proteins; Signal Transduction; Sulfonamides

Orai channels have been associated with cell proliferation, survival and metastasis in several cancers. The present study investigates the expression and the role of Orai3 in cell proliferation in non-small cell lung cancer (NSCLC). We show that Orai3 is over-expressed in cancer tissues as compared to the non-tumoral ones. Furthermore, Orai3 staining is stronger in high grade tumors. Pharmacological inhibition or knockdown of Orai3 significantly reduced store operated calcium entry (SOCE), inhibited cell proliferation and arrested cells of two NSCLC cell lines in G0/G1 phase. These effects were concomitant with a down-regulation of cyclin D1, cyclin E, CDK4 and CDK2 expression. Moreover, Orai3 silencing decreased Akt phosphorylation levels. In conclusion, Orai3 constitutes a native SOCE pathway in NSCLC that controls cell proliferation and cell cycle progression likely via Akt pathway.

Topics: Adenocarcinoma; Adenocarcinoma of Lung; Aged; Calcium; Calcium Channels; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin E; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase 4; Female; G1 Phase Cell Cycle Checkpoints; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Male; Middle Aged; Neoplasm Staging; Phosphorylation; Proto-Oncogene Proteins c-akt; Resting Phase, Cell Cycle; RNA, Small Interfering; Signal Transduction

miR-206, a member of the so-called myomiR family, is largely acknowledged as a specific, positive regulator of skeletal muscle differentiation. A growing body of evidence also suggests a tumor suppressor function for miR-206, as it is frequently downregulated in various types of cancers. In this study, we show that miR-206 directly targets cyclin D1 and contributes to the regulation of CCND1 gene expression in both myogenic and non-muscle, transformed cells. We demonstrate that miR-206, either exogenous or endogenous, reduces cyclin D1 levels and proliferation rate in C2C12 cells without promoting differentiation, and that miR-206 knockdown in terminally differentiated C2C12 cells leads to cyclin D1 accumulation in myotubes, indicating that miR-206 might be involved in the maintenance of the post-mitotic state. Targeting of cyclin D1 might also account, at least in part, for the tumor-suppressor activity suggested for miR-206 in previous studies. Accordingly, the analysis of neoplastic and matched normal lung tissues reveals that miR-206 downregulation in lung tumors correlates, in most cases, with higher cyclin D1 levels. Moreover, gain-of-function experiments with cancer-derived cell lines and with in vitro transformed cells indicate that miR-206-mediated cyclin D1 repression is directly coupled to growth inhibition. Altogether, our data highlight a novel activity for miR-206 in skeletal muscle differentiation and identify cyclin D1 as a major target that further strengthens the tumor suppressor function proposed for miR-206.

Topics: Adenocarcinoma; Cell Differentiation; Cell Line, Transformed; Cell Line, Tumor; Cell Transformation, Neoplastic; Cyclin D1; Humans; Lung Neoplasms; MicroRNAs; Muscle, Skeletal; Organ Specificity

Epidermal growth factor receptor (EGFR) and vascular endothelial growth factor receptor 2 (VEGFR2) have emerged as two effective clinical targets for non-small-cell lung cancer (NSCLC). In the present study, we found that delphinidin, an anthocyanidin, present in pigmented fruits and vegetables, is a potent inhibitor of both EGFR and VEGFR2 in NSCLC cells that overexpress EGFR/VEGFR2. Using these cells, we next determined the effects of delphinidin on cell growth and apoptosis in vitro and on tumor growth and angiogenesis in vivo. Delphinidin (5-60 µM) treatment of NSCLC cells inhibited the activation of PI3K, and phosphorylation of AKT and MAPKs. Additionally, treatment of NSCLC cells with delphinidin resulted in inhibition of cell growth without having significant toxic effects on normal human bronchial epithelial cells. Specifically, treatment of NCI-H441 and SK-MES-1 cells with delphindin (5-60 µM) resulted in (i) cleavage of PARP protein, (ii) activation of caspase-3 and -9, (iii) downregulation of anti-apoptotic proteins (Bcl2, Bcl-xL and Mcl-1), (iv) upregulation of pro-apoptotic proteins (Bax and Bak), and (v) decreased expression of PCNA and cyclin D1. Furthermore, in athymic nude mice subcutaneously implanted with human NSCLC cells, delphinidin treatment caused a (i) significant inhibition of tumor growth, (ii) decrease in the expression of markers for cell proliferation (Ki67 and PCNA) and angiogenesis (CD31 and VEGF), and (iii) induction of apoptosis, when compared with control mice. Based on these observations, we suggest that delphinidin, alone or as an adjuvant to current therapies, could be used for the management of NSCLC, especially those that overexpress EGFR and VEGFR2.

Topics: Animals; Anthocyanins; Antineoplastic Agents, Phytogenic; Apoptosis; Carcinoma, Non-Small-Cell Lung; Caspases; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Disease Models, Animal; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Lung Neoplasms; Mice; Mitogen-Activated Protein Kinases; Neovascularization, Pathologic; Phosphatidylinositol 3-Kinases; Phosphorylation; Poly(ADP-ribose) Polymerases; Proliferating Cell Nuclear Antigen; Proteolysis; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-bcl-2; Signal Transduction; Tumor Burden; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factor Receptor-2; Xenograft Model Antitumor Assays

Targeted inactivation of Runx3 in mouse lung induced mucinous and nonmucinous adenomas and markedly shortened latency of adenocarcinoma formation induced by oncogenic K-Ras. RUNX3 was frequently inactivated in K-RAS mutated human lung adenocarcinomas. A functional genetic screen of a fly mutant library and molecular analysis in cultured cell lines revealed that Runx3 forms a complex with BRD2 in a K-Ras-dependent manner in the early phase of the cell cycle; this complex induces expression of p14(ARF)/p19(Arf) and p21(WAF/CIP). When K-Ras was constitutively activated, the Runx3-BRD2 complex was stably maintained and expression of both p14(ARF) and p21(WAF/CIP) was prolonged. These results provide a missing link between oncogenic K-Ras and the p14(ARF)-p53 pathway, and may explain how cells defend against oncogenic K-Ras.

Topics: Acetylation; Adenocarcinoma; Adenocarcinoma of Lung; ADP-Ribosylation Factors; Alveolar Epithelial Cells; Animals; Carcinogenesis; Cell Differentiation; Cell Line, Tumor; Core Binding Factor Alpha 3 Subunit; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Gene Expression; Gene Knockout Techniques; HEK293 Cells; Histone Deacetylases; Humans; Lung Neoplasms; Mice; Mice, Transgenic; Protein Processing, Post-Translational; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins p21(ras); ras Proteins; Repressor Proteins; Respiratory Mucosa; Transcription Factors

To investigate the correlation between expression of A-kinase anchoring protein 95 (AKAP95) and protein expression of cyclin E1 and cyclin D1 in lung cancer tissue.. Fifty-one cases of lung cancer were included in the study. The protein expression of AKAP95, cyclin E1, and cyclin D1 were measured by immunohistochemistry.. The protein expression of cyclin E1 in lung cancer tissues was significantly higher than that in para-cancerous tissues (positive rate: 75.56%vs 20%, P < 0.01); its expression showed no relationship with histopathological type, lymph node metastasis, and cellular differentiation (P > 0.05). The protein expression of cyclin D1 in lung cancer tissues was higher than that in para-cancerous tissues (positive rate: 69.39% vs 14.29%); its expression showed a significant relationship with histopathological type (P < 0.05). The expression of AKAP95 was correlated with the protein expression of cyclin E1 and cyclin D1 in lung cancer tissues (P < 0.01).. Cyclin E1 and cyclin D1 are highly expressed in lung cancer tissue, suggesting that they play an important role in the development and progression of lung cancer. The protein expression of cyclin E1 has no relationship with cellular differentiation, lymph node metastasis, and histopathological type of lung cancer, and the protein expression of cyclin D1 has a significant relationship with histopathological type. The expression of AKAP95 is correlated with the protein expression of cyclin E1 and cyclin D1 in lung cancer tissue.

Topics: A Kinase Anchor Proteins; Adult; Aged; Cyclin D1; Cyclin E; Humans; Lung; Lung Neoplasms; Middle Aged; Oncogene Proteins

Metastasis, a multistep process, is a major cause of mortality in cancer patients. Thus, it is hoped that inhibition of metastasis at any step, such as proliferation, migration, or invasion, using small-molecule inhibitors will reduce this mortality. Recent study suggests that the Janus kinase/signal transducer and activator of transcription 3 signal transduction pathway is a central pathway that regulates tumor progression and metastasis and can be blocked using tyrosine kinase inhibitors. In this study we used a synthetic tyrosine kinase inhibitor, AG490, to block the constitutive activation of the Janus kinase/signal transducer and activator of transcription 3 pathway in A549 lung carcinoma and A375 melanoma cell lines. Our results show that AG490 at subtoxic doses can effectively suppress tumor cell proliferation by limiting the expression of cyclin D1. Furthermore, AG490 is seen to induce apoptosis, inhibit cellular migration by disrupting actin organization, and suppress matrix metalloproteinase 2 activity. Taken together, these data demonstrate that AG490 can exert antimetastatic activity by inhibiting cellular proliferation, invasion, and migration.

Topics: Actins; Apoptosis; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin D1; Dose-Response Relationship, Drug; Enzyme Inhibitors; Humans; In Vitro Techniques; Janus Kinase 2; Lung Neoplasms; Matrix Metalloproteinase 2; Melanoma; Protein-Tyrosine Kinases; STAT3 Transcription Factor; Tyrphostins

Crk-Like (CRKL) is an adapter protein that has crucial roles in multiple biological processes, including cell proliferation, adhesion, and migration. Amplification of CRKL gene was found in non-small cell lung cancer (NSCLC). However, the expression pattern of CRKL protein and its clinical significance in human NSCLC have not been well characterized to date. In this study, expression of CRKL was evaluated in 131 NSCLC tissues by immumohistochemistry. CRKL protein was up-regulated in the lung carcinomas compared with adjacent normal lung tissue. Overexpression of CRKL was found in 58 of 131 (44.3%) NSCLC samples and correlated with poor tumor differentiation (P = 0.0042), histological type (adenocarcinoma; P = 0.001), advanced p-TNM stage (P = 0.0004), nodal metastasis (P = 0.0273), high proliferation index (P = 0.0062) and poor overall survival (P = 0.0084). Further univariate and multivariate analysis showed a significant association of CRKL overexpression and worse overall survival in lung cancer patients. In addition, overexpression of CRKL in HBE and H1299 cell lines promoted cell proliferation by facilitating cell cycle progression. Further analysis of cell cycle related molecules showed that CRKL induced cyclin D1, cyclin B1 expression, and increased Rb phosphorylation. In conclusion, this study demonstrated overexpression of CRKL correlated with poor prognosis and lung cancer proliferation by cell cycle regulation.

Topics: Adaptor Proteins, Signal Transducing; Carcinoma, Non-Small-Cell Lung; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase 6; Female; Gene Expression Regulation, Neoplastic; Humans; Lung; Lung Neoplasms; Male; Middle Aged; Nuclear Proteins; Prognosis; Up-Regulation

Genistein (GEN) is a molecule of great interest as a potent chemopreventive agent against atherosclerosis and cancer. However, the bioavailability of GEN is very low in vivo. Our previous study showed that a GEN derivative, 7-difluoromethyl-5,4'-dimethoxygenistein (dFMGEN) has a better bioavailability than GEN in vivo. In this study, we further evaluated the efficacy of dFMGEN as a candidate for cancer therapy. We demonstrated that dFMGEN treatment decreased the viability of A549 cells in a concentration- and time-dependent manner and induced cell-cycle arrest at the G(1) phase. G(1) phase arrest was correlated with a significant reduction of Cdk4 and cyclin D1 protein level. Further studies showed that cyclin-dependent kinase (Cdk)4 and cyclin D1 protein-level decrease was caused by Cdk inhibitors p15, p21, and p27 level increase, and decreased protein level directly suppressed Rb protein phosphorylation and E2F-1 expression, then cell-cycle progression was arrested. Finally, we also found that dFMGEN has a dosage effect in suppressing tumor growth in vivo, and that dFMGEN was well tolerated by animals. In summary, our results suggest that dFMGEN has therapeutic potential for the treatment of human lung cancer.

Topics: Animals; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase 4; Dose-Response Relationship, Drug; G1 Phase Cell Cycle Checkpoints; Genistein; Humans; Lung Neoplasms; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Phosphorylation; Retinoblastoma Protein; Time Factors; Xenograft Model Antitumor Assays

The purposes of this study were to investigate the effects of phosphatidylethanolamine-binding protein 4 (PEBP4) on the cell growth, proliferation, apoptosis, and invasion of non-small cell lung cancer (NSCLC) cells and to provide evidence for future treatment options for NSCLC. Western blot assays were performed to examine PEBP4 protein expression levels in NSCLC cell lines (HCC827, A549, NCI-H661, NCI-H292, and 95-D) and a normal human bronchial epithelial (HBE) cell line. A PEBP4 shRNA expression vector was constructed and transfected into HCC827 cells. Subsequently, the effects of PEBP4 on the cell viability, cell cycle distribution, apoptosis levels, and invasion properties of HCC827 cells were analyzed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays, flow cytometry analyses, and transwell invasion assays. In addition, the effects of PEBP4 on the expression of proteins including cyclin D1, p53, Bcl-2, MMP-2, and MMP-9 were investigated. PEBP4 was highly expressed in lung cancer cells (HCC827, A549, NCI-H661, NCI-H292, and 95-D), but its expression was low in HBE cells. Cell viability, cell proliferation, and invasion of HCC827 cells in the PEBP4 knockdown group were significantly lower than that in the negative control and blank control groups (p < 0.05), and there were no significant differences between the negative and blank control groups in terms of cell viability, cell proliferation, apoptosis, and invasion. In HCC827 cells, the expression levels of cyclin D1, Bcl-2, MMP-2, and MMP-9 in the PEBP4 knockdown group were significantly lower (p < 0.05), and the expression of p53 protein was significantly higher than that in the negative and blank control groups (p < 0.05). There were no significant differences between the negative and blank control groups in the expression levels of cyclin D1, p53, Bcl-2, MMP-2, and MMP-9. In conclusion, PEBP4 enhanced HCC827 cell proliferation and invasion ability and inhibited apoptosis. Decreased PEBP4 expression may play a role in the reduced invasion ability and increased apoptosis of the human NSCLC cell line HCC827.

Topics: Apoptosis; Carcinoma, Non-Small-Cell Lung; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cyclin D1; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Neoplasm Invasiveness; Phosphatidylethanolamine Binding Protein; Proto-Oncogene Proteins c-bcl-2; RNA Interference; RNA, Small Interfering; Tumor Suppressor Protein p53

Tumor necrosis factor receptor-associated factor 6 (TRAF6) is a unique adaptor protein of the tumor necrosis factor receptor-associated factor family that mediates both tumor necrosis factor receptor and interleukin-1 receptor/Toll-like receptor signaling. A recent study showed that TRAF6 played an important role in tumorigenesis and invasion through activation of nuclear factor kappa B (NF-κB). However, the biological role of TRAF6 remains unknown in lung cancer up to now. To address the expression of TRAF6 in lung cancer cells, four lung cancer cell lines (A549, HCC827, NCI-H292, and 95-D) and human bronchial epithelial cells were used to detect the expression of TRAF6 protein by western blotting. Results indicated that TRAF6 displayed an upregulation in human lung cancer cell lines. To investigate the effects of TRAF6 on the biological behavior of human lung adenocarcinoma cell, we generated human lung adenocarcinoma A549 cell line in which TRAF6 was depleted. The results showed that downregulation of TRAF6 could decrease cell viability, suppress cell proliferation and invasion, and promote cell apoptosis. At the same time, we explored the effects of TRAF6 on the expression of the following proteins: phosphor-NF-κB (p-p65), cyclin D1, caspase-3, and matrix metalloproteinase 9 (MMP9). Downregulation of TRAF6 could decrease the expression of p-p65, cyclin D1, and MMP9 and increase the expression of caspase-3. All these results suggested that TRAF6 might be involved in the potentiation of growth, proliferation, and invasion of A549 cell line, as well as the inhibition of A549 cell apoptosis by the activation of NF-κB. To make a long story short, the overexpression of TRAF6 might be related to the tumorigenesis and invasion of lung cancer.

Topics: Adenocarcinoma; Apoptosis; Caspase 3; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cell Transformation, Neoplastic; Cyclin D1; Down-Regulation; Humans; Lung Neoplasms; Matrix Metalloproteinase 9; Neoplasm Invasiveness; NF-kappa B; Signal Transduction; TNF Receptor-Associated Factor 6; Transcription Factor RelA; Up-Regulation

Deferasirox is an orally effective iron (Fe) chelator currently used for the treatment of iron-overload disease and has been implemented as an alternative to the gold standard chelator, desferrioxamine (DFO). Earlier studies demonstrated that DFO exhibits anticancer activity due to its ability to deplete cancer cells of iron. In this investigation, we examined the in vitro and in vivo activity of deferasirox against cells from human solid tumors. To date, there have been no studies to investigate the effect of deferasirox on these types of tumors in vivo. Deferasirox demonstrated similar activity at inhibiting proliferation of DMS-53 lung carcinoma and SK-N-MC neuroepithelioma cell lines compared with DFO. Furthermore, deferasirox was generally similar or slightly more effective than DFO at mobilizing cellular (59)Fe and inhibiting iron uptake from human transferrin depending on the cell type. However, deferasirox potently inhibited DMS-53 xenograft growth in nude mice when given by oral gavage, with no marked alterations in normal tissue histology. To understand the antitumor activity of deferasirox, we investigated its effect on the expression of molecules that play key roles in metastasis, cell cycle control, and apoptosis. We demonstrated that deferasirox increased expression of the metastasis suppressor protein N-myc downstream-regulated gene 1 and upregulated the cyclin-dependent kinase inhibitor p21(CIP1/WAF1) while decreasing cyclin D1 levels. Moreover, this agent increased the expression of apoptosis markers, including cleaved caspase-3 and cleaved poly(ADP-ribose) polymerase 1. Collectively, we demonstrate that deferasirox is an orally effective antitumor agent against solid tumors.

Topics: Administration, Oral; Animals; Antigens, CD; Antineoplastic Agents; Apoptosis; Benzoates; Cell Cycle; Cell Line, Tumor; Copper; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Deferasirox; Female; Humans; Iron; Iron Chelating Agents; Lung Neoplasms; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Metastasis; Neoplasm Transplantation; Neuroectodermal Tumors, Primitive, Peripheral; Protein Serine-Threonine Kinases; Receptors, Transferrin; Small Cell Lung Carcinoma; Transplantation, Heterologous; Triazoles; Zinc

The prognostic significance of cyclin A2 overexpression in non-small cell lung cancer (NSCLC) is controversial.. To understand the effect of cyclin A2 on recurrence in NSCLC, we retrospectively analyzed the expression of Bcl-2, cyclin A2, E-cadherin, Ki-67, and p53 using immunohistochemistry in 635 NSCLCs.. Overexpression of cyclin A2 was found in 466 (73%) of 635 NSCLCs, and recurrence occurred in 291 (46%) of 635 NSCLCs with a median follow-up of 5.4 years. The relationship between recurrence and cyclin A2 overexpression was not homogenous by pathologic stage (Breslow-Day test for homogeneity, P = 0.007). Overexpression of cyclin A2 was associated with poor recurrence-free survival (RFS) in 374 stage I NSCLCs (P = 0.02), and RFS was worse in patient with negative expression of Bcl-2 than those with positive expression of Bcl-2. Cox proportional hazard analysis showed that stage I NSCLC patients with overexpression of cyclin A2 and negative expression of Bcl-2 had poorer RFS (hazard ratio = 3.86, 95% confidence interval = 1.07-15.77; P = 0.03) than those with normal expression of cyclin A2 and Bcl-2, after adjusting for age, adjuvant radiotherapy, and histology. Neural network and generalized linear model including cyclin A2 and Bcl-2 showed best performance in the prediction of recurrence; error rates for neural network and generalized linear model were 15% and 12%, respectively.. Negative effect of cyclin A2 on RFS in stage I NSCLC was aggravated by negative expression of Bcl-2.

Topics: Adenocarcinoma; Biomarkers, Tumor; Cadherins; Carcinoma, Non-Small-Cell Lung; Carcinoma, Squamous Cell; Cyclin A2; Cyclin D1; Female; Follow-Up Studies; Humans; Immunoenzyme Techniques; Ki-67 Antigen; Lung Neoplasms; Male; Middle Aged; Neoplasm Recurrence, Local; Neoplasm Staging; Prognosis; Proto-Oncogene Proteins c-bcl-2; Retrospective Studies; Survival Rate; Tissue Array Analysis; Tumor Suppressor Protein p53

Identification of secreted proteins of lung cancer could provide new candidates of serum biomarkers for cancer diagnosis or targets for therapeutic intervention. In this study, we developed a novel strategy that combined functional monoclonal antibody library screening technique and mass spectrometry to identify functional secreted proteins. BALB/c mice were immunized with cancer cells isolated from fresh human lung cancer tissues. The monoclonal antibody library containing 1160 mAbs was established with the mouse spleen cells, whose serum had most anti-proliferative effect on lung cancer cells. Monoclonal antibodies were subjected to an immunoreactive and functional screen and monoclonal antibodies that reacted strongly with secreted proteins in condition medium and lung cancer tissues with high inhibotion of cell proliferation were selected. Antigens that recognized by antibodies were obtained by immunoprecipitation and then identified by mass spectrometry. Mac-2-binding protein (Mac-2BP), the antigen of 13H3 antibody, was identified using this approach. Functional studies demonstrated that the 13H3 antibody suppressed lung cancer cell lines ANIP-973 and A549 proliferation in vitro and inhibit ANIP973 xenograft tumors growth in vivo by inducing cell-cycle arrest at G1 phase, with up-regulation of p27 and down-regulation of cyclin D1. Moreover, the serum level of Mac-2BP was significantly higher in lung cancer patients than healthy controls. At a cutoff value of 6 μg/ml, Mac-2BP might be a diagnostic biomarker of lung cancer, especially for SCLC. Mac-2BP concentrations of 6 μg/ml or higher was associated with poor overall survival in univariate analysis, and was an independent predictor in the multivariate COX analysis. Together, these results firstly demonstrated that Mac-2BP can be used as a therapeutic target and potential biomarker for lung cancer. Our strategy is feasible, which may facilitate the identification of novel secreted biomarkers of lung cancer.

Topics: Aged; Animals; Antibodies, Monoclonal; Antigens, Neoplasm; Biomarkers, Tumor; Carcinoma, Squamous Cell; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Female; Gene Expression Regulation, Neoplastic; Humans; Immunoprecipitation; Lung Neoplasms; Male; Mass Spectrometry; Membrane Glycoproteins; Mice; Mice, Inbred BALB C; Middle Aged; Neoplasm Proteins; Peptide Library; Proliferating Cell Nuclear Antigen; Xenograft Model Antitumor Assays

Deeper mechanistic understanding of lung adenocarcinoma (non-small cell lung carcinoma, or NSCLC), a leading cause of cancer-related deaths overall, may lead to more effective therapeutic strategies. In analyzing NSCLC clinical specimens and cell lines, we discovered a uniform decrease in miR-186 (MIR186) expression in comparison with normal lung tissue or epithelial cell lines. miR-186 expression correlated with patient survival, with median overall survival time of 63.0 or 21.5 months in cases exhibiting high or low levels of miR-186, respectively. Enforced overexpression of miR-186 in NSCLC cells inhibited proliferation by inducing G(1)-S checkpoint arrest. Conversely, RNA interference-mediated silencing miR-186 expression promoted cell-cycle progression and accelerated the proliferation of NSCLC cells. Cyclin D1 (CCND1), cyclin-dependent kinase (CDK)2, and CDK6 were each directly targeted for inhibition by miR-186 and restoring their expression reversed miR-186-mediated inhibition of cell-cycle progression. The inverse relationship between expression of miR-186 and its targets was confirmed in NSCLC tumor xenografts and clinical specimens. Taken together, our findings established a tumor-suppressive role for miR-186 in the progression of NSCLC.

Topics: Adenocarcinoma; Adenocarcinoma of Lung; Cell Cycle; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin D2; Cyclin-Dependent Kinase 6; Down-Regulation; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; MicroRNAs; RNA, Small Interfering

We have demonstrated that aryl hydrocarbon receptor (AhR) is overexpressed in lung adenocarcinoma (AD). AhR is usually associated with heat shock protein 90 (Hsp90) in the cytoplasm. 17-Allylamino-17-demethoxygeldanamycin (17-AAG), an Hsp90 inhibitor, is currently under evaluation for its anticancer activity in clinical trials. Here we investigated whether AhR plays a role in 17-AAG-mediated anticancer activity by functioning as a downstream target or by modulating its anticancer efficacy. AhR expression in lung AD cells was modulated by siRNA interference or overexpression. Tumor growth was determined with colony formation in vitro or in vivo. Anticancer activity of 17-AAG was determined by measuring cell viability, cell cycle distribution, and expression of cell cycle regulators. Proteins and mRNA levels were examined by immunoblotting and the real-time reverse transcription-polymerase chain reaction, respectively. In this study, AhR overexpression positively modulated growth of lung AD cells, at least partially, via RelA-dependent mechanisms. Although treatment with 17-AAG reduced AhR levels and AhR-regulated gene expression in lung AD cells, AhR expression increased anticancer activity of 17-AAG. In addition, 17-AAG treatment reduced cell viability, CDK2, CDK4, cyclin E, cyclin D1, and phosphorylated Rb levels in AhR-expressing lung AD cells. NAD(P)H:quinone oxidoreductase (NQO1), which is regulated by AhR, was shown to increase anticancer activity of 17-AAG in cells. Knockdown of NQO1 expression attenuated the reduction of cell cycle regulators by 17-AAG treatment in AhR overexpressed cells. We demonstrated that AhR protein not only functions as a downstream target of 17-AAG, but also enhances anticancer activity of 17-AAG in lung AD cells.

Topics: Adenocarcinoma; Adenocarcinoma of Lung; Animals; Antineoplastic Agents; Benzoquinones; Cell Cycle; Cell Line, Tumor; Cell Survival; Cyclin D1; Cyclin E; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase 4; Humans; Lactams, Macrocyclic; Lung Neoplasms; Male; Mice; Mice, Inbred BALB C; Mice, Nude; NAD(P)H Dehydrogenase (Quinone); Receptors, Aryl Hydrocarbon; RNA, Messenger; Xenograft Model Antitumor Assays

The purposes of this study were to elucidate the effects of ARHI (aplysia ras homolog I) on several biological features of lung cancer cells, including growth, proliferation and invasion, to collect experimental evidence for the future biological treatment of human lung cancer. The eukaryotic expression vector, pcDNA3.1-ARHI, was constructed and transfected into the human lung cancer cell line SK-MES-1. The biological properties of the resulting ARHI-expressing lung cancer cell line were evaluated using methyl thiazolyl tetrazolium assay, flow cytometry, and a Transwell invasion assay. Additionally, the influence of ARHI on the gene expression levels of cyclin D1, p27(KIP1), death-associated protein kinase 1 (DAPK1), and matrix metalloproteinases1/2 (MMP-1/2) was determined. Compared to the non-transfected SK-MES-1 cells and the cells transfected with the empty pcDNA3.1 plasmid, the ARHI-transfected cells displayed significantly reduced growth rates and decreased viability (P < 0.05). The ARHI-transfected cells also displayed a significantly higher percentage of cells in G1 phase (P < 0.05) and a lower percentage of cells in S phase (P < 0.05); a higher percentage of apoptosis (P < 0.05); and finally, a notable reduction in the basement membrane-penetration rate in the Transwell invasion assay (P < 0.05). Furthermore, it was determined that ARHI is capable of inhibiting the expression of cyclin D1, MMP-1, and MMP-2; however, ARHI promotes the expression of both p27(KIP1) and DAPK1 in SK-MES-1 cells. In conclusion, overexpression of ARHI gene might be associated with the inhibition of lung cancer cell growth, proliferation and invasion, and the promotion of apoptosis.

Topics: Apoptosis; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p27; Death-Associated Protein Kinases; Gene Expression; Humans; Lung Neoplasms; Matrix Metalloproteinase 1; Matrix Metalloproteinase 2; Neoplasm Invasiveness; rho GTP-Binding Proteins

Lung cancer is the major cause of mortality worldwide. Major signalling pathways that could play significant role in lung cancer therapy include (1) Growth promoting pathways (Epidermal Growth Factor Receptor/Ras/ PhosphatidylInositol 3-Kinase) (2) Growth inhibitory pathways (p53/Rb/P14ARF, STK11) (3) Apoptotic pathways (Bcl-2/Bax/Fas/FasL). Insilico strategy was implemented to solve the mystery behind selected lung cancer pathway by applying comparative modeling and molecular docking studies.. YASARA [v 12.4.1] was utilized to predict structural models of P16-INK4 and RB1 genes using template 4ELJ-A and 1MX6-B respectively. WHAT CHECK evaluation tool demonstrated overall quality of predicted P16-INK4 and RB1 with Z-score of -0.132 and -0.007 respectively which showed a strong indication of reliable structure prediction. Protein-protein interactions were explored by utilizing STRING server, illustrated that CDK4 and E2F1 showed strong interaction with P16-INK4 and RB1 based on confidence score of 0.999 and 0.999 respectively. In order to facilitate a comprehensive understanding of the complex interactions between candidate genes with their functional interactors, GRAMM-X server was used. Protein-protein docking investigation of P16-INK4 revealed four ionic bonds illustrating Arg47, Arg80,Cys72 and Met1 residues as actively participating in interactions with CDK4 while docking results of RB1 showed four hydrogen bonds involving Glu864, Ser567, Asp36 and Arg861 residues which interact strongly with its respective functional interactor E2F1.. This research may provide a basis for understanding biological insights of P16-INK4 and RB1 proteins which will be helpful in future to design a suitable drug to inhibit the disease pathogenesis as we have determined the interacting amino acids which can be targeted in order to design a ligand in-vitro to propose a drug for clinical trials. Protein -protein docking of candidate genes and their important interacting residues likely to be provide a gateway for developing computer aided drug designing.

Topics: Cyclin D1; Cyclin-Dependent Kinase Inhibitor p16; E2F1 Transcription Factor; Humans; Lung Neoplasms; Molecular Docking Simulation; Protein Binding; Retinoblastoma Protein; Signal Transduction

Markedly pleomorphic epithelioid cells with high mitotic activity, giant cell formation, very large atypical nuclei, multiple nucleoli and abundant cytoplasm characterize 'monster' cells and may indicate aggressive tumor behavior. Very rare reports of melanomas comprised of 'monster cells' or cells with comparable histomorphological features, found in tissue samples from skin, lymph nodes, CNS, oral cavity and ileum have been published in the literature. This case is the first such description in the lung, and it is characterized with a battery of immunohistochemical stains; BRAF mutation status was negative, and fluorescence in situ hybridization analysis revealed increased copy number gains in 11q (cyclin D1), which is associated with poor prognosis in melanoma. The presence of monster cells in melanoma was associated with aggressive behavior in the reported patient.

Topics: Cyclin D1; Fatal Outcome; Female; Giant Cells; Humans; Lung Neoplasms; Melanoma; Middle Aged; Skin Neoplasms

GDK-100017, a 2,3,6-trisubstituted quinoxaline derivative, reduced β-catenin-T-cell factor/lymphoid enhancer factor (TCF/LEF)-dependent transcriptional activity and inhibited cell proliferation in a dose-dependent manner with an IC₅₀ value of about 10 μM in A549/Wnt2 cells. GDK-100017 down-regulated the expression of Wnt/β-catenin pathway target genes such as cyclin D1 and Dkk1 but not c-myc or survivin. GDK-100017 inhibited cell proliferation by arresting the cell cycle in the G1 phase not only in A549/wnt2 cells but also in SW480 colon cancer cells. In addition to its wnt signaling inhibitory properties, GDK-100017 also enhanced the radiosensitivity of the A549 human NSCLC line. These results suggest that GDK-100017 possesses potential anti-cancer activity by inhibiting the Wnt/β-catenin signal pathway, blocking the β-catenin-TCF/LEF interaction, and enhancing radiosensitivity.

Topics: Antineoplastic Agents; beta Catenin; Carcinoma, Non-Small-Cell Lung; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Colonic Neoplasms; Cyclin D1; Humans; Lung Neoplasms; Quinoxalines; Radiation Tolerance; Radiation-Sensitizing Agents; Respiratory Mucosa; Wnt Proteins; Wnt Signaling Pathway

Nomilin is a triterpenoid present in common edible citrus fruits with putative anticancer properties. In this study, the authors investigated the antimetastatic potential of nomilin and its possible mechanism of action. Metastasis was induced in C57BL/6 mice through the lateral tail vein using highly metastatic B16F-10 melanoma cells. Administration of nomilin inhibited tumor nodule formation in the lungs (68%) and markedly increased the survival rate of the metastatic tumor-bearing animals. These results correlated with the biochemical parameters and histopathological analysis. Nomilin showed an inhibition of tumor cell invasion and activation of matrix metalloproteinases. Treatment with nomilin induced apoptotic response, characterized by an increase in the sub-G1 fraction of cells with chromatin condensation and membrane blebbing, a typical ladder of DNA fragmentation, and detection of apoptotic cells by TUNEL assay. Nomilin treatment also exhibited a downregulated Bcl-2 and cyclin-D1 expression and upregulated p53, Bax, caspase-9, caspase-3, p21, and p27 gene expression in B16F-10 cells. Proinflammatory cytokine production and gene expression were found to be downregulated in nomilin-treated cells. The study also reveals that nomilin could inhibit the activation and nuclear translocation of antiapoptotic transcription factors such as nuclear factor (NF)-κB, CREB, and ATF-2 in B16F-10 cells.

Topics: Activating Transcription Factor 2; Animals; Antineoplastic Agents, Phytogenic; Apoptosis; bcl-2-Associated X Protein; Benzoxepins; Caspase 3; Caspase 9; Cell Cycle; Cell Line, Tumor; Cyclic AMP Response Element-Binding Protein; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase Inhibitor p27; DNA Fragmentation; Down-Regulation; Limonins; Lung Neoplasms; Male; Matrix Metalloproteinase Inhibitors; Matrix Metalloproteinases; Melanoma, Experimental; Mice; Mice, Inbred C57BL; Neoplasm Invasiveness; Neoplasm Metastasis; NF-kappa B; Plant Extracts; Proto-Oncogene Proteins c-bcl-2; Survival Rate; Transcription Factors; Tumor Suppressor Protein p53; Up-Regulation

Interleukin-7 is a potent regulator of lymphocyte proliferation, but it inducing growth of solid tumors is few known. We study the relationship between Interleukin-7 and the regulator of the cell cycle, cyclin D1 and the mechanism of Interleukin-7 regulating cell growth in human lung cancer. We detected expression of cyclin D1 and its impact on the prognosis of lung cancer patients. Using Western blot, reverse transcriptase-PCR, Co-Immunoprecipitation, and Chromatin Immunoprecipitation, we investigated how Interleukin-7 regulated cyclin D1 in vitro and in nude mice. We found that, in lung cancer cell lines and in nude mice, Interleukin-7/Interleukin-7 receptor increased the expression of cyclin D1 and phosphorylation of c-Fos/c-Jun, induce c-Fos and c-Jun heterodimer formation, and enhanced c-Fos/c-Jun DNA-binding activity to regulate cyclin D1. In addition, lymph node metastasis, tumor stage, and cyclin D1 were the strongest predictors of survival in 100 human non-small cell lung cancer specimens analyzed. Taken together, our results provided evidence that Interleukin-7/Interleukin-7 receptor induced cyclin D1 up-regulation via c-Fos/c-Jun pathway to promote proliferation of cells in lung cancer.

Topics: Animals; Carcinoma, Non-Small-Cell Lung; Cell Growth Processes; Cell Line, Tumor; Cyclin D1; Female; Humans; Immunohistochemistry; Interleukin-7; JNK Mitogen-Activated Protein Kinases; Lung Neoplasms; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Middle Aged; Promoter Regions, Genetic; Proto-Oncogene Proteins c-fos; Receptors, Interleukin-7; RNA, Small Interfering; Transcription Factor AP-1; Transfection; Transplantation, Heterologous; Up-Regulation

A number of histone demethylases have been identified and biochemically characterized, yet their biological functions largely remain uncharacterized, particularly in the context of human diseases such as cancer. In this study, we describe important roles for the histone demethylase KDM3A, also known as JMJD1A, in human carcinogenesis. Expression levels of KDM3A were significantly elevated in human bladder carcinomas compared with nonneoplastic bladder tissues (p < 0.0001), when assessed by real-time PCR. We confirmed that some other cancers including lung cancer also overexpressed KDM3A, using cDNA microarray analysis. Treatment of cancer cell lines with small interfering RNA targeting KDM3A significantly knocked down its expression and resulted in the suppression of proliferation. Importantly, we found that KDM3A activates transcription of the HOXA1 gene through demethylating histone H3 at lysine 9 di-methylation by binding to its promoter region. Indeed, expression levels of KDM3A and HOXA1 in several types of cancer cell lines and bladder cancer samples were statistically correlated. We observed the down-regulation of HOXA1 as well as CCND1 after treatment with KDM3A siRNA, indicating G(1) arrest of cancer cells. Together, our results suggest that elevated expression of KDM3A plays a critical role in the growth of cancer cells, and further studies may reveal a cancer therapeutic potential in KDM3A inhibition.

Topics: Cell Line, Tumor; Cell Proliferation; Cyclin D1; G1 Phase Cell Cycle Checkpoints; Gene Expression Regulation, Neoplastic; Histones; Homeodomain Proteins; Humans; Jumonji Domain-Containing Histone Demethylases; Lung Neoplasms; Methylation; Oligonucleotide Array Sequence Analysis; RNA Interference; RNA, Small Interfering; Transcription Factors; Transcription, Genetic; Urinary Bladder Neoplasms

The different mechanisms involved in p120-catenin (p120ctn) isoforms' 1/3 regulation of cell cycle progression are still not elucidated to date.. We found that both cyclin D1 and cyclin E could be effectively restored by restitution of p120ctn-1A or p120ctn-3A in p120ctn depleted lung cancer cells. When the expression of cyclin D1 was blocked by co-transfection with siRNA-cyclin D1 in p120ctn depleted cells restoring p120ctn-1A or 3A, the expression of cyclin E was slightly decreased, not increased, implying that p120ctn isoforms 1 and 3 cannot up-regulate cyclin E directly but may do so through up-regulation of cyclin D1. Interestingly, overexpression of p120ctn-1A increased β-catenin and cyclin D1 expression, while co-transfection with siRNA targeting β-catenin abolishes the effect of p120ctn-1A on up-regulation of cyclin D1, suggesting a role of β-catenin in mediating p120ctn-1A's regulatory function on cyclin D1 expression. On the other hand, overexpression of p120ctn isoform 3A reduced nuclear Kaiso localization, thus decreasing the binding of Kaiso to KBS on the cyclin D1 promoter and thereby enhancing the expression of cyclin D1 gene by relieving the repressor effect of Kaiso. Because overexpressing NLS-p120ctn-3A (p120ctn-3A nuclear target localization plasmids) or inhibiting nuclear export of p120ctn-3 by Leptomycin B (LMB) caused translocation of Kaiso to the nucleus, it is plausible that the nuclear export of Kaiso is p120ctn-3-dependent.. Our results suggest that p120ctn isoforms 1 and 3 up-regulate cyclin D1, and thereby cyclin E, resulting in the promotion of cell proliferation and cell cycle progression in lung cancer cells probably via different protein mediators, namely, β-catenin for isoform 1 and Kaiso, a negative transcriptional factor of cyclin D1, for isoform 3.

Topics: beta Catenin; Blotting, Western; Catenins; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Chromatin Immunoprecipitation; Cyclin D1; Cyclin E; Delta Catenin; Flow Cytometry; Humans; Lung Neoplasms; Reverse Transcriptase Polymerase Chain Reaction; RNA, Small Interfering; Transcription Factors

Cyclin D1, as a regulatory factor in cell cycle, is highly expressed in many tumors, such as lung cancer, breast cancer and thyroid cancer. The aim of the present study was to study the role of Cyclin D1 in invasion and metastasis of lung cancer cells. Lung adenocarcinoma cell line A549 and squamous cell line SK-MES-1 were selected as the objects, because A549 expresses Cyclin D1 highly, and SK-MES-1 expresses lowly. Nude mice were injected with A549 or SK-MES-1 via tail vein, and were sacrificed after 4 weeks for cancer tissue isolation. The harvested cancer cells were reinjected into another nude mouse. After one more time of such seeding, highly metastatic lung cancer model was established. After A549 and SK-MES-1 were transfected with Cyclin D1 RNAi and expression vector respectively, transwell migration assay was used to analyze transferring capacity of lung cancer cells. Western blot was used to detect Cyclin D1 and WNT/TCF pathway proteins expressions in parental cell lines and cancer tissue from metastasis model animals. The results showed that, along with the increase of seeding times, lung cancer cells from model animals, no matter A549 or SK-MES-1, exhibited augmented metastasis activity and up-regulated Cyclin D1 expression. The transferring capacity was weakened significantly in A549 cells where the Cyclin D1 was interfered by RNAi, and it was enhanced significantly in SK-MES-1 cells which were transfected with the expression vector of Cyclin D1. The expressions of WNT/TCF pathway proteins, including β-catenin, lymphoid enhancer-binding factor (LEF) and T cell factor (TCF), increased significantly in highly metastatic model animals. The parental cell lines showed lower expressions of WNT/TCF pathway proteins compared with cancer tissue from metastasis model animals. These results suggest that Cyclin D1 is closely related with the invasion and metastasis of lung cancer cells, and the WNT/TCF signal pathway may promote the expression of Cyclin D1.

Topics: Adenocarcinoma; Animals; Carcinoma, Squamous Cell; Cell Line, Tumor; Cyclin D1; Female; Humans; Lung Neoplasms; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasm Transplantation; RNA Interference; Transfection; Wnt Signaling Pathway

Rsf-1 (HBXAP) was recently reported to be overexpressed in various cancers and associated with the malignant behavior of cancer cells. However, the expression of Rsf-1 in primary lung cancer and its biological roles in non-small cell lung cancer (NSCLC) have not been reported. The molecular mechanism of Rsf-1 in cancer aggressiveness remains ambiguous. In the present study, we analyzed the expression pattern of Rsf-1 in NSCLC tissues and found that Rsf-1 was overexpressed at both the mRNA and protein levels. There was a significant association between Rsf-1 overexpression and TNM stage (p=0.0220) and poor differentiation (p=0.0013). Furthermore, knockdown of Rsf-1 expression in H1299 and H460 cells with high endogenous Rsf-1 expression resulted in a decrease of colony formation ability and inhibition of cell cycle progression. Rsf-1 knockdown also induced apoptosis in these cell lines. Further analysis showed that Rsf-1 knockdown decreased cyclin D1 expression and phospho-ERK levels. In conclusion, Rsf-1 is overexpressed in NSCLC and contributes to malignant cell growth by cyclin D1 and ERK modulation, which makes Rsf-1 a candidate therapeutic target in lung cancer.

Topics: Adenocarcinoma; Adenocarcinoma of Lung; Aged; Aged, 80 and over; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Extracellular Signal-Regulated MAP Kinases; Humans; Lung Neoplasms; Male; Middle Aged; Neoplasm Staging; Nuclear Proteins; RNA, Messenger; Trans-Activators

The objective of the study was to investigate the impact of the B cell translocation gene 2 (BTG2) on lung cancer cell growth, proliferation, metastasis, and other biological characteristics and to provide experimental evidence for the biological treatment of human lung cancer. A pcDNA3.1-BTG2 eukaryotic expression vector was constructed and transfected into the human lung cancer cell line A549. The biological changes in the BTG2-expressing cells were analyzed using growth curves, the MTT (tetrazolium) assay, propidium iodide (PI) staining, and the Transwell invasion chamber. Additionally, Western blotting was used to determine the impact of BTG2 on the protein expression of cyclin D1, MMP-1, and MMP-2. Compared to the empty vector-transfected A549 cells or the mock-transfected A549 cells, the pcDNA3.1-BTG2-transfected A549 cells grew significantly slower. No significant differences were detected between the empty vector-transfected group and the mock-transfected A549 cells. The growth curve analysis and the PI staining showed that the pcDNA3.1-BTG2-transfected cells grew significantly slower than the empty vector-transfected A549 cells (P < 0.05). The cell invasion assay results suggested that the invasion rate of the pcDNA3.1-BTG2-transfected A549 cells was significantly slower than the invasion rate of the empty vector-transfected group and the mock-transfected group (P < 0.05). The overexpression of BTG2 may inhibit the protein expression of cyclin D1, MMP-1, and MMP-2 in A549 cells. The overexpression of BTG2 may inhibit the growth, proliferation, and invasiveness of the A549 human lung cancer cell line.

Topics: Blotting, Western; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cell Survival; Cyclin D1; Gene Expression Regulation, Neoplastic; Humans; Immediate-Early Proteins; Lung Neoplasms; Matrix Metalloproteinase 1; Matrix Metalloproteinase 2; Reverse Transcriptase Polymerase Chain Reaction; Time Factors; Transfection; Tumor Suppressor Proteins

Cyclin D1, an oncogenic G1 cyclin which can be induced by environmental carcinogens and whose over-expression may cause dysplasia and carcinoma, has been shown to be a target for cancer chemoprevention and therapy. In this study, we investigated the effects and underlying mechanisms of action of a polyprenylated xanthone, gambogenic acid (GEA) on gefitinib-sensitive and -resistant lung cancer cells. We found that GEA inhibited proliferation, caused G1 arrest and repressed colony-forming activity of lung cancer cells. GEA induced degradation of cyclin D1 via the proteasome pathway, and triggered dephosphorylation of GSK3β which was required for cyclin D1 turnover, because GSK3β inactivation by its inhibitor or specific siRNA markedly attenuated GEA-caused cyclin D1 catabolism. GEA induced autophagy of lung cancer cells, possibly due to activation of GSK3β and inactivation of AKT/mTOR signal pathway. These results indicate that GEA is a cyclin D1 inhibitor and a GSK3β activator which may have chemopreventive and therapeutic potential for lung cancer.

Topics: Antineoplastic Agents, Phytogenic; Autophagy; Cell Line, Tumor; Cyclin D1; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase Inhibitor p27; Enzyme Activation; G1 Phase Cell Cycle Checkpoints; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Humans; Lung Neoplasms; Proto-Oncogene Proteins c-akt; Signal Transduction; Terpenes; TOR Serine-Threonine Kinases; Xanthenes; Xanthones

MicroRNAs (miRNAs) are small noncoding RNAs that play critical roles in regulating various cellular functions by transcriptional silencing. miRNAs can function as either oncogenes or tumor suppressors (oncomirs), depending on cancer types. In our study, using miRNA microarray, we observed that downregulation of the Notch-1 pathway, by delta-tocotrienol, correlated with upregulation of miR-34a, in nonsmall cell lung cancer cells (NSCLC). Moreover, re-expression of miR-34a by transfection in NSCLC cells resulted in inhibition of cell growth and invasiveness, induction of apoptosis and enhanced p53 activity. Furthermore, cellular mechanism studies revealed that induction of miR-34a decreased the expression of Notch-1 and its downstream targets including Hes-1, Cyclin D1, Survivin and Bcl-2. Our findings suggest that delta-tocotrienol is a nontoxic activator of mir-34a which can inhibit NSCLC cell proliferation, induce apoptosis and inhibit invasion, and thus offering a potential starting point for the design of novel anticancer agents.

Topics: Apoptosis; Basic Helix-Loop-Helix Transcription Factors; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Down-Regulation; Gene Expression Regulation, Neoplastic; Homeodomain Proteins; Humans; Inhibitor of Apoptosis Proteins; Lung Neoplasms; MicroRNAs; Neoplasm Invasiveness; Proto-Oncogene Proteins c-bcl-2; Receptor, Notch1; Signal Transduction; Survivin; Transcription Factor HES-1; Transfection; Tumor Suppressor Protein p53; Up-Regulation; Vitamin E

To guide clinicians in selecting treatment options for patients with non-small cell lung cancer (NSCLC), it is desirable to have reliable markers predicting clinical outcome. This study analyzed the correlation between signal transducer and activator of transcription 3 (STAT3) and cyclin D1 in NSCLC and their association with clinicopathological features and survival.. We investigated 65 specimens of NSCLC tissues by immunohistochemistry using STAT3 and cyclin D1 antibodies. First we determined the correlation between STAT3 and cyclin D1 expression and the clinicopathological features of the tumor. Then we assessed the prognostic relevance of STAT3 and cyclin D1.. A significant correlation was found between high levels of STAT3 expression and the degree of tumor differentiation. Additionally, a significant positive correlation was found between the expression of STAT3 and cyclin D1 (r=0.405, p=0.001). The overexpression of STAT3 and the presence of metastasis were significantly associated with shorter overall survival in univariate analysis (p=0.028 and p=0.036, respectively). Multivariate analysis confirmed that STAT3 expression was an independent prognostic factor (p=0.001).. STAT3 might be correlated with tumor differentiation, and its elevated expression may be an adverse prognostic indicator for patients with NSCLC. Activation of the STAT3/cyclin D1 signaling pathway may be attributed to the malignant transformation of NSCLC and may represent a possible target for therapy.

Topics: Aged; Biomarkers, Tumor; Carcinoma, Non-Small-Cell Lung; Cell Transformation, Neoplastic; Cyclin D1; Humans; Immunohistochemistry; Lung Neoplasms; Middle Aged; Prognosis; Signal Transduction; STAT3 Transcription Factor

The purposes of this study were to investigate the effects of the SASH1 gene on the growth, proliferation, apoptosis, invasiveness, and metastatic potential of lung cancer cells and explore the potential use of SASH1 for the treatment of human lung cancer. The SASH1 gene was cloned into the pcDNA3.1 eukaryotic expression vector, and SASH1 shRNA were designed and constructed. The resulting constructs were transfected into A549 human lung cancer cells, and the changes in the relevant biological characteristics of the cells overexpressing SASH1 and cells with downregulated expression of SASH1 were analyzed using the MTT assay, transwell invasion assay, and flow cytometry. The effects of the SASH1 gene on the expression of cyclin D1, Bcl-2, and MMP-2/9 were also concurrently examined. In the A549 cells from the pcDNA3.1-SASH1 transfected group, cell viability, proliferation, and migration were significantly reduced compared to the control cells (p = 0.039, p = 0.013), and a cell cycle arrest in G1 was observed. The A549 cells transfected with the SASH1 shRNA demonstrated significantly higher cell viabilities, proliferation, and migration compared to the control cells (p = 0.012, p = 0.045). Additionally, the percentage of A549 cells undergoing apoptosis was significantly higher in the pcDNA3.1-SASH1 transfected cells and significantly lower in the SASH1 shRNA transfected cells compared to the control cells (p = 0.010, p = 0.000). The cyclin D1, Bcl-2, and MMP-9/2 protein expression levels were significantly lower in the pcDNA3.1-SASH1-transfected cells and were significantly higher in the SASH1 shRNA-transfected cells than that in the control cells. The SASH1 gene may inhibit A549 cell growth and proliferation as well as promote cellular apoptosis. The overexpression of the SASH1 gene may also be related to the decreased migration of A549 human lung cancer cells.

Topics: Apoptosis; Cell Cycle; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin D1; Gene Expression; Humans; Lung Neoplasms; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Proto-Oncogene Proteins c-bcl-2; Tumor Suppressor Proteins

MicroRNAs have been reported to be closely related to the development of human lung cancers. However, the functions of microRNAs in non-small cell lung cancer (NSCLC) remain largely undefined. Here, we investigated the role of microRNA-193b (miR-193b) in NSCLC. Our data showed that miR-193b was markedly down-regulated in NSCLC cancer tissues compared with adjacent normal tissues. The NSCLC cell line (A549) transfected with the miR-193b exhibited significantly decreased proliferation, migration, and invasion capacities when compared with the control cells. In contrast, inhibition of miR-193b increased the proliferation, migration, and invasion of A549 cells. Moreover, miR-193b repressed the expressions of cyclin D1 and urokinase-type plasminogen activator in A549 cells. These data suggest that miR-193b is a tumor suppressor in NSCLC.

Topics: Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin D1; Down-Regulation; G1 Phase Cell Cycle Checkpoints; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; MicroRNAs; Neoplasm Invasiveness; Urokinase-Type Plasminogen Activator

Histone deacetylase 2 (HDAC2) is crucial for embryonic development, affects cytokine signaling relevant for immune responses, and is often significantly overexpressed in solid tumors, but little is known of its role in human lung cancer. In this study, we demonstrated the aberrant expression of HDAC2 in lung cancer tissues and investigated oncogenic properties of HDAC2 in human lung cancer cell lines. HDAC2 inactivation resulted in regression of tumor cell growth and activation of cellular apoptosis via p53 and Bax activation and Bcl2 suppression. In cell cycle regulation, HDAC2 inactivation caused induction of p21WAF1/CIP1 expression, and simultaneously suppressed the expressions of cyclin E2, cyclin D1, and CDK2, respectively. Consequently, this led to the hypophosphorylation of pRb protein in G1/S transition and thereby inactivated E2F/DP1 target gene transcriptions of A549 cells. In addition, we demonstrated that HDAC2 directly regulated p21WAF1/CIP1 expression in a p53-independent manner. However, HDAC1 was not related to p21WAF1/CIP1 expression and tumorigenesis of lung cancer. Lastly, we observed that sustained-suppression of HDAC2 in A549 lung cancer cells attenuated in vitro tumorigenic properties and in vivo tumor growth of the mouse xenograft model. Taken together, we suggest that the aberrant regulation of HDAC2 and its epigenetic regulation of gene transcription in apoptosis and cell cycle components play an important role in the development of lung cancer.

Topics: Animals; Apoptosis; Apoptosis Regulatory Proteins; bcl-2-Associated X Protein; bcl-Associated Death Protein; Cell Cycle Proteins; Cell Line, Tumor; Cyclin D1; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; E2F Transcription Factors; Gene Expression Regulation, Neoplastic; Histone Deacetylase 2; Humans; Lung Neoplasms; Mice; Mice, Nude; Phosphorylation; Retinoblastoma Protein; RNA Interference; RNA, Small Interfering; Transcription, Genetic; Transplantation, Heterologous; Tumor Suppressor Protein p53

Compared to well-known function of cyclin D1 in lung cancer, the role of cyclin D2 is not clear. This study was aimed at understanding the clinicopathological significance of cyclin D2 in primary non-small cell lung cancer (NSCLC).. We retrospectively analyzed expression statuses of cyclin D1, cyclin D2, p16, p21, p27, Ki-67, and phospho-pRb (Ser-807/811) using immunohistochemistry in 626 NSCLCs.. Cyclin D2 was expressed in normal lung tissue, and its expression was reduced in 170 (27%) of 626 NSCLCs with a median duration of follow-up of 64 months. Mean phospho-pRb (Ser-807/811) levels were not associated with expression levels of cyclin D2 (P=0.15). The relationship between recurrence and the reduced expression of cyclin D2 was not homogenous by stage (Breslow-Day test for homogeneity, P=0.04). Reduced expression of cyclin D2 was not associated with patient's prognosis in 370 stage I, 112 stage II, and 18 stage IV NSCLCs. However, for 126 stage III NSCLCs, reduced expression of cyclin D2 was adversely associated with recurrence-free survival (RFS) (hazard ratio [HR]=3.71, 95% CI=1.54-13.17; P=0.01), independent of histology and expression of cyclin D1. The reduced expression of cyclin D2 was not associated with the overexpression of cyclin D1 (P=0.65).. The present study suggests that reduced expression of cyclin D2 in stage III NSCLC may be associated with poor RFS. And, cyclin D2 may have a distinct role from cyclin D1 in NSCLC.

Topics: Aged; Biomarkers, Tumor; Carcinoma, Non-Small-Cell Lung; Cyclin D1; Cyclin D2; Female; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Lung Neoplasms; Male; Middle Aged; Neoplasm Staging; Phosphorylation; Prognosis; Recurrence; Retinoblastoma Protein; Survival Analysis

Here we have shown the alteration of transcription factors STAT3, NF-κB and downstream associated molecules much before the appearance of lung tumor and their response to antitumor agent, inositol hexaphosphate. Histological examination revealed the pathophysiology of the lung tissues and the onset or progression of tumor from 4 or 9 to 24 weeks in terms of tumor volume and the number. Over expression of NF-κB (p50/Rel A), COX-2, STAT3, pSTAT3 (Tyr 705), IL-6 and cyclin D1 also progressed from the time of no tumor to the time of tumor appearance and was reduced in mice drinking 2%IP6. We suggest that the alterations of STAT3, NF-κB and downstream associated molecules are critical in the development of lung tumors and can be exploited as possible mechanisms after the exposure. Status of these altered genes before the tumor development suggests their possible use as targets for the tumor control in the predisposed conditions.

Topics: Animals; Carcinogens; Cyclin D1; Cyclooxygenase 2; Female; Interleukin-6; Lung Neoplasms; Mice; Mice, Inbred BALB C; NF-kappa B; RNA, Messenger; STAT3 Transcription Factor; Urethane

Caveolin-1 (CAV-1), one component of caveolae, involves in multiple cellular processes and signal transductions. We previously showed that the expression of CAV-1 gene in NCI-H446 cells inhibited cell proliferation and promoted cell metastasis. Here we explore the function of CAV-1 on tumor growth and metastasis by using NCI-H460 in vitro. First, we established NCI-H460 cell line, which CAV-1 was stably knockdown. Then we investigated the effects of CAV-1 on the morphology, proliferation, cell cycle and metastasis potential for NCI-H460 cell by crystal violet stains, CCK-8, colony formation, flow cytometry, scratch-wound assay and transwell assay. Western blot was used to examine the expression changes of cyclin D1, PCNA, E-cadherin and β-catenin. Our results showed stable knockdown of CAV-1 inhibited the proliferation of NCI-H460 cells. Cell cycle of the transfected cells was arrested in G1/S phase and the expressions of cyclin D1 and PCNA protein were downregulated. Downregulation of CAV-1 promoted the migration and invasion abilities of NCI-H460 cells in vitro. The expression of β-catenin increased and the level of E-cadherin decreased. In summary, our findings provide experimental evidence that CAV-1 may function as a proproliferative and antimetastatic gene in NCI-H460 cell line.

Topics: Antigens, CD; beta Catenin; Cadherins; Carcinoma, Large Cell; Caveolin 1; Cell Cycle; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cell Shape; Cyclin D1; Gene Expression Regulation, Neoplastic; Gene Silencing; Humans; Lung Neoplasms; Neoplasm Proteins; Proliferating Cell Nuclear Antigen; Recombinant Proteins; RNA, Messenger; RNA, Small Interfering; Tumor Stem Cell Assay

Cancer is a public health problem in the world accounting for most of the deaths. Currently, common treatment of cancer such as chemotherapy works by killing fast-growing cancer cells. Unfortunately, chemotherapy cannot tell the difference between cancer cells and fast-growing healthy cells, including red and white blood cells. As a result, one of the most serious potential side effects of some types of chemotherapy is a low white blood cell count that makes it unreliable (Parkin et al. [34]; Pauk et al. [3]). Even though intense research has been going on in recent years, successful therapeutic targets against this disease have been elusive. In this study, we evaluate the anti-proliferative activity of Euphorbia mauritanica and Kedrostis hirtella in lung cancer. In our assessment it was observed that E. mauritanica and K. hirtella were able to induce cell death at 5 μg/ml in A549 cells over 22 h and at 10 μg/ml over 24 h in the Lqr1 cell line. Molecular analysis of DNA fragmentation and Annexin V were used to examine the type of cell death induced by E. mauritanica and K. hirtella extracts. These results showed an increase in necrotic and apoptotic characteristics with both nuclear DNA fragmentation and smear. Therefore, these results suggest that E. mauritanica and K. hirtella may play a role in inducing cell death in lung cancer cells. However, further studies need to be conducted to ascertain these results.

Topics: Apoptosis; Carrier Proteins; Cell Line, Tumor; Cell Proliferation; Cell Survival; Chromatography, Thin Layer; Chrysobalanaceae; Cyclin D1; DNA Fragmentation; DNA-Binding Proteins; Drug Screening Assays, Antitumor; Euphorbia; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Necrosis; Neoplasms, Squamous Cell; Plant Extracts; Staurosporine; Tumor Suppressor Protein p53; Ubiquitin-Protein Ligases

Non-small cell lung carcinoma (NSCLC) is a major killer in cancer related human death. Its therapeutic intervention requires superior efficient molecule(s) as it often becomes resistant to present chemotherapy options. Here we report that vapor of volatile oil compounds obtained from Litsea cubeba seeds killed human NSCLC cells, A549, through the induction of apoptosis and cell cycle arrest. Vapor generated from the combined oils (VCO) deactivated Akt, a key player in cancer cell survival and proliferation. Interestingly VCO dephosphorylated Akt at both Ser(473) and Thr(308); through the suppression of mTOR and pPDK1 respectively. As a consequence of this, diminished phosphorylation of Bad occurred along with the decreased Bcl-xL expression. This subsequently enhanced Bax levels permitting the release of mitochondrial cytochrome c into the cytosol which concomitantly activated caspase 9 and caspase 3 resulting apoptotic cell death. Impairment of Akt activation by VCO also deactivated Mdm2 that effected overexpression of p53 which in turn upregulated p21 expression. This causes enhanced p21 binding to cyclin D1 that halted G1 to S phase progression. Taken together, VCO produces two prong effects on lung cancer cells, it induces apoptosis and blocked cancer cell proliferation, both occurred due to the deactivation of Akt. In addition, it has another crucial advantage: VCO could be directly delivered to lung cancer tissue through inhalation.

Topics: Apoptosis; Carcinoma, Non-Small-Cell Lung; Caspases; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Survival; Cyclin D1; Humans; Litsea; Lung Neoplasms; Oils, Volatile; Phosphorylation; Plant Extracts; Plant Oils; Proto-Oncogene Proteins c-akt; Seeds; Signal Transduction

To study the mechanism of interleukin 7/interleukin 7 receptor (IL-7/IL-7R) in promoting cell proliferation and inducing lymphangiogenesis of non-small cell lung cancer (NSCLC) in vivo and in vitro.. Immunohistochemical study for IL-7, IL-7R, cyclin D1 and vascular endothelial growth factor-D (VEGF-D) was carried out in NSCLC tissues from 95 patients. The relationship between IL-7/IL-7R expression and various parameters was analyzed. The mechanism of IL-7/IL-7R in promoting cell proliferation and inducing lymphangiogenesis was studied by methylthiazolyldiphenyl-tetrazolium bromide, fluorescence-activated cell sorting, reverse transcriptase-PCR, Western blot, co-immunoprecipitation, chromatin immunoprecipitation and nude mice experiments with xenograft tumors.. IL-7 (63.2%, 60/95), IL-7R (61.1%, 58/95), cyclin D1 (52.6%, 50/95) and VEGF-D (58.9%, 56/95) showed that high level of expression in NSCLC. IL-7/IL-7R over-expression correlated with cyclin D1 expression (P < 0.01, P < 0.01), VEGF-D expression (P < 0.01, P < 0.01), increased lymphovascular density (P = 0.005, P = 0.013), advanced clinical stage (P = 0.008, P = 0.005) and presence of lymph node metastasis (P < 0.01, P < 0.01). IL-7/IL-7R could promote proliferation of A549 cell, increase cyclin D1 and VEGF-D expression, and enhance c-Fos/c-Jun expression and phosphorylation, resulting in formation of heterodimer. Furthermore, IL-7/IL-7R could induce binding of c-Fos/c-Jun to cyclin D1/VEGF-D promoters and regulate their transcription. IL-7/IL-7R could also promote proliferation and lymphangiogenesis of lung cancer xenograft tumors.. IL-7/IL-7R promotes c-Fos/c-Jun expression and activity in NSCLC. This further facilitates cyclin D1 expression and accelerates proliferation of cells and VEGF-D-induced lymphovascular formation.

Topics: Animals; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Female; Humans; Interleukin-7; Lung Neoplasms; Lymphangiogenesis; Lymphatic Metastasis; Male; Mice; Mice, Nude; Middle Aged; Neoplasm Staging; Neoplasm Transplantation; Proto-Oncogene Proteins c-fos; Proto-Oncogene Proteins c-jun; Receptors, Interleukin-7; Vascular Endothelial Growth Factor D

Epigenetic silencing is a common mechanism to inactivate tumor suppressor genes during carcinogenesis. Enhancer of Zeste 2 (EZH2) is the histone methyltransferase subunit in polycomb repressive complex 2 which mediates transcriptional repression through histone methylation. EZH2 overexpression has been linked to aggressive phenotypes of certain cancers. However, the mechanism that EZH2 played in promoting malignancy in non-small cell lung cancer (NSCLC) remains unclear. In addition, the correlation of EZH2 overexpression and the prognosis of NSCLC patients in non-Asian cohort need to be determined.. Up-regulation of EZH2 was found in NSCLC cells compared with normal human bronchial epithelial cells by western blot assay. Upon EZH2 knockdown using small interfering RNA (siRNA), the proliferation, anchorage-independent growth and invasion of NSCLC cells were remarkably suppressed with profound induction of G1 arrest. Furthermore, the expression of cyclin D1 was notably reduced whereas p15(INK4B), p21(Waf1/Cip1) and p27(Kip1) were increased in NSCLC cells after EZH2-siRNA delivery. To determine whether EZH2 expression contributes to disease progression in patients with NSCLC, Taqman quantitative real-time RT-PCR was used to measure the expression of EZH2 in paired tumor and normal samples. Univariate analysis revealed that patients with NSCLC whose tumors had a higher EZH2 expression had significantly inferior overall, disease-specific, and disease-free survivals compared to those whose tumors expressed lower EZH2 (P = 0.005, P = 0.001 and P = 0.003, respectively). In multivariate analysis, EZH2 expression was an independent predictor of disease-free survival (hazard ratio = 0.450, 95% CI: 0.270 to 0.750, P = 0.002).. Our results demonstrate that EZH2 overexpression is critical for NSCLC progression. EZH2 mRNA levels may serve as a prognostic predictor for patients with NSCLC.

Topics: Bronchi; Carcinoma, Non-Small-Cell Lung; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Enhancer of Zeste Homolog 2 Protein; Epithelial Cells; Female; Humans; Lung Neoplasms; Male; Polycomb Repressive Complex 2; Prognosis; RNA, Messenger; Up-Regulation

Cyclin D1 (CCND1) is a key regulatory protein of the cell cycle. The purpose of our study was to assess the role of CCND1 genetic variants influencing the genetic susceptibility of non-small cell lung cancer (NSCLC). We conducted a study of 1234 individuals, including 892 controls and 342 cases. Individuals carrying two G-alleles have a 2-fold increased risk for the development of NSCLC and the waiting time for onset of NSCLC in these patients was 2 years earlier in comparison with other individuals. Our results may be important in contributing to the knowledge of the mechanisms involved in lung carcinogenesis.

Topics: Aged; Carcinoma, Non-Small-Cell Lung; Case-Control Studies; Cyclin D1; Female; Genotype; Humans; Lung Neoplasms; Male; Middle Aged; Polymorphism, Single Nucleotide; Portugal

Primary lung tumors are rare in children, and mucoepidermoid carcinoma (MEC) represents less than 10% of them. Additionally, MEC arising from bronchogenic cysts (BC) is particularly unusual. We describe the clinical and genetic findings on a MEC occurring within a previous location of a BC in an adolescent. This particular association has not been previously reported. The lesion revealed normal karyotype without the typical t(11;19)(q21;p13) translocation. Cyclin D1 overexpression (165-fold increase) was demonstrated by real-time PCR although FISH assessment showed normal hybridization at 11q13. Information on these unusual clinical presentations may present relevant insight on tumorigenesis of infrequent pediatric pulmonary tumors.

Topics: Adolescent; Bronchogenic Cyst; Carcinoma, Mucoepidermoid; Child; Cyclin D1; Female; Gene Expression; Humans; Immunohistochemistry; Lung Neoplasms; Reverse Transcriptase Polymerase Chain Reaction

δ-Catenin is the only member of the p120 catenin (p120ctn) subfamily that its primary expression is restricted to the brain. Since δ-catenin is upregulated in human lung cancer, the effects of δ-catenin overexpression in lung cancer still need to be clarified. Immunohistochemistry was performed to investigate the expression of δ-catenin and Kaiso, a δ-catenin-binding transcription factor, in 151 lung cancer specimens. A correlation between cytoplasmic δ-catenin and Kaiso expression was also associated with high TNM stage, lymph node metastases and poor prognosis. Co-immunoprecipitation assay confirmed the interactions of δ-catenin and Kaiso in lung cancer cells. In addition, gene transfection and RNAi technology were used to demonstrate that increased δ-catenin expression was promoted, whereas its knockdown suppressed its lung cancer invasive ability. In addition, methylation-specific PCR and ChIP assay demonstrated that δ-catenin could regulate MTA2 via Kaiso in a methylation-dependent manner, while it could regulate cyclin D1 and MMP7 expression through Kaiso in a sequence-specific manner. In conclusion, a δ-catenin/Kaiso pathway exists in lung cancer cells. Increased δ-catenin expression is critical for maintenance of the malignant phenotype of lung cancer, making δ-catenin a candidate target protein for future cancer therapeutics.

Topics: Adult; Aged; Carcinoma, Non-Small-Cell Lung; Catenins; Cell Line, Tumor; Cyclin D1; Delta Catenin; Female; Histone Deacetylases; Humans; Lung Neoplasms; Male; Matrix Metalloproteinase 7; Middle Aged; Prognosis; Repressor Proteins; Transcription Factors; Transcription, Genetic; Up-Regulation

Scutellaria baicalensis Georgi is a widely used medicinal herb in several Asian countries including Korea. The various medicinal properties attributed to Scutellaria baicalensis include anti-bacterial, anti-viral, anti-inflammatory and anti-cancer effects. The present study investigated the cytotoxicity of Scutellaria baicalensis water extract (SBWE) on A549 non-small-cell-lung cancer cells and the A549 expression of cyclin D1, cyclin-dependent kinase 4 (CDK4) and matrix metalloproteinase-2 (MMP-2), and the effects of SBWE on cell cycle progression, especially the G1/S phase, and on cell motility.. SBWE cytotoxicity was assessed by a standard colorimetric assay utilizing 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and expression of cyclin D1 and CDK4 protein in SBWE-treated A549 cells was assessed by Western blot analysis. Flow cytometry analysis was performed to determine the effect of SBWE on A549 cell cycle progression. A549 cell MMP-2 activity was examined by zymography. Cell motility and migration was assessed by a scratch wound healing assay.. SBWE was not cytotoxic. The production of Cyclin D1, CDK4 and MMP-2 activity were significantly decreased in a SBWE dose-dependent manner, with maximum inhibition occurring at SBWE concentrations of 250 μg/ml and 500 μg/ml. SBWE inhibited cell cycle progression in the G1/S phase and significantly inhibited the motility of A549 cells.. Cyclin D1 protein may be associated with MMP-2 activity and cell motility. Thus, SBWE promotes a strong protective effect against MMP-2 mediated metastasis and cell proliferation through the down-regulation of cyclin D1. SBWE may be a useful chemotherapeutic agent for lung cancer.

Topics: Antineoplastic Agents, Phytogenic; Carcinoma, Non-Small-Cell Lung; Cell Cycle; Cell Line, Tumor; Cyclin D1; Cyclin-Dependent Kinase 4; Ethnopharmacology; Humans; Lung Neoplasms; Matrix Metalloproteinase 2; Matrix Metalloproteinase Inhibitors; Medicine, Korean Traditional; Phytotherapy; Plant Extracts; Republic of Korea; Scutellaria baicalensis

Ritonavir is a potential therapeutic agent in lung cancer, but its targets in lung adenocarcinoma are unknown, as are candidate biomarkers for its activity.. RNAi was used to identify genes whose expression affects ritonavir sensitivity. Synergy between ritonavir, gemcitabine, and cisplatin was tested by isobologram analysis.. Ritonavir inhibits growth of K-ras mutant lung adenocarcinoma lines A549, H522, H23, and K-ras wild-type line H838. Ritonavir causes G0/G1 arrest and apoptosis. Associated with G0/G1 arrest, ritonavir down-regulates cyclin-dependent kinases, cyclin D1, and retinoblastoma protein phosphorylation. Associated with induction of apoptosis, ritonavir reduces survivin messenger RNA and protein levels more than twofold. Ritonavir inhibits phosphorylation of c-Src and signal transducer and activator of transcription protein 3, which are important events for survivin gene expression and cell growth, and induces cleavage of PARP1. Although knock down of survivin, c-Src, or signal transducer and activator of transcription protein 3 inhibits cell growth, only survivin knock down enhances ritonavir inhibition of growth and survivin overexpression promotes ritonavir resistance. Ritonavir was tested in combination with gemcitabine or cisplatin, exhibiting synergistic and additive effects, respectively. The combination of ritonavir/gemcitabine/cisplatin is synergistic in the A549 line and additive in the H522 line, at clinically feasible ritonavir concentrations (<10 μM).. Ritonavir is of interest for lung adenocarcinoma therapeutics, and survivin is an important target and potential biomarker for its sensitivity. Ritonavir cooperation with gemcitabine/cisplatin might be explained by involvement of PARP1 in repair of cisplatin-mediated DNA damage and survivin in repair of gemcitabine-mediated double-stranded DNA breaks.

Topics: Adenocarcinoma; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Blotting, Western; Cell Cycle; Cell Proliferation; Cisplatin; Cyclin D1; Deoxycytidine; DNA Breaks, Double-Stranded; Drug Therapy, Combination; Gemcitabine; HIV Protease Inhibitors; Humans; Inhibitor of Apoptosis Proteins; Lung Neoplasms; Poly (ADP-Ribose) Polymerase-1; Poly(ADP-ribose) Polymerases; Reverse Transcriptase Polymerase Chain Reaction; Ritonavir; RNA, Messenger; RNA, Small Interfering; Survivin; Tumor Cells, Cultured

Effective clinical management of prostate cancer (PCA) has been challenged by significant intratumoural heterogeneity on the genomic and pathological levels and limited understanding of the genetic elements governing disease progression. Here, we exploited the experimental merits of the mouse to test the hypothesis that pathways constraining progression might be activated in indolent Pten-null mouse prostate tumours and that inactivation of such progression barriers in mice would engender a metastasis-prone condition. Comparative transcriptomic and canonical pathway analyses, followed by biochemical confirmation, of normal prostate epithelium versus poorly progressive Pten-null prostate cancers revealed robust activation of the TGFβ/BMP-SMAD4 signalling axis. The functional relevance of SMAD4 was further supported by emergence of invasive, metastatic and lethal prostate cancers with 100% penetrance upon genetic deletion of Smad4 in the Pten-null mouse prostate. Pathological and molecular analysis as well as transcriptomic knowledge-based pathway profiling of emerging tumours identified cell proliferation and invasion as two cardinal tumour biological features in the metastatic Smad4/Pten-null PCA model. Follow-on pathological and functional assessment confirmed cyclin D1 and SPP1 as key mediators of these biological processes, which together with PTEN and SMAD4, form a four-gene signature that is prognostic of prostate-specific antigen (PSA) biochemical recurrence and lethal metastasis in human PCA. This model-informed progression analysis, together with genetic, functional and translational studies, establishes SMAD4 as a key regulator of PCA progression in mice and humans.

Topics: Animals; Bone Morphogenetic Proteins; Cell Proliferation; Cyclin D1; Disease Progression; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Genes, Tumor Suppressor; Humans; Lung Neoplasms; Lymphatic Metastasis; Male; Mice; Mice, Transgenic; Models, Biological; Neoplasm Invasiveness; Neoplasm Metastasis; Osteopontin; Penetrance; Prognosis; Prostate; Prostate-Specific Antigen; Prostatic Neoplasms; PTEN Phosphohydrolase; Smad4 Protein; Transforming Growth Factor beta

The Wnt signaling pathway plays a major role in cancer development and progression. As a novel anticancer drug can be developed using inhibitors of this pathway, we investigated the clinical significance of the Wnt signaling pathway molecules in non-small cell lung cancer (NSCLC).. Immunohistochemical analysis of a tissue microarray with 262 resected NSCLC specimens was performed to study the expression and subcellular localization of Wnt1 in relation to downstream molecules, including GSK-3β, β-catenin, c-Myc, cyclin D1, and p53. These results were correlated with other clinicopathologic features.. Cytoplasmic Wnt1 overexpression was detected in 36.6% (96 of 262) NSCLCs, and aberrant β-catenin staining was identified in 76% (189 of 262) of NSCLCs. There were significant associations between Wnt1 expression and altered expression of β-catenin (p = 0.034), overexpression of c-Myc (p < 0.001), or overexpression of cyclin D1 (p = 0.018). While there was no significant association between Wnt1 or β-catenin and stage, the 5-year survival was significantly lower in patients with Wnt1- and β-catenin-positive NSCLCs than negative NSCLCs (p < 0.05, respectively). In multivariate analysis, stage and Wnt1+/β-catenin+ expression were independent prognostic factors of overall survival (p < 0.05).. These findings show that Wnt1 expression may be one of the possible mechanisms of the activation of the canonical Wnt/β-catenin signaling pathway in NSCLC, and Wnt1 and altered β-catenin expression are poor prognostic markers, independent of stage.

Topics: Adenocarcinoma; Adult; Aged; Aged, 80 and over; beta Catenin; Carcinoma, Non-Small-Cell Lung; Carcinoma, Squamous Cell; Cyclin D1; Female; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Humans; Immunoenzyme Techniques; Lung Neoplasms; Male; Middle Aged; Neoplasm Invasiveness; Prognosis; Proto-Oncogene Proteins c-myc; Survival Rate; Tissue Array Analysis; Tumor Suppressor Protein p53; Wnt1 Protein; Young Adult

Lung cancer is the leading cause of cancer-related mortality worldwide. Early detection or prevention strategies are urgently needed to increase survival. Hyperplasia is the first morphologic change that occurs in the bronchial epithelium during lung cancer development, followed by squamous metaplasia, dysplasia, carcinoma in situ, and invasive tumor. This study was designed to determine the molecular mechanisms that control bronchial epithelium hyperplasia. Using primary normal human tracheobronchial epithelial (NHTBE) cells cultured by using the 3-dimensional (3D) organotypic method, we found that the epidermal growth factor receptor (EGFR) ligands, EGF, TGF-α, and amphiregulin induced hyperplasia, as determined by cell proliferation and multilayered epithelium formation. We also found that EGF induced increased cyclin D1 expression, which plays a critical role in bronchial hyperplasia; this overexpression was mediated by activating the mitogen-activated protein kinase pathway but not the phosphoinositide 3-kinase/Akt signaling pathway. Erlotinib, an EGFR tyrosine kinase inhibitor, and U0126, a MAP/ERK kinase (MEK) inhibitor, completely inhibited EGF-induced hyperplasia. Furthermore, a promoter analysis revealed that the activator protein-1 transcription factor regulates EGF-induced cyclin D1 overexpression. Activator protein-1 depletion by using siRNA targeting its c-Jun component completely abrogated EGF-induced cyclin D1 expression. In conclusion, we showed that bronchial hyperplasia can be modeled in vitro by using primary NHTBE cells maintained in a 3D organotypic culture. EGFR and MEK inhibitors completely blocked EGF-induced bronchial hyperplasia, suggesting that they have a chemopreventive role.

Topics: Bronchi; Butadienes; Cell Line, Tumor; Cyclin D1; Enzyme Inhibitors; ErbB Receptors; Erlotinib Hydrochloride; Humans; Hyperplasia; Luciferases; Lung Neoplasms; MAP Kinase Kinase Kinases; Models, Biological; Nitriles; Organ Culture Techniques; Quinazolines; RNA, Small Interfering

Lung cancer is the leading cause of cancer death in the majority of developed countries. Uncontrolled cell proliferation is the hallmark of malignant tumours. Cyclins play an important role in cell cycle regulation. The aim of this study was to evaluate the expression of cyclins A, B1, D1 and E in advanced non-small cell lung cancer (stages IIIB-IV) with its prognostic significance.. An immunohistochemical assessment of cyclins A, B1, D1 and E expression was performed in the paraffin-embedded tumor tissues of 19 patients (9 men and 10 women). The mean was age 59 +/- 6.64 years. 9 patients were in IIIB and 10 in IV. The 2-years survival rate was evaluated.. We showed positive cyclin A expression in 13 tumor tissue specimens (68%), cyclin B1 in 3 (16%), cyclin D1 in 9 (47%) and cyclin E in 7 (37%). We analyzed the prognostic value of examinated cyclins in all NSCLC patients and separately in patients with squamous cell lung cancer and adenocarcinoma and in patients in stage IIIB and IV, but we have no found any correlations. We did not find also any differences in examinated cyclins expression depending on stages nor different histopathological types.. We did not observe prognostic value of cyclins A, B1, D1 or E expression in advanced non-small cell lung cancer.

Topics: Adenocarcinoma; Biomarkers, Tumor; Carcinoma, Non-Small-Cell Lung; Carcinoma, Squamous Cell; Cyclin A; Cyclin B1; Cyclin D1; Cyclin E; Cyclins; Female; Humans; Immunohistochemistry; Lung Neoplasms; Male; Middle Aged; Neoplasm Staging; Prognosis; Survival Rate

Exposure to cadmium is associated with the development of pulmonary damage such as emphysema and lung cancer. This metal is also a powerful inducer of different proinflammatory and cell cycle regulatory proteins in many biologic models. Previously, we showed that prolonged exposure of low concentration of cadmium resulted in upregulation of proinflammatory cytokines and cell cycle regulatory molecules in mice lung cell. The present study was undertaken to determine molecular mechanism of inflammation and its relation to cell proliferation in a transformed human lung adenocarcinoma epithelial cell line (A549) in response to cadmium chloride. In comparative studies, we examine that short-duration exposure to lower doses of cadmium significantly increase the growth of A549 cells, whereas higher doses are toxic and cause cell death. We also observed that cadmium induced elevated expression of epidermal growth factor receptor (EGFR) along with different proinflammatory cytokines like interleukin-1 beta (IL-1β), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6). The possible occurrence of cell proliferation events was evaluated via analysis of the physical state of the DNA and the expression of Ki-67 and proliferating cell nuclear antigen (PCNA). We also checked the pattern of expression of different cell cycle regulatory molecules involved in the onset of cell proliferation. Our results indicate that cadmium treatment appears to induce inflammatory and growth responses in transformed A549 cell line by activating EGFR and its downstream modulators. These results may contribute to better understand the toxic mechanism of cadmium; moreover, the expression profile of cadmium-induced regulatory molecules could provide potential biomarkers for cadmium exposure.

Topics: Adenocarcinoma; Antineoplastic Agents; Cadmium Chloride; Cell Cycle; Cell Death; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclooxygenase 2; Dose-Response Relationship, Drug; Environmental Pollutants; ErbB Receptors; Gefitinib; Gene Expression Regulation, Neoplastic; Humans; Ki-67 Antigen; Lethal Dose 50; Lung Neoplasms; Microscopy, Acoustic; Proliferating Cell Nuclear Antigen; Quinazolines; Up-Regulation

Lung cancer has limited effective therapy and no effective prevention. Cytotoxic chemotherapy has not improved when combined with the epidermal growth factor receptor (EGFR) inhibitor erlotinib (standard lung cancer therapy) or with the rexinoid bexarotene. Combining erlotinib and bexarotene, however, to cotarget cyclin D1 via the retinoid X receptor and EGFR was active preclinically in KRAS-driven lung cancer cells derived from transgenic mice and in two clinical studies in lung cancer (including wild-type EGFR tumors, with or without KRAS mutations), as reported in this issue of the journal by Dragnev and colleagues (beginning on page 818). These results, along with closely related clinical results of the BATTLE program, support the promise of this cotargeting approach for lung cancer prevention and therapy and of cyclin D1 as a predictive, personalizing marker for it.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Cyclin D1; Humans; Lung Neoplasms; Mice; Mutation; Proto-Oncogene Proteins; Proto-Oncogene Proteins p21(ras); ras Proteins; Treatment Outcome

To explore the potential of the anti-sense nucleic acid of CyclinD1 in lung cancer therapy, the expression vector containing the anti-sense nucleic acid of CyclinD1 was constructed and named pcDNA3.1-CyclinD1. The A549 cells were transfected with pcDNA3.1-CyclinD1 vectors. After being screened by G418, the stable expression positive clones were obtained. MTT method and flow cytometry technique were used to detect cell proliferation and apoptosis, respectively. The results showed the transfected cells exhibited significantly increased apoptosis and inhibited cell growth, compared with negative control and empty vector groups. To investigate the mechanism for anti-sense nucleic acid of CyclinD1 inducing A549 cells apoptosis, the expression levels of retinoblastoma protein (pRb), adenovirus E2 factor-1 (E2F-1), vascular endothelial growth factor (VEGF), matrix metalloproteinase (MMP)-2 and MMP-9 were detected by Western blot, and the results showed the expressions of these proteins were all decreased significantly in anti-sense nucleic acid of CyclinD transfected group, compared with those in negative control and empty vector groups. In a word, anti-sense nucleic acid of CyclinD1 induces the apoptosis of lung adenocarcinoma cancer cells, and the depressions of pRb, E2F-1, VEGF, MMP-2 and MMP-9 expressions may be the possible mechanism.

Topics: Adenocarcinoma; Apoptosis; Cell Line, Tumor; Cyclin D1; DNA, Antisense; Genetic Vectors; Humans; Lung Neoplasms; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Recombination, Genetic; Retinoblastoma Protein; Transfection; Vascular Endothelial Growth Factor A

The phosphoinositide 3-kinase (PI3K)-mammalian target of rapamycin (mTOR) signaling axis has emerged as a novel target for cancer therapy. Agents that inhibit PI3K, mTOR or both are currently under development. The mTOR allosteric inhibitor, RAD001, and the PI3K/mTOR dual kinase inhibitor, BEZ235, are examples of these agents. We were interested in developing strategies to enhance mTOR-targeted caner therapy. In this study, we found that BEZ235 alone effectively inhibited the growth of rapamycin-resistant cancer cells. Interestingly, the combination of sub-optimal concentrations of RAD001 and BEZ235 exerted synergistic inhibition of the growth of human lung cancer cells along with induction of apoptosis and G1 arrest. Furthermore, the combination was also more effective than either agent alone in inhibiting the growth of lung cancer xenografts in mice. The combination showed enhanced effects on inhibiting mTOR signaling and reducing the expression of c-Myc and cyclin D1. Taken together, our results suggest that the combination of RAD001 and BEZ235 is a novel strategy for cancer therapy.

Topics: Animals; Antineoplastic Agents; Apoptosis; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Down-Regulation; Drug Resistance, Neoplasm; Drug Synergism; Eukaryotic Initiation Factor-4F; Everolimus; Female; G1 Phase; Humans; Imidazoles; Lung Neoplasms; Mice; Proto-Oncogene Proteins c-myc; Quinolines; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Xenograft Model Antitumor Assays

Inorganic arsenic, a ubiquitous environmental contaminant, is associated with an increased risk of cancer. There are several hypotheses regarding arsenic-induced carcinogenesis. The mechanism of action remains obscure, although hyper-proliferation of cells is involved. In the present study, the molecular mechanisms underlying the proliferation and malignant transformation of human embryo lung fibroblast (HELF) cells induced by a low concentration of arsenite were investigated. The results reveal that a low concentration of arsenite induces cell proliferation and promotes cell cycle transition from the G(1) to the S phase. Moreover, arsenite activates the JNK1/c-Jun signal pathway, but not JNK2, which up-regulates the expression of cyclin D1/CDK4 and phosphorylates the retinoblastoma (Rb) protein. Blocking of the JNK1/c-Jun signal pathway suppresses the increases of cyclin D1 expression and Rb phosphorylation, which attenuates cell proliferation, reduces the transition from the G1 to the S phase, and thereby inhibits the neoplastic transformation of HELF cells induced by a low concentration of arsenite. Thus, activation of the JNK1/c-Jun pathway up-regulates the expression of cyclin D1, which is involved in the tumorigenesis caused by a low concentration of arsenite.

Topics: Animals; Anticarcinogenic Agents; Arsenites; Carcinogens, Environmental; Cell Line; Cell Proliferation; Cell Transformation, Neoplastic; Cyclin D1; Fibroblasts; G1 Phase; Humans; Lung; Lung Neoplasms; Mice; Mice, Nude; Mitogen-Activated Protein Kinase 8; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-jun; RNA Interference; RNA, Small Interfering; Signal Transduction; Sodium Compounds; Up-Regulation

Inhibition of cell-cycle progression is a target for the treatment of cancer. 3-Oxoolean-12-en-27-oic acid (3-OOLA) has shown significant anticancer activity towards diverse cancer cells, but has not been investigated for non-small cell lung carcinoma (NSCLC) cells. In this study, we investigated the antiproliferative effect of 3-OOLA in NSCLC cell lines and its underlying mechanism.. The MTT assay, bromodeoxyuridine (BrdU) incorporation assay, and flow cytometry were used for cell proliferation studies, and annexin V staining for apoptotic effects. Western blot analysis was used to evaluate expression of cell-cycle regulatory proteins, such as cyclins and cyclin-dependent kinases (CDKs).. 3-OOLA caused G0/G1 phase cell-cycle arrest without inducing apoptosis in NSCLC cells, and Western blot analyses demonstrated down-regulation of cyclin D1, cyclin E and phosphorylated Rb.. 3-OOLA inhibits cell proliferation of NSCLC cells by inducing cell-cycle arrest at G0/G1 through down-regulation of cyclin D1 and cyclin E.

Topics: Carcinoma, Non-Small-Cell Lung; Cell Growth Processes; Cell Line, Tumor; Cyclin D1; Cyclin E; Down-Regulation; G1 Phase; Humans; Lung Neoplasms; Oleanolic Acid; Resting Phase, Cell Cycle; Retinoblastoma Protein

Two canine mammary gland tumor (MGT) cell lines, CHMp-5b and -13a, were transplanted into nude mice, and their tumor development and metastatic potential were evaluated. In addition, NF-κB, Ki-67 and cyclin D1 expressions in the tumor masses were evaluated, and the relationship between these expressions and malignancy of the tumors developed in nude mice was investigated. Lower activation of NF-κB was positively correlated with a lower potential of metastasis and better prognosis in nude mice xenografted with CHMp-13a compared with CHMp-5b. Then, CHMp-5b cells were treated with BAY11-7082, an inhibitor of NF-κB, and the expressions of I-κBα, p-I-κBα, cyclin D1 and Bcl-2 in these cells were evaluated by Western blot analysis. In addition, BAY11-7082 at a dose of 6 mg/kg was administered intraperitoneally to nude mice xenografted with CHMp-5b, and relationships between tumor growth and lung and lymph node metastasis in nude mice and NF-κB, I-κBα and p-I-κBα expressions in tissues of these mice were evaluated. BAY11-7082 treatment induced decreased expressions of p-I-κBα, cyclin D1 and Bcl-2 in a dose- and time-dependent manner, suggesting that BAY inhibited NF-κB in this cell line. CHMp-5b-xenografted mice treated with BAY11-7082 showed a reduction in tumor growth and metastasis. Therefore, inhibition of the NF-κB signaling pathway may be a promising novel therapeutic approach for canine MGTs.

Topics: Animals; Cell Line, Tumor; Cyclin D1; Dogs; Female; Gene Expression Regulation, Neoplastic; Ki-67 Antigen; Lung Neoplasms; Mammary Neoplasms, Animal; Mice; Mice, Nude; Neoplasms, Experimental; NF-kappa B

High-fat diets (HFDs) are known to cause obesity and are associated with breast cancer progression and metastasis. Because obesity is associated with breast cancer progression, it is important to determine whether dietary fat per se stimulates breast cancer progression in the absence of obesity. This study investigated whether an HFD increases breast cancer growth and metastasis, as well as mortality, in obesity-resistant BALB/c mice.. The 4-week-old, female BALB/c mice were fed HFD (60% kcal fat) or control diet (CD, 10% kcal fat) for 16 weeks. Subsequently, 4T1 mammary carcinoma cells were injected into the inguinal mammary fat pads of mice fed continuously on their respective diets. Cell-cycle progression, angiogenesis, and immune cells in tumor tissues, proteases and adhesion molecules in the lungs, and serum cytokine levels were analyzed with immunohistochemistry, Western blotting, and enzyme-linked immunosorbent assay (ELISA). In vitro studies were also conducted to evaluate the effects of cytokines on 4T1 cell viability, migration, and adhesion.. Spleen and gonadal fat-pad weights, tumor weight, the number and volume of tumor nodules in the lung and liver, and tumor-associated mortality were increased in the HFD group, with only slight increases in energy intake and body weight. HF feeding increased macrophage infiltration into adipose tissues, the number of lipid vacuoles and the expression of cyclin-dependent kinase (CDK)2, cyclin D1, cyclin A, Ki67, CD31, CD45, and CD68 in the tumor tissues, and elevated serum levels of complement fragment 5a (C5a), interleukin (IL)-16, macrophage colony-stimulating factor (M-CSF), soluble intercellular adhesion molecule (sICAM)-1, tissue inhibitors of metalloproteinase (TIMP)-1, leptin, and triggering receptor expressed on myeloid cells (TREM)-1. Protein levels of the urokinase-type plasminogen activator, ICAM-1, and vascular cell adhesion molecule-1 were increased, but plasminogen activator inhibitor-1 levels were decreased in the lungs of the HFD group. In vitro assays using 4T1 cells showed that sICAM-1 increased viability; TREM-1, TIMP-1, M-CSF, and sICAM-1 increased migration; and C5a, sICAM-1, IL-16, M-CSF, TIMP-1, and TREM-1 increased adhesion.. Dietary fat increases mammary tumor growth and metastasis, thereby increasing mortality in obesity-resistant mice.

Topics: Animals; Antigens, CD; Antigens, Differentiation, Myelomonocytic; Body Weight; Cell Movement; Cell Proliferation; Complement C5a; Cyclin A; Cyclin D1; Cyclin-Dependent Kinase 2; Cytokines; Dietary Fats; Energy Intake; Female; Interleukin-16; Ki-67 Antigen; Leptin; Leukocyte Common Antigens; Liver Neoplasms; Lung; Lung Neoplasms; Mammary Neoplasms, Experimental; Mice; Mice, Inbred BALB C; Neovascularization, Pathologic; Obesity; Platelet Endothelial Cell Adhesion Molecule-1

Cyclin D1 (CCND1) is critical in the transition of the cell cycle from G1 to S phase and unbalanced cell cycle regulation is a hallmark of carcinogenesis. The study aimed at investigating the association of CCND1 genotypes with lung cancer risk in Taiwan and examining the interaction between CCND1 genotype and smoking habit.. CCND1 A870G (rs9344) and C1722G (rs678653) genotypes were determined by polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analysis of DNA from the blood of 358 lung cancer patients and 716 cancer-free healthy controls.. The results showed that there were significant differences between lung cancer and control groups in the distribution of the genotypes (p=0.0003) and allelic frequency (p=0.0007) in the CCND1 rs9344 genotype. Individuals who carried AG or GG genotype had 0.59- and 0.52-fold risk, respectively, of developing lung cancer compared to those who carried the AA genotype (95% CI=0.44-0.78 and 0.35-0.79, respectively). There was also an obvious interaction of CCND1 rs9344 genotype with personal smoking habit on lung cancer risk (p=0.0009).. These findings support the conclusion that cell cycle regulation may play a role in lung cancer development and that CCND1 rs9344 polymorphism together with smoking habit maybe a useful biomarker for lung cancer prediction.

Topics: Case-Control Studies; Cyclin D1; Female; Gene Frequency; Genetic Predisposition to Disease; Humans; Lung Neoplasms; Male; Middle Aged; Polymorphism, Single Nucleotide; Smoking; Taiwan

Lung cancer is a major cause of mortality and morbidity worldwide. Galectin-3 is multifunctional protein, which is involved in regulation of cell growth, cell adhesion, cell proliferation, angiogenesis and apoptosis. Cyclin D1 together with other cyclin plays an important role in cell cycle control. Cyclin D1 regulates the G1-to-S phase transition. The aim of this study was the evaluation of correlations between clinicopathological findings and cyclin D1 and galectin-3 expression in non-small cell lung cancer (NSCLC). We wanted also to analyze the prognostic value of cyclin D1 and galectin-3 expression. Moreover we tried to evaluate the correlations between galectin-3 and cyclin D1 expression in tumor tissue.. We used the immunochemistry method to investigate the expression of galectin-3 and cyclin D1 in the paraffin-embedded tumor tissue of 47 patients (32 men and 15 women; mean age 59.34 ± 8.90). years. We used monoclonal antibodies to cyclin D1 (NCL-L-cyclin D1-GM clone P2D11F11 NOVO CASTRA) and to galectin-3 (mouse monoclonal antibody NCL-GAL3 NOVO CASTRA).. Galectin-3 expression was positive in 18 cases (38.29%) and cyclin D1 in 39 (82.97%). We showed only weak trend, that galectin-3 expression was lower in patients without lymph node involvement (p = 0.07) and cyclin D1 expression was higher in this group (p = 0.080). We didn't reveal differences in cyclin D1 and galectin-3 expression in SCC and adenocarcinoma patients. We didn't demonstrated also differences in galectin-3 and cyclin D1 expression depending on disease stage. Moreover we analyzed the prognostic value of cyclin D1 expression and galectin-3 in all examinated patients and separately in SCC and in adenocarcinoma and in all stages, but we didn't find any statistical differences. We demonstrated that in galectin-3 positive tumors cyclin D1 expression was higher (96.55% vs 61.11%, Chi2 Yatesa 7.53, p = 0.0061) and we revealed negative correlation between cyclin D1 and galectin-3 expression (R Spearman -0.458, p = 0.0011). In squamous cell lung cancer we didn't observed correlations between these both examinated markers (R = -0.158, p = 0.460), and in adenocarcinoma the negative correlation was very strong (R = -0.829 p = 0.000132).. We didn't reveal any important correlations between clinicopathological findings and galectin-3 and cyclin D1 expression and in non small cell lung cancer. We didn't observed also prognostic value of cyclin D1 or galectin-3 expression. But we showed higher cyclin D1 expression in galectin-3 negative tumor tissues. We revealed also differences in correlations between galectin-3 and cyclin D1 expression in two main histopathological types of NSCLC.

Topics: Aged; Carcinoma, Non-Small-Cell Lung; Cyclin D1; Female; Galectin 3; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Male; Middle Aged; Neoplasm Staging; Prognosis

Cyclin D1 overexpression exists in multiple types of cancer and is a potential chemopreventive or therapeutic target.. Non-small cell lung cancer and mesothelioma cells were incubated with antisense oligonucleotides (ASO) to cyclin D1 (CD1) and evaluated for effects on cellular proliferation, apoptosis, expression of cell cycle-specific proteins, and protein phosphorylation states.. ASO to CD1 inhibited proliferation of non-small lung cancer cells and mesothelioma cells. ASO induced apoptosis as determined by TUNEL assay. Western blot analysis of cell lysate showed that ASO inhibited the de novo synthesis of CD1, CD3, and CDK2 in multiple cell lines. Immunoprecipitation and immunoblotting with phosphoantibodies demonstrated that CD1, CD3, and CDK2 exist in a phosphorylated state.. The work demonstrates that non-small cell lung cancer and mesothelioma cells respond to ASO-mediated cellular growth inhibition. These findings make ASO to CD1 attractive as a potential therapeutic for mesothelioma and non-small cell lung cancer.

Topics: Apoptosis; Blotting, Western; Carcinoma, Non-Small-Cell Lung; Cell Cycle; Cell Cycle Proteins; Cell Proliferation; Cyclin D1; Humans; Immunoprecipitation; Lung Neoplasms; Mesothelioma; Oligonucleotides, Antisense; Phosphorylation; Tumor Cells, Cultured

The role of estrogens in the increased risk of lung adenocarcinoma in women remains uncertain. We reported that lung adenocarcinoma cell lines from female, but not male, patients with non-small cell lung cancer respond proliferatively and transcriptionally to estradiol (E(2)), despite equal protein expression of estrogen receptors (ER) alpha and beta. To test the hypothesis that nuclear localization of ER alpha corresponds to genomic E(2) activity in lung adenocarcinoma cells from females, cell fractionation, immunoblot, and confocal immunohistochemical microscopy were performed. We report for the first time that E(2) increases phospho-serine-118-ER alpha (P-ser118-ER alpha) and cyclin D1 (CCND1) nuclear colocalization in H1793, but not A549 lung adenocarcinoma cells, derived from a female and male patient, respectively. ER beta was primarily in the cytoplasm and mitochondria, independent of E(2) treatment, and showed no difference between H1793 and A549 cells. E(2) induced higher transcription of endogenous ER alpha-regulated CCND1 in H1793 than in A549 cells. Likewise, higher rapid, non-genomic E(2)-induced extracellular signal-regulated kinase 1/2 activation was detected in H1793 compared with A549 cells, linking extracellular signal-regulated kinase activation to increased P-ser118-ER alpha. Furthermore, E(2) increased cyclin D1 and P-ser118-ER alpha nuclear localization in H1793, but not A549 cells. Together, our results indicate that nuclear localization of P-ser118-ER alpha provides one explanation for sex-dependent differences in E(2)-genomic responses in lung adenocarcinoma cell lines.

Topics: Adenocarcinoma; Cell Line, Tumor; Cell Nucleus; Cyclin D1; Enzyme Activation; Estradiol; Estrogen Receptor alpha; Estrogen Receptor beta; Female; Fluorescence; Fulvestrant; Genome, Human; Humans; Lung Neoplasms; Male; Microscopy, Confocal; Mitogen-Activated Protein Kinases; Mutant Proteins; Phosphoserine; Protein Transport; Sex Characteristics; Signal Transduction; Subcellular Fractions; Tamoxifen

The CT120 gene had been proven to be a novel gene closely related to pulmonary carcinogenesis and cancer progression. Our aim was to explore the mechanism of growth suppression caused by silencing CT120.. CT120 was detected in lung cancer tissues and the cell line A549, and the cell clones for silencing CT120 were obtained. Then the target genes were detected and the downstream proteins from the silencing of CT120 were separated and identified.. The expression of CT120 was higher in lung cancer tissues and A549 cells. Silencing of CT120 was shown to inhibit cell growth, reduce the expression of cyclin D1 and Cdk4, and increase the expression of p53 and caspase-3. The differential proteins were related to carcinogenesis, invasiveness, and metastasis.. CT120 may play an important role in tumor progression, and the down-regulation of CT120 expression could be a new drug target candidate in the treatment of lung cancer.

Topics: Apoptosis; Caspase 3; Cell Division; Cell Line, Tumor; Cyclin D1; Cyclin-Dependent Kinase 4; Disease Progression; Gene Silencing; Humans; Lung Neoplasms; Membrane Proteins; Neoplasm Proteins; Oligoribonucleotides, Antisense; Tumor Suppressor Protein p53

Indole-3-carbinol (I3C) has anti-tumor effects in various cancer cell lines. However, the anti-tumor effect of I3C on human lung cancers has been rarely reported. We investigated the anti-tumor effects and its mechanism of I3C on human lung carcinoma A549 cell line. Treatment of the A549 cells with I3C significantly reduced cell proliferation, increased formations of fragmented DNA and apoptotic body, and induced cell cycle arrest at G0/G1 phase. I3C increased not only the protein levels of cyclin D1, phosphorylated p53, and p21 but also the expression of Fas mRNA. Cleavage of caspase-9, -8, -3 and PARP also was increased by I3C. Treatment with wortmannin significantly suppressed both I3C-induced Ser15 phosphorylation and accumulation of p53 protein. The inhibition of caspase-8 by z-IETD-FMK significantly decreased cleavage of procaspase-8,-3 and PARP in I3C-treated A549 cells. Taken together, these results demonstrate that I3C induces cell cycle arrest at G0/G1 through the activation of p-p53 at Ser 15 and induces caspase-8 mediated apoptosis via the Fas death receptor. This molecular mechanism for apoptotic effect of I3C on A549 lung carcinoma cells may be a first report and suggest that I3C may be a preventive and therapeutic agent against lung cancer.

Topics: Anticarcinogenic Agents; Apoptosis; Blotting, Western; Caspase 8; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cyclin D1; DNA Repair; Enzyme Activation; Fas Ligand Protein; Flow Cytometry; Genes, p53; Humans; Indicators and Reagents; Indoles; Lung Neoplasms; Phosphorylation; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction

Wnt/beta-catenin signaling plays an important role not only in cancer, but also in cancer stem cells. In this study, we found that beta-catenin and OCT-4 was highly expressed in cisplatin (DDP) selected A549 cells. Stimulating A549 cells with lithium chloride (LiCl) resulted in accumulation of beta-catenin and up-regulation of a typical Wnt target gene cyclin D1. This stimulation also significantly enhanced proliferation, clone formation, migration and drug resistance abilities in A549 cells. Moreover, the up-regulation of OCT-4, a stem cell marker, was observed through real-time PCR and Western blotting. In a reverse approach, we inhibited Wnt signaling by knocking down the expression of beta-catenin using RNA interference technology. This inhibition resulted in down-regulation of the Wnt target gene cyclin D1 as well as the proliferation, clone formation, migration and drug resistance abilities. Meanwhile, the expression of OCT-4 was reduced after the inhibition of Wnt/beta-catenin signaling. Taken together, our study provides strong evidence that canonical Wnt signaling plays an important role in lung cancer stem cell properties, and it also regulates OCT-4, a lung cancer stem cell marker.

Topics: beta Catenin; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin D1; Drug Resistance, Neoplasm; Gene Knockdown Techniques; Humans; Lithium Chloride; Lung Neoplasms; Neoplastic Stem Cells; Octamer Transcription Factor-3; Signal Transduction; Wnt Proteins

Toona sinensis is a traditional Chinese herb, and the extracts of T. sinensis leaf possess a variety of biological functions. This study attempted to test the antiproliferative effect of TSL-1 (a bioactive fraction of T. sinensis) in H441 cells (lung adenocarcinoma). The data showed that the antiproliferative effect of TSL-1 on H441 cells is prominent using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. TSL-1-induced apoptosis was confirmed by cell morphology, sub-G(1) peak accumulation, cleavage of poly(ADP)-ribose polymerase, and propidium iodide-annexin V double staining. Furthermore, decreased Bcl-2 accompanied by increased Bax (in western blotting) was found with TSL-1 treatment of H441 cells. TSL-1 treatment-induced G(1) arrest was concurrent with the down-regulation of protein levels of cyclin D1 and cyclin-dependent kinase 4 in H441 cells. Peroral and intraperitoneal administrations of TSL-1 were performed to evaluate the therapeutic efficacy, and peroral administration of TSL-1 was also used to elucidate the therapeutic efficacy in the H441 cell xenograft model in vivo. The data revealed that TSL-1 treatment inhibited H441 tumor growth in both therapeutic and preventive experiments. Taken together, these results demonstrate that TSL-1 possesses the capability of preventing and alleviating lung cancer proliferation in vitro and in vivo with proven nephrological and hepatic safety and has the potential to be developed as an anti-lung cancer drug.

Topics: Adenocarcinoma; Animals; Antineoplastic Agents, Phytogenic; Apoptosis; bcl-2-Associated X Protein; Cedrela; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase 4; Down-Regulation; Humans; Lung Neoplasms; Mice; Mice, Inbred BALB C; Mice, Nude; Phytotherapy; Plant Extracts; Plant Leaves; Proto-Oncogene Proteins c-bcl-2; Xenograft Model Antitumor Assays

Hypoxia-inducible factor (HIF) and cyclin D1 are both key mediators of cell growth and proliferation in normal and cancer cells. However, the interrelation between HIF and cyclin D1 remains unclear. In the present study, we observed the inverse correlation between cyclin D1 and HIF-1 in hypoxia condition. Overexpression of the dominant negative mutant of HIF-1alpha (DN-HIF) significantly enhanced cyclin D1 expression upon hypoxia or arsenite exposure, suggesting the negative regulation of cyclin D1 by HIF-1. Furthermore, we found that the impairment of HIF-1 increased cyclin D1 expression in A549 pulmonary cancer cells, which in turn promoted G1-S cell cycle transition and cell proliferation. Cyclin D1 expression was increased in s.c. xenograft of DN-HIF stably transfected A549 cells in nude mice compared with that of control cells. Chromatin immunoprecipitation assay revealed that HIF-1 was able to directly bind to the promoter region of cyclin D1, which indicates that the negative regulation of cyclin D1 by HIF-1 is through a direct mechanism. Inhibition of histone deacetylase (HDAC) by pretreatment of cells with trichostatin A or specific knockdown of HDAC7 by its shRNA antagonized the suppression of cyclin D1 by HIF-1, suggesting that HDAC7 is required for HIF-1-mediated cyclin D1 downregulation. Moreover, we found that 5-fluorouracil-triggered apoptosis of DN-HIF-transfected A549 cells was reduced by sicyclin D1 (cyclin D1-specific interference RNA) introduction, suggesting that clinical observation of HIF-1 overexpression-associated chemoresistance might be, at least partially, due to the negative regulation of cyclin D1.

Topics: Animals; Apoptosis; Cell Growth Processes; Cell Hypoxia; Cell Line, Tumor; Cyclin D1; Fibroblasts; Fluorouracil; G1 Phase; Humans; Hypoxia-Inducible Factor 1; Lung Neoplasms; Mice; S Phase

Although anticancer effect of gambogic acid (GA) and its potential mechanisms were well documented in past decades, limited information is available on the anticancer effect of gambogenic acid (GNA), another major active component of Gamboge. Here we performed a study to determine whether GNA possesses anticancer effect and find its potential mechanisms. The results suggested that GNA significantly inhibited the proliferation of several tumor cell lines in vitro and in vivo. Treatment with GNA dose and time dependently induced A549 cells apoptosis, arrested the cells to G0/G1 phase in vitro and down-regulated the expression of cyclin D1 and cyclooxygenase (COX)-2 in mRNA level. In addition, anticancer effect was further demonstrated by applying xenografts in nude mice coupled with the characteristic of apoptosis in the GNA treated group. Taken together, these observations might suggest that GNA inhibits tumor cell proliferation via apoptosis-induction and cell cycle arrest.

Topics: Adenocarcinoma; Animals; Antineoplastic Agents, Phytogenic; Apoptosis; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclooxygenase 2; Dose-Response Relationship, Drug; Down-Regulation; Female; Garcinia; Gene Expression Regulation; Humans; Lung Neoplasms; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Phytotherapy; Plant Extracts; Resins, Plant; RNA, Messenger; Terpenes; Xanthenes; Xanthones; Xenograft Model Antitumor Assays

Anticancer effects of beta-lapachone (beta-lap) are due to generation of ROS and metabolic catastrophes as a result of NAD(P)H:quinone oxidoreductase (NQO1)-mediated futile cycling between the oxidized and reduced forms of beta-lap. It has been shown that NQO1 is also essential for the TNF-induced activation of NF-kappaB and that beta-lap suppresses the TNF-induced NF-kappaB activation. We investigated whether or not NQO1 is involved and beta-lap suppresses the radiation-induced NF-kappaB activation using A549 human lung cancer cells and NQO1-knock down A549 cells (shNQO1 A549 cells). Irradiation with 4 Gy markedly increased the DNA binding activity of NF-kappaB in A549 cells, but not in the shNQO1 A549 cells, thus demonstrating that NQO1 plays a pivotal role in irradiation-induced NF-kappaB activation. Treatment with 10 micronM beta-lap for 4 h almost completely abrogated the radiation-induced increase in NF-kappaB activation and the transcription of NF-kappaB target genes such as bcl2, gadd45beta and cyclinD1. Moreover, beta-lap markedly suppressed the activation of IkappaB kinase gamma (IKKgamma) and the subsequent phosphorylation of IkappaBalpha, thereby inhibiting NF-kappaB activation. It is concluded that beta-lap suppresses the radiation-induced activation of NF-kappaB by interrupting the involvement of NQO1 in the activation of NF-kappaB, thereby inhibiting the transcription of survival signals. The radiosensitization caused by beta-lap may, in part, be attributed to beta-lap-induced suppression of NF-kappaB activation.

Topics: Antigens, Differentiation; Cell Line, Tumor; Cell Survival; Cyclin D1; Humans; I-kappa B Kinase; Lung Neoplasms; NAD(P)H Dehydrogenase (Quinone); Naphthoquinones; NF-kappa B; Proto-Oncogene Proteins c-bcl-2; Radiation-Sensitizing Agents; Radiation, Ionizing

Correct diagnosis of pleural effusion (PE) as either benign or malignant is crucial, although conventional cytological evaluation is of limited diagnostic accuracy, with relatively low sensitivity rates.. We identified biological markers accurately detected in a simple PE examination. We analysed data from 19 patients diagnosed with lung cancer (nine adeno-Ca, five non-small-cell Ca (not specified), four squamous-cell Ca, one large-cell Ca) and 22 patients with benign inflammatory pathologies: secondary to trauma, pneumonia or TB.. Pleural effusion concentrations of seven analysed biological markers were significantly lower in lung cancer patients than in benign inflammatory patients, especially in matrix metalloproteinase (MMP)-9, MMP-3 and CycD1 (lower by 65% (P<0.000003), 40% (P<0.0007) and 34% (P<0.0001), respectively), and in Ki67, ImAnOx, carbonyls and p27. High rates of sensitivity and specificity values were found for MMP-9, MMP-3 and CycD1: 80 and 100%; 87 and 73%; and 87 and 82%, respectively.. Although our results are of significant merit in both the clinical and pathogenetic aspects of lung cancer, further research aimed at defining the best combination for marker analysis is warranted. The relative simplicity in analysing these markers in any routine hospital laboratory may result in its acceptance as a new diagnostic tool.

Topics: Adenocarcinoma; Aged; Biomarkers; Carcinoma, Large Cell; Carcinoma, Non-Small-Cell Lung; Carcinoma, Squamous Cell; Cyclin D1; Female; Humans; Ki-67 Antigen; Lung Neoplasms; Male; Matrix Metalloproteinase 3; Matrix Metalloproteinase 9; Middle Aged; Pleural Effusion; Pneumonia

Overexpression of p70S6K in breast cancer patients is associated with aggressive disease and poor prognosis. Recent studies showed that patients with breast cancer with increased p70S6K phosphorylation had poor survival and increased metastasis. The purpose of our study was to determine whether knockdown of p70S6K would inhibit cell growth, invasion, and metastasis in breast cancer. We therefore stably knocked down p70S6K expression in MDA-231, a highly metastatic breast cancer cell line, using a lentiviral short hairpin RNA (shRNA) based approach. Inhibition of p70S6K led to inhibition of cell growth, migration, and invasion in vitro. To determine the role of p70S6K in breast cancer tumorigenesis and metastasis, we used an MDA-231 orthotopic and metastatic animal model. In the orthotopic model, mice injected with MDA-231-p70S6K shRNA cells developed significantly smaller tumors than control mice injected with MDA-231 control shRNA cells (P < 0.01). No metastasis was observed in the p70S6K downregulated group, whereas lung metastasis was detected in all mice in the control group. To determine the role of p70S6K on growth and invasion, we tested downstream signaling targets by Western blot analysis. Knockdown of p70S6K inhibited phosphorylation of focal adhesion kinase, tissue transglutaminase 2, and cyclin D1 proteins, which promote cell growth, survival, and invasion. In addition, downregulation of p70S6K induced expression of PDCD4, a tumor-suppressor protein. In conclusion, we showed that p70S6K plays an important role in metastasis by regulating key proteins like cyclin D1, PDCD4, focal adhesion kinase, E-cadherin, beta-catenin, and tissue transglutaminase 2, which are essential for cell attachment, survival, invasion, and metastasis in breast cancer.

Topics: Animals; Antineoplastic Agents; beta Catenin; Breast Neoplasms; Cadherins; Carcinoma; Cyclin D1; Drug Delivery Systems; Female; Focal Adhesion Protein-Tyrosine Kinases; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Mice; Mice, Nude; Protein Glutamine gamma Glutamyltransferase 2; Protein Kinase Inhibitors; Ribosomal Protein S6 Kinases, 70-kDa; RNA, Small Interfering; Xenograft Model Antitumor Assays

Two primary prevention trials unexpectedly showed adverse effects of supplemental beta-carotene on lung cancer incidence in cigarette smokers. To elucidate the molecular mechanisms that might underlie these effects, we studied the immunohistochemical expression of cytochrome P450 1A1, 1A2, and 2E1, retinoic acid receptor beta, activated protein-1 elements, cyclin D1, and Ki67 in lung tumors and, when available, adjacent normal tissues obtained from incident cases in the Alpha-Tocopherol, Beta-Carotene Cancer Prevention Study. Archival lung tissue was available from 52 men randomized to receive 20 mg of beta-carotene per day and 30 men randomized to the placebo arm, all of whom were diagnosed with incident non-small-cell lung carcinoma during the course of the trial and subsequently underwent radical pulmonary resection. In normal-appearing bronchial epithelium, positive staining for cyclin D1 was observed in 23% of cases in the beta-carotene group and 0% of cases in the placebo group (based on only 3 of 13 versus 0 of 11 cases staining positively, however; P = 0.04), with no differences in expression noted in lung tumor tissue (P = 0.48). There were no statistically significant differences in Ki67 expression in normal or cancerous lung tissue between intervention groups, although a small increase in staining in tumors was noted among cases in the beta-carotene versus placebo group (88% versus 71% of cases stained positive, respectively; P = 0.13). Contrary to expectation, beta-carotene supplementation had no apparent effect on retinoic acid receptor-beta expression. These findings suggest that male smokers supplemented with beta-carotene may have had an increased risk of lung cancer due to aberrant cell growth, although our results are based on a relatively small number of cases and require confirmation in other completed trials of beta-carotene supplementation.

Topics: Aged; alpha-Tocopherol; beta Carotene; Biomarkers, Tumor; Carcinoma, Non-Small-Cell Lung; Cocarcinogenesis; Cyclin D1; Cytochromes; Dietary Supplements; Double-Blind Method; Humans; Ki-67 Antigen; Lung; Lung Neoplasms; Male; Middle Aged; Multicenter Studies as Topic; Neoplasm Proteins; Randomized Controlled Trials as Topic; Receptors, Retinoic Acid; Retrospective Studies; Smoking

MicroRNAs (miRNAs) are a group of small noncoding RNAs with modulator activity of gene expression. Deregulation of miRNA genes was found in several types of cancers. To explore the role of the miRNAs in Chinese lung squamous cell carcinoma (SCC), the expression profile of 711 miRNAs in SCC was analyzed. Total RNAs were used for hybridization on a commercially available array (miRCURY LNA array v.10.0), which contains 1,200 probes in tetramer, corresponding to 711 human miRNA genes. The results of miRNA microarray analysis were confirmed with quantitative real-time polymerase chain reaction. Seven human miRNAs (miR-126, miR-193a-3p, miR-30d, miR-30a, miR-101, let-7i, and miR-15a) were found to be significantly downregulated in lung SCC (P < 0.05), compared with normal lung tissues. The miRNAs miR-185 * and miR-125a-5p were significantly upregulated in lung SCC (P < 0.05), compared with normal lung tissues. The miRNA let-7i was downregulated in 9 of the 20 SCC samples, and miR-126 was downregulated in 16 of 20. The deregulation of some miRNAs in lung SCC suggests their possible involvement in the development and progression of SCC.

Topics: Carcinoma, Squamous Cell; Cyclin D1; Gene Expression Profiling; Humans; Lung Neoplasms; MicroRNAs; Oligonucleotide Array Sequence Analysis; Proto-Oncogene Proteins c-bcl-2; Reverse Transcriptase Polymerase Chain Reaction

A large group of interacting molecular factors, involved in epithelial-mesenchymal transition, epidermal growth factor receptor (EGFR) signaling, and G1 mitotic phase, are shown to play an important role in cancerogenesis and progression of non-small cell lung cancer (NSCLC). Since success concerning potential correlations, structural and numeric gene aberrations, and biological risk assessment of these molecular factors are still lacking, combined analysis of a multitude of intertwined factors is currently a promising approach.. Cyclins (D1, D2, D3, and E), p21, p27, EGFR, Snail, E-cadherin, beta-catenin, phosphatidylinositol-3' kinase, phosphatase and tensin homologue, phosphorylated Akt, and phosphorylated signal transducer, and activator of transcription-3 were analyzed by immunohistochemistry in 405 surgically resected NSCLC, using a standardized tissue microarray platform. In addition, the gene status of EGFR and cyclin D1 was examined by fluorescence in situ hybridization. Extensive clinical data were acquired, enabling detailed clinicopathologic correlation during a postoperative follow-up period of up to 14 years.. The protein overexpressions of nuclear p27, cyclin D1, cyclin D3, E-cadherin, and EGFR as assessed by immunohistochemistry were all associated with a significant reduction in overall survival time. In addition, cyclin D1 proved especially important, being the only independent molecular tumor-related factor with prognostic significance by multivariable analysis. In analogy to EGFR, recurrent numeric gene aberrations, particularly high-level amplifications, of cyclin D1 were obvious.. The results emphasize that deregulation of controlling factors of the early G1 phase is of significant oncogenic relevance and may represent a potential treatment target in NSCLC.

Topics: Adenocarcinoma; Adult; Aged; Aged, 80 and over; Carcinoma, Large Cell; Carcinoma, Non-Small-Cell Lung; Carcinoma, Squamous Cell; Cyclin D1; Cyclin D3; Cyclin-Dependent Kinase Inhibitor p27; Female; Humans; Immunoenzyme Techniques; In Situ Hybridization, Fluorescence; Intracellular Signaling Peptides and Proteins; Lung Neoplasms; Male; Middle Aged; Mitosis; Prognosis; Retrospective Studies; Survival Rate; Tissue Array Analysis; Young Adult

Stage IIIA non-small cell lung cancer (NSCLC) with ipsilateral mediastinal lymph node metastases (N2) is a heterogeneous disease with differing prognoses. In this study, we retrospectively investigated the prognostic value of the expression of 10 molecular markers in 87 patients with stage IIIA pN2 NSCLC treated with radical surgery.. Primary tumor tissue microarrays (TMAs) were constructed and sections used for immunohistochemical analysis of epidermal growth factor receptor, ErbB-2, c-kit, cyclooxygenase-2, survivin, bcl-2, cyclin D1, cyclin B1, metalloproteinase (MMP)-2, and MMP-9. Univariate and multivariate analyses and unsupervised hierarchical clustering analysis of clinical pathologic and immunostaining data were performed.. Bcl-2 (p < 0.0001) and cyclin D1 (p = 0.015) were more highly expressed in squamous cell carcinoma (SCC), whereas MMP-2 (p = 0.009), MMP-9 (p = 0.005), and survivin (p = 0.032) had increased expression in other histologic subtypes. In univariate analysis, SCC histology and cyclin D1 expressions were favorable prognostic factors (p = 0.015 and p < 0.0001, respectively); by contrast, MMP-9 expression was associated with worse prognosis (p = 0.042). In multivariate analysis, cyclin D1 was the only positive prognostic factor (p < 0.0001). Unsupervised hierarchical clustering analysis of TMA immunostaining data identified five distinct clusters. They formed two subsets of patients with better (clusters 1 and 2) and worse (clusters 3, 4, and 5) prognoses, and median survival of 51 and 10 months, respectively (p < 0.0001). The better prognosis subset mainly comprised patients with SCC (80%).. Hierarchical clustering of TMA immunostaining data using a limited set of markers identifies patients with stage IIIA pN2 NSCLC at high risk of recurrence, who may benefit from more aggressive treatment.

Topics: Adenocarcinoma; Adult; Aged; Biomarkers, Tumor; Carcinoma, Large Cell; Carcinoma, Non-Small-Cell Lung; Carcinoma, Squamous Cell; Cluster Analysis; Cyclin B1; Cyclin D1; Cyclooxygenase 2; ErbB Receptors; Female; Humans; Immunoenzyme Techniques; Inhibitor of Apoptosis Proteins; Lung Neoplasms; Male; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Microtubule-Associated Proteins; Middle Aged; Neoplasm Staging; Prognosis; Prospective Studies; Proto-Oncogene Proteins c-bcl-2; Receptor, ErbB-2; Retrospective Studies; Survival Rate; Survivin; Tissue Array Analysis

Improved understanding of lung cancer development and progression, including insights from studies of animal models, are needed to combat this fatal disease. Previously, we found that mice with a knockout (KO) of G-protein coupled receptor 5A (Gprc5a) develop lung tumors after a long latent period (12 to 24 months).. To determine whether a tobacco carcinogen will enhance tumorigenesis in this model, we administered 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) i.p. to 2-months old Gprc5a-KO mice and sacrificed groups (n=5) of mice at 6, 9, 12, and 18 months later. Compared to control Gprc5a-KO mice, NNK-treated mice developed lung tumors at least 6 months earlier, exhibited 2- to 4-fold increased tumor incidence and multiplicity, and showed a dramatic increase in lesion size. A gene expression signature, NNK-ADC, of differentially expressed genes derived by transcriptome analysis of epithelial cell lines from normal lungs of Gprc5a-KO mice and from NNK-induced adenocarcinoma was highly similar to differential expression patterns observed between normal and tumorigenic human lung cells. The NNK-ADC expression signature also separated both mouse and human adenocarcinomas from adjacent normal lung tissues based on publicly available microarray datasets. A key feature of the signature, up-regulation of Ube2c, Mcm2, and Fen1, was validated in mouse normal lung and adenocarcinoma tissues and cells by immunohistochemistry and western blotting, respectively.. Our findings demonstrate that lung tumorigenesis in the Gprc5a-KO mouse model is augmented by NNK and that gene expression changes induced by tobacco carcinogen(s) may be conserved between mouse and human lung epithelial cells. Further experimentation to prove the reliability of the Gprc5a knockout mouse model for the study of tobacco-induced lung carcinogenesis is warranted.

Topics: Adenocarcinoma; Animals; Blotting, Western; Carcinogens; Cyclin D1; Flap Endonucleases; Genomics; Humans; Immunohistochemistry; In Vitro Techniques; Lung Neoplasms; Mice; Mice, Knockout; Nitrosamines; Nuclear Proteins; Oligonucleotide Array Sequence Analysis; Receptors, G-Protein-Coupled; Ubiquitin-Conjugating Enzymes

Topics: Antineoplastic Combined Chemotherapy Protocols; Biomarkers, Tumor; Breast Neoplasms; Carcinoma, Non-Small-Cell Lung; Clinical Trials as Topic; Cyclin D1; ErbB Receptors; Female; Humans; Lung Neoplasms; Mutation; Neoplasms; Proto-Oncogene Proteins; Proto-Oncogene Proteins B-raf; Proto-Oncogene Proteins p21(ras); ras Proteins; Research Design; Retinoid X Receptors; Vascular Endothelial Growth Factor A

Mutational activation of KRAS is a common event in human tumors. Identification of the key signaling pathways downstream of mutant KRAS is essential for our understanding of how to pharmacologically target these cancers in patients. We show that PD0325901, a small-molecule MEK inhibitor, decreases MEK/ERK pathway signaling and destabilizes cyclin D1, resulting in significant anticancer activity in a subset of KRAS mutant tumors in vitro and in vivo. Mutational activation of PIK3CA, which commonly co-occurs with KRAS mutation, provides resistance to MEK inhibition through reactivation of AKT signaling. Genetic ablation of the mutant PIK3CA allele in MEK inhibitor-resistant cells restores MEK pathway sensitivity, and re-expression of mutant PIK3CA reinstates the resistance, highlighting the importance of this mutation in resistance to therapy in human cancers. In KRAS mutant tumors, PIK3CA mutation restores cyclin D1 expression and G(1)-S cell cycle progression so that they are no longer dependent on KRAS and MEK/ERK signaling. Furthermore, the growth of KRAS mutant tumors with coexistent PIK3CA mutations in vivo is profoundly inhibited with combined pharmacologic inhibition of MEK and AKT. These data suggest that tumors with both KRAS and phosphoinositide 3-kinase mutations are unlikely to respond to the inhibition of the MEK pathway alone but will require effective inhibition of both MEK and phosphoinositide 3-kinase/AKT pathway signaling.

Topics: Alleles; Animals; Benzamides; Cell Growth Processes; Cell Line, Tumor; Class I Phosphatidylinositol 3-Kinases; Colonic Neoplasms; Cyclin D1; Diphenylamine; Extracellular Signal-Regulated MAP Kinases; Gene Knockout Techniques; HCT116 Cells; Humans; Lung Neoplasms; MAP Kinase Kinase Kinases; MAP Kinase Signaling System; Mice; Mutation; Pancreatic Neoplasms; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins p21(ras); ras Proteins

cyclin D1 is a member of the cyclin family, and it has been proven that it plaied an important role in tumorigenesis, invasion and metastasis. We performed a retrospective study on the cyclin D1 expression in non-small cell lung cancer (NSCLC) according to the clinical characteristics.. One hundred fifteen postsurgical NSCLC patients were investigated. Immunohistochemistry was used to evaluate the cyclin D1 expression.. Overall survival was significantly lower in patients with cyclin D1-high expression of tumors than those with cyclin D1 low expression of tumors (Chi-square=5.132, P=0.023). In early stage patients (stage I, II), the overall survival was significantly lower in patients with cyclin D1-high expression of tumors than those with cyclin D1-low expression of tumors (Chi-square=6.863, P=0.009). cyclin D1 status (hazard ratio=0.630; P=0.035), differentiation (hazard ratio=0.399; P<0.001), and pTNM (hazard ratio=1.576; P<0.001) to be independent prognostic factors for NSCLC patients. Specifically, the cyclin D1 status (hazard ratio=0.188; P=0.008) was a significant prognostic factor for patients with stage I NSCLCs.. cyclin D1 expression is an independent prognosis factor for postoperative patient in stage I, II NSCLCs.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Carcinoma, Non-Small-Cell Lung; Cyclin D1; Female; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Male; Middle Aged; Neoplasm Metastasis; Prognosis; Retrospective Studies; Young Adult

Lung cancer is the most lethal cancer and almost 90% of lung cancer is due to cigarette smoking. Even though nicotine, one of the major ingredients of cigarette smoke and the causative agent for addiction, is not a carcinogen by itself, several investigators have shown that nicotine can induce cell proliferation and angiogenesis. We observed that the proliferative index of nicotine is different in the lung cancer cell lines H1299 (p53-/-) and A549 (p53+/+) which indicates that the mode of up-regulation of survival signals by nicotine might be different in cells with and without p53.. While low concentrations of nicotine induced activation of NF-κB, Akt, Bcl2, MAPKs, AP1 and IAPs in H1299, it failed to induce NF-κB in A549, and compared to H1299, almost 100 times higher concentration of nicotine was required to induce all other survival signals in A549. Transfection of WT-p53 and DN-p53 in H1299 and A549 respectively, reversed the mode of activation of survival signals. Curcumin down-regulated all the survival signals induced by nicotine in both the cells, irrespective of their p53 status. The hypothesis was confirmed when lower concentrations of nicotine induced NF-κB in two more lung cancer cells, Hop-92 and NCI-H522 with mutant p53 status. Silencing of p53 in A549 using siRNA made the cells susceptible to nicotine-induced NF-κB nuclear translocation as in A549 DN-p53 cells.. The present study reveals a detrimental role of nicotine especially in lung cancer patients with impaired p53 status and identifies curcumin as a potential chemopreventive.

Topics: Cell Line, Tumor; Cell Proliferation; Curcumin; Cyclin D1; Cyclooxygenase 2; Down-Regulation; Humans; Lung Neoplasms; Nicotine; Phosphorylation; Proto-Oncogene Proteins c-akt; Signal Transduction; Transcription Factor AP-1; Tumor Suppressor Protein p53

Activation to a large extent of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway and mutations in the p53 gene are involved in lung cancer therapeutic resistance. The mammalian target of rapamycin (mTOR) acts as a downstream effector for Akt. Activation of the Akt/mTOR signal is a contributing factor to decreased radiation sensitivity. The purpose of this study was to examine whether the effect of rapamycin on radiation sensitivity is affected by cellular p53 gene status. Cellular radiation sensitivity was evaluated by using two human non-small cell lung cancer (NSCLC) cell lines with the same genetic background except for their p53 gene status (H1299/wtp53 and H1299/mp53). The cells were treated with rapamycin and/or radiation. Cell viability, cell proliferation, apoptosis, cell cycle and Akt/mTOR signaling activity were explored. Rapamycin synergistically enhanced the cytotoxicity of radiation, promoting the induction of apoptosis. Moreover, the combined treatment augmented the cytostatic effects of radiation regardless of cellular p53 gene status. Rapamycin in combination with radiation increased G1 arrest and suppressed progression to S phase in both cell lines. Furthermore, the combined treatment conduced to a prominent p53-independent down-regulation of the mTOR signal and pro-survival molecule, cyclin D1. Rapamycin can enhance the effect of radiation through the repression of pro-survival signals and the reduction in the apoptotic threshold. Taken together, inhibition of the mTOR signal may be a promising strategy for radiosensitization with no relevance to p53 gene status from the aspects of cell lethality and cell growth depression.

Topics: Apoptosis; Carcinoma, Non-Small-Cell Lung; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cyclin D1; Dose-Response Relationship, Radiation; Humans; Lung Neoplasms; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Radiation Tolerance; Radiation-Sensitizing Agents; Signal Transduction; Sirolimus; Time Factors; TOR Serine-Threonine Kinases; Transfection; Tumor Suppressor Protein p53

Survivin, a member of the inhibitor of apoptosis protein (IAP) family, overexpresses in tumor cells and not expresses in terminally differentiated adult tissues. This study aimed to investigate the effects of survivin-specific siRNA on cell proliferation, apoptosis and chemosensitivity to cisplatin in vitro and in vivo and explore the mechanisms about decreasing expression of survivin in reversing cancer cells resistance to chemotherapeutic drug.. Survivin-specific siRNA was transfected into A549/DDP cells. The expression of survivin and lung resistance-related protein (LRP) mRNA levels were determined by RT-PCR, chemosensitivity of A549/DDP (cisplatin) cells to cisplatin was determined by MTT assay, and apoptosis and cell cycle were determined by flow cytometry (FCM). The protein expression levels of survivin, LRP, cyclin-D(1), caspase-3 and bcl-2 were determined by Western blotting analyses. The effect of survivin siRNA inhibition on tumor growth was studied in athymic nude mice in vivo.. Survivin-specific siRNA efficiently down-regulated survivin expression. The cell cycle was arrested at G2/M phase, and apoptosis was obviously found. Inhibition of survivin expression could make the IC50 and drug-resistant index of cisplatin decrease, and enhance the cancer cells sensitivity to cisplatin. After transfection by survivin-specific siRNA, expression of LRP and cyclin-D1 were downregulated, caspase-3 expression was upregulated, bcl-2 expression had no obvious change. The animal experiment confirmed knockdown of survivin could inhibit the tumor growth.. Survivin-specific siRNA can efficiently suppress the expression of survivin, increase apoptosis, inhibit cells proliferation and enhance the chemosensitivity to cisplatin in vitro and in vivo. Suppression of survivin expression helping to reverse drug-resistance may have relationship with downregulation of LRP and upregulation of caspase-3. Anti-tumor strategies based on the inhibition of survivin may be useful in targeting lung adenocarcinomas.

Topics: Adenocarcinoma; Animals; Apoptosis; Caspase 3; Cell Line, Tumor; Cisplatin; Cyclin D1; Drug Resistance, Neoplasm; Female; Humans; Inhibitor of Apoptosis Proteins; Lung Neoplasms; Male; Mice; Mice, Inbred BALB C; Microtubule-Associated Proteins; Proto-Oncogene Proteins c-bcl-2; RNA, Messenger; RNA, Small Interfering; Survivin; Vault Ribonucleoprotein Particles

The discovery of new genes with important regulatory functions on cellular level, allowed the investigation of many biological and molecular changes and defining their value as potential prognostic factors.. The study included 122 patients with non-small cell lung cancer. We examined the expression of cyclin D1 and p53.. The 5-years survival rate was 26% for the group with strong nuclear expression of cyclin D1, and 80% for the group with moderate expression of the marker. We found 5-years survival rate of 100% for the group with poor expression of p53, and 24.55% for the group with strong expression.. The increased levels of cyclin D1 and p53 are negative prognostic factors for the patients with non-small cell lung cancer.

Topics: Adult; Aged; Carcinoma, Non-Small-Cell Lung; Cyclin D1; Female; Humans; Lung Neoplasms; Male; Middle Aged; Prognosis; Survival Rate; Tumor Suppressor Protein p53

The modulatory influence of tea polyphenols (epigallocatechin gallate, epicatechin gallate and theaflavin) on benzo[a]pyrene (B[a]P)-induced lung carcinogenesis in mice was analyzed using histopathological and molecular parameters. Progression of lung lesions was restricted at the hyperplastic stage by tea polyphenols. A significant reduction in cellular proliferative index and an increase in apoptotic index were noted in the restricted lung lesions. High expression of H-ras, c-myc, cyclin D1 and p53 genes was seen at the inflammatory stage (9th week) and in subsequent premalignant lesions, but down-regulation of H-ras at the hyperplastic stage (17th week). Expression of bcl-2 was high in hyperplastic lesions, whereas the expression of mdm2 and bcl-xl increased only at the moderately dysplastic stage (36th week). The tea polyphenols inhibited inflammatory response in the lung lesions on the 9th week, when decreased expression of H-ras and c-myc and increased expression of bax were noted. Prolonged treatment (>9th week) with tea polyphenols resulted in changes in the expression of some additional genes, such as reduced expression of cyclin D1 (from the 17th week), bcl-2 (from the 26th week; mild dysplasia) and p21 (on the 36th week), and high expression of p53 (from the 17th week) and p27 (on the 36th week). These observations indicate that the tea polyphenols can restrict B[a]P-induced lung carcinogenesis by differential modulation of the expression of p53 and its associated genes such as bax, bcl-2, mdm2, p21 and p27, along with H-ras, c-myc and cyclin D1, at different time points.

Topics: Animals; Apoptosis; Benzo(a)pyrene; Biflavonoids; Catechin; Cell Proliferation; Cell Transformation, Neoplastic; Cyclin D1; Female; Flavonoids; Lung; Lung Neoplasms; Male; Mice; Phenols; Polyphenols; Proto-Oncogene Proteins c-myc; ras Proteins; Tea; Tumor Suppressor Protein p53

In androgen sensitive LNCaP prostate cancer cells, the proliferation induced by the epidermal growth factor (EGF) involves a cross-talk between the EGF receptor (EGFR) and the androgen receptor (AR). In lung cancer the role of the EGF-EGFR transduction pathway has been documented, whereas androgen activity has received less attention. Here we demonstrate that in LNCaP and A549 non-small cell lung cancer (NSCLC), AR and EGFR are required for either 5alpha-dihydrotestosterone (DHT) or EGF-stimulated cell growth. Only EGF activated ERK signaling and up-regulated early gene expression, while DHT triggered the expression of classical AR-responsive genes with the exception of the EGF-induced PSA transcript in A549 cells. DHT and EGF up-regulated cyclinD1 (CD1) at both mRNA and protein levels in A549 cells, while in LNCaP cells each mitogen increased only CD1 protein expression. In both cell contexts, CD1 up-regulation was prevented by selective inhibitors as well as by knock-down of either AR or EGFR and also inhibiting p38MAPK and the mammalian target of rapamycin (mTOR) pathways. Interestingly, p38MAPK and mTOR repression prevented the activation of the mTOR target ribosomal p70S6 kinase induced by DHT and EGF, indicating that p38MAPK acts as an upstream mTOR regulator. In addition, the proliferative effects promoted by both DHT and EGF in LNCaP and A549 cancer cells were no longer observed blocking either p38MAPK or mTOR activity. Hence, our data suggest that p38MAPK-dependent activation of the mTOR/CD1 pathway may represent a mechanism through which AR and EGFR cross-talk contributes to prostate and lung cancer progression.

Topics: Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cyclin D1; ErbB Receptors; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Male; p38 Mitogen-Activated Protein Kinases; Prostatic Neoplasms; Protein Kinases; Receptor Cross-Talk; Receptors, Androgen; Signal Transduction; TOR Serine-Threonine Kinases

Inactivation and silencing of PTEN have been observed in multiple cancers, including follicular thyroid carcinoma. PTEN (phosphatase and tensin homologue deleted from chromosome 10) functions as a tumour suppressor by opposing the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) signalling pathway. Despite correlative data, how deregulated PTEN signalling leads to thyroid carcinogenesis is not known. Mice harbouring a dominant-negative mutant thyroid hormone receptor beta (TRbeta(PV/PV) mice) spontaneously develop follicular thyroid carcinoma and distant metastases similar to human cancer. To elucidate the role of PTEN in thyroid carcinogenesis, we generated TRbeta(PV/PV) mice haploinsufficient for Pten (TRbeta(PV/PV)Pten(+/-) mouse). PTEN deficiency accelerated the progression of thyroid tumour and increased the occurrence of metastasis spread to the lung in TRbeta(PV/PV)Pten(+/-) mice, thereby significantly reducing their survival as compared with TRbeta(PV/PV)Pten(+/+) mice. AKT activation was further increased by two-fold in TRbeta(PV/PV)Pten(+/-) mice thyroids, leading to increased activity of the downstream mammalian target of rapamycin (mTOR)-p70S6K signalling and decreased activity of the forkhead family member FOXO3a. Consistently, cyclin D1 expression was increased. Apoptosis was decreased as indicated by increased expression of nuclear factor-kappaB (NF-kappaB) and decreased caspase-3 activity in the thyroids of TRbeta(PV/PV)Pten(+/-) mice. Our results indicate that PTEN deficiency resulted in increased cell proliferation and survival in the thyroids of TRbeta(PV/PV)Pten(+/-) mice. Altogether, our study provides direct evidence to indicate that in vivo, PTEN is a critical regulator in the follicular thyroid cancer progression and invasiveness.

Topics: Animals; Apoptosis; Carrier Proteins; Caspase 3; Cell Proliferation; Cell Survival; Chromosomes, Mammalian; Cyclin D1; Disease Models, Animal; Enzyme Activation; Forkhead Box Protein O3; Forkhead Transcription Factors; Lung Neoplasms; Mice; Mice, Mutant Strains; Mice, Transgenic; Neoplasm Invasiveness; Neoplasm Metastasis; NF-kappa B; Phosphatidylinositol 3-Kinases; Phosphotransferases (Alcohol Group Acceptor); Proto-Oncogene Proteins c-akt; PTEN Phosphohydrolase; Ribosomal Protein S6 Kinases, 70-kDa; Signal Transduction; Thyroid Hormone Receptors beta; Thyroid Neoplasms; TOR Serine-Threonine Kinases

Arsenic is an established lung carcinogen, however, the carcinogenic mechanisms are currently under investigation. Phosphorylation of the epidermal growth factor receptor (EGFR) has been reported with arsenic exposure in bladder cells. EGFR is a tyrosine kinase transmembrane receptor that regulates important processes in carcinogenesis, including cell survival, cell cycle progression, tumor invasion, and angiogenesis. We investigated the mechanisms of EGFR pathway activation by levels of arsenic relevant to human exposure scenarios both in vitro using cultured lung epithelial cells, and in lung tumors samples from New England Lung Cancer Study participants. Toenail arsenic levels were used as an internal biomarker of arsenic exposure. Our in vitro data suggest that arsenic increases levels of the EGFR ligand, heparin binding-EGF, and activate EGFR phosphorylation in the lung. Downstream of EGFR, arsenic exposure increased pERK and cyclin D1 levels. These effects were inhibited by treatment of cultured cells with the EGFR tyrosine kinase inhibitor, Tarceva (erlotinib). In a consecutive series of human lung tumor specimens, pEGFR protein levels were higher in subjects with elevated toenail arsenic levels compared to those with low exposure (odds ratio adjusted for other factors, OR 4.1 (95% confidence interval 1.1-15.6) (p = 0.04). These data suggest that arsenic exposure may stimulate EGFR pathway activation in the lung. Moreover, the tumors that arise in arsenic-exposed individuals also exhibit signs of EGFR pathway dysregulation. Further work is needed to assess the clinical utility of targeting the EGFR pathway in subgroups of lung cancer patients who have been exposed to elevated levels of arsenic.

Topics: Adult; Aged; Amphiregulin; Analysis of Variance; Arsenic; Arsenites; Biomarkers; Bronchi; Cells, Cultured; Cyclin D1; Cycloheximide; EGF Family of Proteins; Epidermal Growth Factor; Epithelial Cells; ErbB Receptors; Erlotinib Hydrochloride; Gene Expression; Glycoproteins; Heparin-binding EGF-like Growth Factor; Humans; Intercellular Signaling Peptides and Proteins; Lung; Lung Neoplasms; Middle Aged; Nails; Phosphorylation; Protein Kinase Inhibitors; Quinazolines; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Sodium Compounds

The ras oncogene needs a second factor to induce transformation and tumorigenicity of the cell. In this study, we show that mouse fibroblast 7-4-Stat3C cells overexpressing both Ha-ras(val12) oncogene and active-form Stat3 (Stat3C) showed higher colony formation in soft agar and xenograft tumor growth in BALB/c mice. Further studies show that both serine-727 and tyrosine-705 of Stat3 were phosphorylated while Ha-ras was overexpressed. Interleukin-6 (IL-6)-induced phosphorylation of tyrosine-705 and serine-727, as well as DNA-binding and transcriptional activity of Stat3 were further enhanced by Ha-ras overexpression. In addition, overexpression of Stat3C in 7-4-Stat3C cells prevented the cells from morphological change and apoptosis triggered by the Ha-ras oncogene under serum-depleted conditions. We demonstrate that Ha-ras and Stat3 acting together synergistically induce Stat3 phosphorylation at serine-727 phosphorylation and cyclin D1 expression and further enhance transformation and tumorigenicity of the cell. Ha-ras-induced Stat3 phosphorylation at serine-727 plays a pivotal role in transcriptional activation of cyclin D1 and suppression of cell apoptosis. The effect of Ha-ras on Stat3 phosphorylation at serine-727 was also detected in human bladder (T24) and lung (H460) cancer cells. Stat3 phosphorylation at serine-727 is important in Ras-related tumorigenesis.

Topics: Amino Acid Sequence; Animals; Apoptosis; Cell Line, Tumor; Cell Transformation, Neoplastic; Cyclin D1; Genes, ras; Humans; Interleukin-6; Lung Neoplasms; Mice; Neoplasm Transplantation; Phosphorylation; Serine; STAT3 Transcription Factor; Transcriptional Activation; Up-Regulation; Urinary Bladder Neoplasms

DAS (diallyl sulfide), DADS (diallyl disulfide), and DATS (diallyl trisulfide) are major oil-soluble allyl sulfides (OAS) that represent major garlic constituents. The anticarcinogenic and antimutagenic effects of these substances have been extensively studied during the last decades. Previous reports suggest that induction of apoptosis by OASs might contribute to their chemopreventive effects. In this study, we report that OASs DADS and DATS induce significant apoptosis in human lung adenocarcinoma A549 cells, whereas DAS does not. Differential modulation of reactive oxygen intermediates (ROI) and mitochondria membrane potential (MMP) may account for the apoptotic effects of DADS and DATS. The underlying molecular mechanisms of apoptosis induction by both compounds include activation of C-Jun N-terminal kinase (JNK), up-regulation of p53, and down-regulation of bcl-2 expression. In our test series, up-regulation of extracellular signal-regulated protein kinase (ERK) was dispensable for apoptosis induction; DAS, DADS, or DATS did not modify expression of MAPK p38, bax, and bcl-xL. Further investigation revealed that the specific JNK inhibitor SP600125 and the antioxidant NAC blocked DADS and DATS-induced apoptosis, whereas ERK inhibitors did not. Additionally, our data provide the first evidence that Fas-mediated cell death pathway is partly involved in DADS but not DATS-mediated cell death. Taken together, our work has elucidated the triggers, important modulators, and signal transduction pathways in DADS and DATS-mediated apoptosis.

Topics: Adenocarcinoma; Allyl Compounds; Anticarcinogenic Agents; Apoptosis; Cell Cycle; Cell Line, Tumor; Cyclin D1; Dose-Response Relationship, Drug; fas Receptor; Garlic; Humans; JNK Mitogen-Activated Protein Kinases; Lung Neoplasms; Membrane Potential, Mitochondrial; Oils; Reactive Oxygen Species; Solubility; Sulfides; Tumor Suppressor Protein p53

In a previous small series of surgically treated non-small cell lung cancer patients (NSCLC), we found that higher apoptotic index (AI) negatively influenced survival (Dworakowska D, Jassem E, Jassem J, Karmolinski A, Dworakowski R, Wirth T, et al. Clinical significance of apoptotic index in non-small cell lung cancer: correlation with p53, mdm2, pRb and p21WAF1/CIP1 protein expression. J Cancer Res Clin Oncol 2005; 131:617-623.). In this study we attempted to verify our previous finding in larger group of 170 NSCLC cases, additionally correlating AI to selected cell cycle regulators as well as a proliferation marker. Apoptosis was assessed with the use of the TUNEL technique, whereas the expression of p53, pRb, mdm2, p21(WAF1/CIP1), cyclin D1 and PCNA were assessed immunohistochemically. The mean and the median AI was 12 and 8, respectively. The expression of p53, pRb, mdm2, p21(WAF1/CIP1) proteins and cyclin D1 was found in 47%, 71%, 37%, 65% and 40% of cases, respectively. The mean and the median PCNA labeling index (PCNA LI) was 34 and 35, respectively. AI was not correlated with any patient characteristic or other tumor markers. In uni- and multivariate analysis AI, analysed separately or jointly with cell cycle regulators and PCNA LI, did not influence disease-free or over-all survival. However, patients with "very high AI/very high PCNA LI" had a particularly poor prognosis (P=0.001). Patients with "very low AI/negative pRb" phenotype survived for a shorter time in comparison to others (P=0.04). In addition, patients with the highest PCNA LI had a worse outcome in comparison to patients with the lowest PCNA LI (P=0.04), especially those with concomitant p53 protein expression (P=0.026) or lacking pRb protein expression (P=0.04). This study demonstrates that joint analysis of several factors involved in apoptosis, proliferation and cell cycle regulation, but not AI alone, might provide additional prognostic information in NSCLC patients.

Topics: Adult; Aged; Apoptosis; Carcinoma, Non-Small-Cell Lung; Cell Cycle Proteins; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Female; Humans; Immunohistochemistry; In Situ Nick-End Labeling; Lung Neoplasms; Male; Middle Aged; Prognosis; Proliferating Cell Nuclear Antigen; Proto-Oncogene Proteins c-mdm2; Survival Analysis; Tumor Suppressor Protein p53

To identify clinical and biometric features associated with overall survival of patients with advanced refractory non-small-cell lung cancer (NSCLC) treated with gefitinib.. One hundred and nine diagnostic NSCLC samples were analysed for EGFR mutation status, EGFR immunohistochemistry, histologic morphometry and quantitative immunofluorescence of 15 markers. Support vector regression modelling using the concordance index was employed to predict overall survival.. Tumours from 4 of 87 patients (5%) contained EGFR tyrosine kinase domain mutations. A multivariate model identified ECOG performance status, and tumour morphometry, along with cyclin D1, caspase-3 activated, and phosphorylated KDR to be associated with overall survival, concordance index of 0.74 (hazard ratio (HR) 5.26, p-value 0.0002).. System-based models can be used to identify a set of baseline features that are associated with reduced overall survival in patients with NSCLC treated with gefitinib. This is a preliminary study, and further analyses are required to validate the model in a randomised, controlled treatment setting.

Topics: Aged; Antineoplastic Agents; Biomarkers, Tumor; Carcinoma, Non-Small-Cell Lung; Caspase 3; Cyclin D1; ErbB Receptors; Female; Gefitinib; Humans; Immunohistochemistry; Lung Neoplasms; Male; Middle Aged; Mutation; Prognosis; Proportional Hazards Models; Quinazolines; Survival Rate

Eukaryotic initiation factor 4E (eIF4E) and cyclin D1, two important factors in cell-cycle progression, play key roles in the carcinogenesis of varied human cancers. However, eIF4E expression in non-small-cell lung cancer (NSCLC) and its association with cyclin D1 has received little investigation. One hundred forty-seven subjects with primary NSCLC, with long-term follow-up and essential clinicopathologic parameters (including age, sex, tumor grade, tumor stage, smoking history, performance status, weight loss, histology grade, and survival data) were evaluated based on expression of eIF4E and cyclin D1. Immunohistochemical analysis was performed using monoclonal antibodies against eIF4E and cyclin D1. While 134 of 147 cases (91%) were positive for eIF4E, 82 of 136 cases (63%) were positive for cyclin D1. Western blot results were consistent with those illustrated by immunohistochemistry. While eIF4E(+) correlated with significantly shorter patient survival (P = .03), cyclin D1(+) correlated with longer patient survival (P = .01). Assessment of coexpression of cyclin D1 and eIF4E shows greater value in determining the prognosis of NSCLC: patients with eIF4E(+)/cyclin D1(-) have poorer outcome, those with eIF4E(-)/cyclin D1(+) have a more favorable outcome, and those with eIF4E(+)/cyclin D1(+) have an intermediate outcome (P = .02). The negative effect on survival in patients with eIF4E(+) suggests its potential prognostic role in NSCLC. These results warrant further investigation to explore the value of eIF4E in identifying patients with aggressive disease for adjuvant treatments.

Topics: Adult; Aged; Aged, 80 and over; Blotting, Western; Carcinoma, Non-Small-Cell Lung; Cyclin D1; Eukaryotic Initiation Factor-4E; Female; Follow-Up Studies; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Lung Neoplasms; Male; Middle Aged; Prognosis; Retrospective Studies; Survival Rate

To observe whether cyclin D1 siRNA-mediated inhibition of cyclin D1 represents a promising antigrowth and antimetastatic strategy for cancer gene therapy, particularly for non-small cell lung cancers. To stably transfect the A549 cell line with a cyclin D1-targeted siRNA to downregulate cyclin D1 expression and observe the effects on protein expression, and tumor growth in vitro and in vivo. Expression of cyclin D1-targeted siRNA resulted in a decrease in cyclin D1, MMP-2, RhoA, and Rac1 protein levels, as detected by Western blot and immunofluorescence studies. Transfected cells also exhibited a marked decrease in the rate of cell growth, and decreased invasive capacity, compared to cells transduced with a scrambled siRNA plasmid and untransduced A549 cells. siRNA-mediated inhibition of cyclin D1 expression represents a promising antigrowth and antimetastatic strategy for cancer gene therapy, particularly for non-small cell lung cancers. It is the reason for inhibiting tumor growth so that cyclin D1 siRNA can inhibit the cell cycle progression. In addition, the mechanism of inhibiting tumor metastasis was related to the decrease in the expression of MMP-2, RhoA, and Rac1 after cyclin D1 was decreased by cyclin D1 siRNA.

Topics: Animals; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Genetic Therapy; Humans; Lung Neoplasms; Matrix Metalloproteinase 2; Matrix Metalloproteinase Inhibitors; Mice; Mice, Nude; rac1 GTP-Binding Protein; rhoA GTP-Binding Protein; RNA, Small Interfering; Transfection; Xenograft Model Antitumor Assays

Described herein is an unusual case of mantle cell lymphoma (MCL) histologically mimicking marginal zone lymphoma (MZL). An 83-year-old man presented with multiple adenopathies and a hilar mass encroaching on the right lung. A transbronchial biopsy showed small blue cells suspicious for small cell carcinoma. On further analysis the cells were predominantly small cleaved and CD20 positive, suggesting follicular lymphoma, grade 2. An axillary lymph node biopsy showed germinal centers surrounded by monocytoid B cells. Flow cytometry was negative for CD5 and CD23 and the diagnosis of MZL was considered. Because of the aggressive clinical behavior, including extensive necrosis on imaging studies, immunohistochemistry for cyclin D-1 was performed and was positive. Bone marrow was extensively involved and it showed t(11;14), in addition to other complex cytogenetic abnormalities. Differentiating MCL from MZL has prognostic and therapeutic implications, particularly when considering the potential role of targeted therapy and cell cycle modulators.

Topics: Aged, 80 and over; Biomarkers, Tumor; CD5 Antigens; Cyclin D1; Diagnosis, Differential; Flow Cytometry; Humans; Immunohistochemistry; Lung Neoplasms; Lymph Nodes; Lymphoma, B-Cell, Marginal Zone; Lymphoma, Mantle-Cell; Male; Small Cell Lung Carcinoma

Previous studies implicate that activation of thromboxane A(2) receptor (TP) induced cell proliferation and transformation in several cell lines. We report here that the activation of TP by its agonist, [1S-[1alpha, 2alpha (Z), 3beta (1E, 3S*), 4alpha]]-7-[3-[3-hydroxy-4-(4-iodophenoxy)-1-butenyl]-7-oxabicyclo [2.2.1] hept-2-yl]-5-heptenoic acid (I-BOP), induced Nurr1 expression and stimulated proliferation of human lung cancer cells. Nurr1, an orphan nuclear receptor in the nuclear receptor subfamily 4A subfamily, has been implicated in cell proliferation, differentiation and apoptosis. I-BOP markedly induced Nurr1 messenger RNA and protein levels as compared with other subfamily members, Nur77 and Nor-1. The signaling pathways of I-BOP-induced Nurr1 expression were examined by using various inhibitors of signaling molecules. The induction of Nurr1 expression by I-BOP appeared to be mediated through protein kinase A (PKA)/cAMP response element binding (CREB), protein kinase C and mitogen-activated protein kinase/extracellular signal-regulated kinase pathways and not related to epidermal growth factor receptor and prostaglandin E(2) pathways. Transcriptional activation of Nurr1 gene by I-BOP was further investigated at the promoter level in H157 cells. 5'-Deletion analysis, site-directed mutagenesis and luciferase reporter assay demonstrated that Nurr1 expression was induced by I-BOP in a PKA/CREB-dependent manner. Further studies have revealed that Nurr1 may mediate cyclin D1 expression and I-BOP-induced cell proliferation in H157 cells since small interfering RNA of Nurr1 blocked I-BOP-induced cyclin D1 expression and cell proliferation and also decreased cell growth rate. These results provide strong evidence that Nurr1 plays a significant role in cell proliferation and may mediate TP agonist-induced proliferation in lung cancer cells.

Topics: Bridged Bicyclo Compounds, Heterocyclic; Cell Line, Tumor; Cell Proliferation; Cyclic AMP Response Element-Binding Protein; Cyclic AMP-Dependent Protein Kinases; Cyclin D1; Dinoprostone; DNA-Binding Proteins; Extracellular Signal-Regulated MAP Kinases; Fatty Acids, Unsaturated; Gene Expression Regulation; Humans; Lung Neoplasms; MAP Kinase Signaling System; NF-kappa B; Nuclear Receptor Subfamily 4, Group A, Member 2; Phosphorylation; Protein Kinase C; Receptors, Thromboxane A2, Prostaglandin H2; Signal Transduction; Transcription Factors

Krüppel-like factor 4 (KLF4) is a zinc-finger protein that plays important roles in stem cells and the development of gastric cancers. However, the role of KLF4 in primary lung cancer is unknown. The purpose of this study is to determine possible roles of KLF4 in lung cancer.. The KLF4 expression in primary lung cancer tissues and case-matched normal lung tissues were determined by protein and mRNA analyses. The effects of KLF4 on cell proliferation, clonogenic formation, and cell cycle progression were determined in cultured lung cancer cells or bronchial epithelial cells after enforced KLF4 overexpression or small interfering RNA knockdown. The in vivo antitumor activity of KLF4 was evaluated by using stably transfected lung cancer cells and by adenovector-mediated gene delivery. The effect of KLF4 in regulating p21 and cyclin D1 was also evaluated.. KLF4 protein and mRNA levels were dramatically decreased in most primary lung tumors compared with in case-matched normal lung tissues. Enforced expression of KLF4 resulted in marked inhibition of cell growth and clonogenic formation. The tumor-suppressive effect of KLF4 was associated with its role in up-regulating p21 and down-regulating cyclin D1, leading to cell cycle arrest at the G(1)-S checkpoint. Knockdown of KLF4 promoted cell growth in immortalized human bronchial epithelial cells. The enforced expression of KLF4 gene to lung cancer cells by ex vivo transfection or adenovector-mediated gene transfer suppressed tumor growth in vivo.. Our results suggest that KLF4 plays an important role in suppressing the growth of lung carcinoma.

Topics: Animals; Cell Cycle; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Gene Expression Regulation, Neoplastic; Humans; Kruppel-Like Factor 4; Kruppel-Like Transcription Factors; Lung Neoplasms; Mice; Mice, Nude; RNA, Messenger; Tumor Cells, Cultured

Real-time reverse transcription polymerase chain reaction and immunohistochemistry were used to evaluate the messenger RNA (mRNA) and protein expression levels of total cyclin D1 and its splice variants (cyclin D1a and cyclin D1b) in 102 paired malignant and nonmalignant tissues from patients with non-small cell lung cancer, respectively. The expression levels of total cyclin D1 and its splice variants were significantly up-regulated in malignant tissues than in nonmalignant tissues at both mRNA and protein levels. Although the expression levels of cyclin D1a were higher than those of cyclin D1b, the relative expression ratios of cyclin D1b mRNA between malignant and nonmalignant lung tissues were obviously higher than those of cyclin D1a mRNA. Analysis of variance showed that cyclin D1b mRNA expression was significantly associated with the histologic grade, lymph node metastasis, distant metastasis, and tumor stage of patients, whereas cyclin D1a mRNA expression was not related to clinicopathologic characteristics except sex. Patients with cyclin D1b mRNA expression above the median value had shorter survival than those below the median value (P = .033). Similarly, cyclin D1b immunopositivity was also associated with histologic grade, and patients with immunostaining positivity for cyclin D1b showed poor survival (P = .005). Multivariate analysis demonstrated that cyclin D1b immunopositivity was an independent risk factor in survival of patients with non-small cell lung cancer (P = .018). Our data show that cyclin D1b, rather than canonical cyclin D1a, might contribute to the development of non-small cell lung cancer. Cyclin D1b would be a better prognostic indicator for non-small cell lung cancer as compared to total cyclin D1 or cyclin D1a.

Topics: Alternative Splicing; Biomarkers, Tumor; Carcinoma, Non-Small-Cell Lung; Cell Nucleus; China; Cyclin D1; Female; Gene Expression Regulation, Neoplastic; Humans; Immunoenzyme Techniques; Lung Neoplasms; Lymphatic Metastasis; Male; Middle Aged; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Survival Rate; Up-Regulation

Various molecular changes characterizing organ-specific carcinogenesis have been identified in human tumors; however, the molecular mechanisms of the genomic changes specific for each cancer are not well defined. A transgenic (Tg) mouse model with constitutive expression of the nucleotide-editing enzyme, activation-induced cytidine deaminase (AID), develops tumors in various organs as a result of the mutagenic activities of AID. This phenotypic character of AID Tg mice allowed us to analyze the organ-specific genetic changes in tumor-related genes commonly triggered by AID-mediated mutagenesis. Among the 80 AID Tg mice analyzed, 11 mice developed hepatocellular carcinomas, and 7 developed lung cancers. In addition, 1 developed the gastric cancer and 3 developed gastric adenomas. Organ-specific preferences for nucleotide changes were observed in some of the tumor-related genes in each epithelial tissue of the AID Tg mice. Of note, the c-myc and K-ras genes were the preferential targets of the mutagenic activity of AID in lung and stomach cancers, respectively, whereas mutations in the p53 and beta-catenin genes were commonly observed in all 3 organs. Quantitative RT-PCR analyses revealed that alpha-fetoprotein, insulin-like growth factor-2 and cyclin D1 genes were specifically upregulated in HCC, whereas upregulation of the matrix metalloproteinase-7 gene was more marked in lung cancer. Our findings suggest that AID, a DNA mutator that plays a critical role linking inflammation to human cancers, might be involved in the generation of organ-specific genetic diversity in oncogenic pathways during cancer development.

Topics: alpha-Fetoproteins; Animals; beta Catenin; Cyclin D1; Cytidine Deaminase; Enzyme Activation; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Genes, myc; Genes, ras; Insulin-Like Growth Factor II; Liver Neoplasms, Experimental; Lung Neoplasms; Matrix Metalloproteinase 7; Mice; Mice, Transgenic; Mutation; Neoplasms, Experimental; Organ Specificity; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Stomach Neoplasms; Tumor Suppressor Protein p53; Up-Regulation

The Wnt gene family is involved in embryogenesis and tumourigenesis. We investigated the clinical significance of Wnt1 expression in non-small cell lung cancer (NSCLC).. We studied 216 NSCLC patients. Immunohistochemistry was performed to investigate the Wnt1 expression in relation to the expression of beta-catenin and Wnt-targets, including c-Myc, Cyclin D1, VEGF-A and MMP-7. The Ki-67 proliferation index and the intratumoural microvessel density (IMD) were also evaluated.. The ratio of tumours with an aberrant beta-catenin expression was significantly higher in Wnt1-positive tumours than in Wnt1-negative tumours (p<0.0001). The Wnt1 expression significantly correlated with the expression of c-Myc (p<0.0001), Cyclin D1 (p<0.0001), VEGF-A (p=0.0160), MMP-7 (p<0.0001), the Ki-67 index (p=0.0048) and the IMD (p=0.0267). Furthermore, the Wnt1 status was a significant prognostic factor for NSCLC patients (p=0.0127).. The Wnt1 overexpression is associated with the expression of tumour-associated Wnt-targets, tumour proliferation, angiogenesis and a poor prognosis in NSCLCs.

Topics: Adult; Aged; Aged, 80 and over; beta Catenin; Carcinoma, Non-Small-Cell Lung; Cell Proliferation; Cyclin D1; Humans; Immunohistochemistry; Lung Neoplasms; Male; Matrix Metalloproteinase 7; Microcirculation; Middle Aged; Neovascularization, Pathologic; Prognosis; Proto-Oncogene Proteins c-myc; Vascular Endothelial Growth Factor A; Wnt1 Protein

Cyclin D1 is found on 11q13, which is a region frequently amplified in several tumor types. The CCND1 locus gives rise to at least two protein isoforms of D1 (D1a and D1b). A common G/A polymorphism (G/A870) is thought to influence the expression levels of D1a and D1b. D1b has been suggested to be increased in the presence of the A allele and more oncogenic than D1a. Furthermore, the A allele has been reported to correlate with increased risk of carcinoma in several tumor types, suggesting that this polymorphism and D1b are important in tumor progression. However, contradictory data about the polymorphism, D1 variant expression, and correlation with survival have been reported. We explored the relationship between gene amplification, G/A870 genotype, D1a and D1b expression, and overall survival in esophageal adenocarcinoma and non-small cell lung cancer.. DNA and RNA were isolated from 54 esophageal adenocarcinoma samples and 89 non-small cell lung cancer samples and were analyzed for gene amplification, genotype at the polymorphism, gene expression, and association with overall survival.. The D1 variant expression did not correlate with amplification, genotype, or overall survival in either tumor type. The total D1 expression correlated with decreased patient survival. Several other genes on 11q13 also seem to be overexpressed and correlated with decreased survival.. We report that the G/A870 polymorphism does not correlate with patient survival, or with D1a or D1b expression. However, the total D1 expression and the expression of several other genes on 11q13 seem to be associated with esophageal adenocarcinoma patient survival.

Topics: Adenocarcinoma; Adult; Aged; Aged, 80 and over; Alleles; Carcinoma, Non-Small-Cell Lung; Cyclin D1; Esophageal Neoplasms; Female; Gene Amplification; Gene Expression; Genotype; Humans; Kaplan-Meier Estimate; Lung Neoplasms; Male; Middle Aged; Polymorphism, Restriction Fragment Length; Polymorphism, Single Nucleotide; Protein Isoforms; Reverse Transcriptase Polymerase Chain Reaction

UBE1L is the E1-like ubiquitin-activating enzyme for the IFN-stimulated gene, 15-kDa protein (ISG15). The UBE1L-ISG15 pathway was proposed previously to target lung carcinogenesis by inhibiting cyclin D1 expression. This study extends prior work by reporting that UBE1L promotes a complex between ISG15 and cyclin D1 and inhibited cyclin D1 but not other G1 cyclins. Transfection of the UBE1L-ISG15 deconjugase, ubiquitin-specific protein 18 (UBP43), antagonized UBE1L-dependent inhibition of cyclin D1 and ISG15-cyclin D1 conjugation. A lysine-less cyclin D1 species was resistant to these effects. UBE1L transfection reduced cyclin D1 protein but not mRNA expression. Cycloheximide treatment augmented this cyclin D1 protein instability. UBE1L knockdown increased cyclin D1 protein. UBE1L was independently retrovirally transduced into human bronchial epithelial and lung cancer cells. This reduced cyclin D1 expression and clonal cell growth. Treatment with the retinoid X receptor agonist bexarotene induced UBE1L and reduced cyclin D1 immunoblot expression. A proof-of-principle bexarotene clinical trial was independently examined for UBE1L, ISG15, cyclin D1, and Ki-67 immunohistochemical expression profiles in pretreatment versus post-treatment tumor biopsies. Increased UBE1L with reduced cyclin D1 and Ki-67 expression occurred in human lung cancer when a therapeutic bexarotene intratumoral level was achieved. Thus, a mechanism for UBE1L-mediated growth suppression was found by UBE1L-ISG15 preferentially inhibiting cyclin D1. Molecular therapeutic implications are discussed.

Topics: Anticarcinogenic Agents; Bexarotene; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cycloheximide; Cytokines; Humans; Ki-67 Antigen; Lung Neoplasms; Models, Biological; Plasmids; Retinoid X Receptors; Tetrahydronaphthalenes; Ubiquitin-Activating Enzymes; Ubiquitins

BHLHB3 is a basic helix-loop-helix (bHLH) domain-containing protein that acts as a transcriptional repressor. We found that BHLHB3 transcript levels were low in three human lung cancer cell lines and downregulated in human lung adenocarcinomas as compared to normal lung tissue. BHLHB3 gene overexpression inhibited colony formation of A549, NCI-H520 and NCI-H596 lung cancer cells. The reduced colony growth was likely due to inhibition of cell proliferation as suggested by the downregulation of cyclin D1 (CCND1) expression in NCI-H520 cells transfected to overexpress the BHLHB3 gene; no evidence of apoptosis was observed. These results point to the potential role of the BHLHB3 protein as a tumor suppressor for lung cancer.

Topics: Basic Helix-Loop-Helix Transcription Factors; Cell Line, Tumor; Cholesterol 7-alpha-Hydroxylase; Cyclin D1; Humans; Lung Neoplasms; Promoter Regions, Genetic; RNA, Messenger; Tumor Suppressor Proteins

Sulindac has been reported to be effective in suppressing tumor growth through the induction of p21WAF1/cip1 in human, animal models of colon cancer and colon cancer cells. In this study, we treated human breast cancer cell line MCF-7 and lung cancer cell line A549 as well as colon cancer cell line SW620 with sulindac to observe the effects of sulindac in other tissue sites. In all cell lines, proliferation was significantly inhibited by sulindac after 24 and 72 h of treatment. Apoptosis was induced by sulindac in both lung cancer cells and colon cancer cells but was not induced in breast cancer cells. Western blots showed that p21 protein level were induced by sulindac in lung cancer cells and colon cancer cells, but not in breast cancer cells. However, the suppression of beta-catenin, a key mediator of Wnt signaling pathway, was seen in all three cell lines with sulindac administration. Further studies revealed that transcriptional activities of beta-catenin were significantly inhibited by sulindac and that the inhibition was sulindac dosage-dependent. The transcriptional targets of beta-catenin, c-myc, cyclin D1 and cdk 4 were also dramatically downregulated. In conclusion, our data demonstrated that the efficacy of sulindac in the inhibition of cell proliferation (rather than the induction of apoptosis) might be through the suppression of beta-catenin pathway in human cancer cells.

Topics: Anti-Inflammatory Agents, Non-Steroidal; Apoptosis; beta Catenin; Blotting, Western; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Colorectal Neoplasms; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase Inhibitor p21; Gene Expression Regulation, Neoplastic; Genes, Reporter; Humans; Luciferases; Lung Neoplasms; Proto-Oncogene Proteins c-myc; Sulindac; Transcription, Genetic; Transfection

ING4, as a novel candidate tumor suppressor gene, has been implicated in several human malignances by tumor growth inhibition and apoptosis enhancement. The mechanism of ING4 remains largely unknown. The purpose of this study was to investigate the inhibitory tumor growth effects of ING4 on lung adenocarcinoma, and its mechanism, by ING4 cDNA transduction into A549 cells. Furthermore, the expression level of ING4 in lung adenocarcinoma tissues was examined. The expression of ING4 was markedly reduced in human lung adenocarcinoma tissues. Overexpression of ING4 can induce growth inhibition in A549 cells both in vitro and in vivo, and also induce up-regulation of p27, down-regulation of cyclinD1, SKP2, and Cox2, and inactivation of the Wnt-1/beta-catenin pathway. Moreover, overexpression of ING4 can enhance the sensitivity of A549 cells to radiotherapy and chemotherapy. Thus, ING4 may play an inhibitory role on A549 cell proliferation and tumor growth in lung adenocarcinoma by up-regulation or down-regulation of cell proliferation-regulating proteins such as p27, cyclinD1, SKP2, and Cox2 by means of inactivation of Wnt-1/beta-catenin signaling.

Topics: Adenocarcinoma; beta Catenin; Cell Cycle Proteins; Cell Line, Tumor; Cell Proliferation; Cloning, Molecular; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p27; Cyclooxygenase 2; DNA, Complementary; Gene Expression; Homeodomain Proteins; Humans; Lung Neoplasms; Plasmids; S-Phase Kinase-Associated Proteins; Signal Transduction; Tumor Suppressor Proteins; Wnt Proteins

To verify the suppressive effect of berberine on the proliferation of the human pulmonary giant cell carcinoma cell line PG and to demonstrate the mechanisms behind the antitumoral effects of berberine.. The proliferative effects of PG cells were detected by 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide colorimetry. The cell cycle was examined by flow cytometry. The expression level of cyclin D1 was detected by RT-PCR. The activities of the activating protein-1 (AP-1) and NF-kappaB signaling pathways related to cyclin D1 were examined by luciferase assay. The cytoplasmic level of c-Jun was detected by Western blot analysis. An electrophoretic mobility shift assay was used to examine the binding of transcription factors to the cyclin D1 gene (CCND1) AP-1 motif.. The results showed that the proliferation of PG cells treated with different concentrations (10, 20, and 40 microg/mL) of berberine for 24 and 48 h was suppressed significantly compared to the control group. After treatment with berberine, the proportion of PG cells at the G0/G1 phase increased, while cells at the S and G2/M phases decreased. Berberine could inhibit the expression of cyclin D1 in PG cells. Berberine inhibited the activity of the AP-1 signaling pathway, but had no significant effect on the NF-kappaB signaling pathway. Berberine suppressed the expression of c-Jun and decreased the binding of transcription factors to the CCND1 AP-1 motif.. Berberine suppresses the activity of the AP-1 signaling pathway and decreases the binding of transcription factors to the CCND1 AP-1 motif. This is one of the important mechanisms behind the antitumoral effects of berberine as a regulator of cyclin D1.

Topics: Amino Acid Motifs; Antineoplastic Agents; Berberine; Carcinoma, Giant Cell; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cyclin D1; Dose-Response Relationship, Drug; Genes, Reporter; Humans; Luciferases; Lung Neoplasms; Time Factors; Transcription Factor AP-1; Transfection

Pin1 isomerizes the bonds of molecules important for numerous oncogenic and cell-signaling pathways, including Bcl-2, p53, c-Jun, beta-catenin, NF-kappaB, cyclin D1, c-Myc and Raf-1. This can cause a change in conformation leading to alterations in catalytic activity, protein-protein interactions, subcellular localization and protein stability. These alterations have been shown to be associated with cell transformation and cancer progression. Pin1 is overexpressed in several different human cancers. This is the first report of Pin1 overexpression in clinical samples of non-small cell lung cancer (NSCLC).. Protein expression levels of Pin1 in tumor and normal lung specimens were analyzed for expression of Pin1, cyclin D1, p53 and MDM2 using immunohistochemistry and compared to several clinicopathological characteristics. The mRNA expression of Pin1 was also analyzed using quantitative real-time RT-PCR and compared to clinicopathological characteristics.. Pin1 protein was shown to be overexpressed in NSCLC tumor samples, and correlated with lymph node positive disease and tumor stage. High expression of MDM2 also correlated with lymph node positive disease and with poorly differentiated tumors. High expression of MDM2 also correlated with lymph node positive disease and with poorly differentiated tumors. High expression levels of Pin1 correlated with high levels of p53 or MDM2 protein, but did not show a correlation with cyclin D1. However, high levels of MDM2 correlated with cyclin D1 overexpression. Pin1 mRNA was expressed significantly more often in the tumors of smokers than of non-smokers. The relationship between the expression of protein and mRNA of Pin1 has obviously showed that protein expression isn't significantly associated with mRNA expression.. Pin1 is overexpressed in many different cancers, including NSCLC, and may possibly be used as a tumor marker or as a target for cancer therapy.

Topics: Aged; Carcinoma, Non-Small-Cell Lung; Cyclin D1; Female; Humans; Immunohistochemistry; Lung Neoplasms; Lymphatic Metastasis; Male; Middle Aged; NIMA-Interacting Peptidylprolyl Isomerase; Peptidylprolyl Isomerase; Proto-Oncogene Proteins c-mdm2; Reverse Transcriptase Polymerase Chain Reaction; Tumor Suppressor Protein p53

Cyclin-dependent kinases (Cdk) and their associated pathways represent some of the most attractive targets for the development of anticancer therapeutics. Based on antitumor activity in animal models, a variety of Cdk inhibitors are undergoing clinical evaluation either as a single agent or in combination with other approved drugs. In our anticancer drug discovery program, a novel series of flavones have been synthesized for evaluation against the activity of Cdk4-D1. This enzyme catalyzes the phosphorylation of retinoblastoma protein, thus inhibiting its function. We have identified a series of potent Cdk4-D1 inhibitors with IC(50) below 250 nmol/L. In this report, we have described the properties of one of the best compound, P276-00 of the flavone's series. P276-00 shows 40-fold selectivity toward Cdk4-D1, compared with Cdk2-E. The specificity toward 14 other related and unrelated kinases was also determined. P276-00 was found to be more selective with IC(50)s <100 nmol/L for Cdk4-D1, Cdk1-B, and Cdk9-T1, as compared with other Cdks, and less selective for non-Cdk kinases. It showed potent antiproliferative effects against various human cancer cell lines, with an IC(50) ranging from 300 to 800 nmol/L and was further compared for its antiproliferative activity against cancer and normal fibroblast cell lines. P276-00 was found to be highly selective for cancer cells as compared with normal fibroblast cells. To delineate its mechanism of action, the effect of P276-00 on cell cycle proteins was studied in human breast cancer cell line (MCF-7) and human non-small cell lung carcinoma (H-460). A significant down-regulation of cyclin D1 and Cdk4 and a decrease in Cdk4-specific pRb Ser(780) phosphorylation was observed. P276-00 produced potent inhibition of Cdk4-D1 activity that was found to be competitive with ATP and not with retinoblastoma protein. The compound also induced apoptosis in human promyelocytic leukemia (HL-60) cells, as evidenced by the induction of caspase-3 and DNA ladder studies. These data suggest that P276-00 has the potential to be developed as an anti-Cdk chemotherapeutic agent.

Topics: Antineoplastic Agents; Breast Neoplasms; Carcinoma, Non-Small-Cell Lung; Caspase 3; Cell Cycle; Cell Proliferation; Cells, Cultured; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinases; Down-Regulation; Enzyme Inhibitors; Fibroblasts; Flavones; HL-60 Cells; Humans; In Vitro Techniques; Lung Neoplasms; Molecular Structure; Phosphorylation; Retinoblastoma Protein

The effect of glycogen synthase kinase 3beta (GSK3beta) has been repeatedly implicated in cell proliferation, but studies on the effect of GSK3beta in different cell lines with different stimuli have drawn different conclusions. To investigate the direct effect of GSK3beta on cell growth in human lung adenocarcinoma cell line A549, we changed its activity by transient transfection with two kinds of GSK3beta mutant plasmids, constitutively active form S9A-GSK3beta and dominant negative form KM-GSK3beta. Twenty-four hours later, cell counting, flow cytometry and Western blot detection were made respectively. The results showed that enhancing GSK3beta activity caused a decrease in cell number, as well as a higher percentage of cells at G(1) phase. Further, the expression of cyclin D1 was down-regulated by GSK3beta. Taken together, our observations suggest that GSK3beta may induce G(1) cell cycle arrest in a cyclin D1-dependent fashion and therefore possibly plays a growth-inhibitory role in A549 cells.

Topics: Adenocarcinoma; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Down-Regulation; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Humans; Lung Neoplasms; Transfection

Topics: Adenocarcinoma; Adult; Aged; Aged, 80 and over; Cyclin D1; Female; Humans; Immunohistochemistry; Lung Neoplasms; Male; Middle Aged; Neoplasm Staging

Double-stranded decoy oligodeoxynucleotide (ODN) is a promising approach for inhibiting gene transcription. Signal transducer and activator of transcription (STAT) 3, a potent transcription factor, is usually constitutively activated in a variety of malignancies, and considered as an attractive drug target. In this study, it was noted that STAT3 was overactivated in human lung cancer cells, and STAT3-decoy ODN, which was high-efficiently transfected into nucleus of cancer cells, significantly inhibited the proliferation of PG cells by inducing apoptosis or cell cycle arrest. The transcription levels of mcl-1, cyclin D1, bcl-xl and survivin were significantly decreased by 64.4, 56.1, 72.8% (P<0.01) and 31.8% (P<0.05), respectively; and the synthesis levels of bcl-xl and cyclin D1 in PG cells showed 64.5% (P<0.01) and 28.6% (P<0.05) decrease, respectively. Our study demonstrated that decoy-ODN targeting at activated STAT3 may potentially be used as an anti-lung cancer therapeutic approach.

Topics: bcl-X Protein; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Down-Regulation; Gene Expression; Humans; Inhibitor of Apoptosis Proteins; Lung Neoplasms; Microtubule-Associated Proteins; Myeloid Cell Leukemia Sequence 1 Protein; Neoplasm Proteins; Oligodeoxyribonucleotides; Proto-Oncogene Proteins c-bcl-2; STAT3 Transcription Factor; Survivin; Transfection

Patients who have pathologic N2 (pN2) nonsmall cell lung cancer (pN2 NSCLC) represent a heterogeneous group with regard to prognosis and treatment. Molecular features of NSCLC seem to be of interest. For the current study, to select an appropriate therapeutic strategy for each patient, patients with N2 NSCLC were stratified into homogenous subgroups according to the expression profiles of cell cycle-related markers.. The expression levels of retinoblastoma protein (pRb), cyclin D1, p16, p53, and p21 proteins and values of the Ki-67 labeling index were evaluated in 61 primary surgically resected tumor specimens from patients with pN2 NSCLC using immunohistochemistry. The prognostic impact of these markers on overall survival was analyzed in both univariate and multivariate analyses.. In univariate analysis, p21, p16, and Ki-67 were correlated significantly with survival. In multivariate analysis, only p21 and p16 influenced survival. Indeed, the group of patients with pN2 NSCLC who were positive for p21 and p16 had the most favorable overall survival (P = .001) and were correlated significantly with the clinical lymph node (cN) status (cN2 disease; P = .008). Moreover, no significant difference in survival was observed between patients with cN0/cN1 disease and patients with cN2 disease within the group (P = .4333).. Loss of control of cell-cycle checkpoints is a common occurrence in pN2 NSCLC. Functional cooperation between different cell-cycle regulators constitutes another level of regulation in cell growth control and tumor suppression. Preoperative patients with pN2 NSCLC, even those with cN2 disease, who have positive p21 and p16 protein expression in their primary tumors are expected to have a favorable postoperative prognosis and may be candidates for primary resection.

Topics: Adenocarcinoma; Biomarkers, Tumor; Carcinoma, Large Cell; Carcinoma, Non-Small-Cell Lung; Carcinoma, Squamous Cell; Cell Cycle Proteins; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p16; Cyclin-Dependent Kinase Inhibitor p21; Female; Gene Expression Regulation, Neoplastic; Humans; Immunoenzyme Techniques; Lung Neoplasms; Male; Middle Aged; Neoplasm Staging; Prognosis; Retinoblastoma Protein; Retrospective Studies; Survival Rate; Tumor Suppressor Protein p53

Lung cancer is the leading cause of cancer mortality in the United States. Despite advances made over the past decades, the overall survival of patients with lung cancer remains dismal. Here we report novel G-quartet oligodeoxynucleotides (GQ-ODN) that were designed to selectively target signal transducer and activator of transcription 3 (Stat3), in the treatment of human non-small cell lung cancer (NSCLC). The objective of this study was to evaluate the effects of two novel GQ-ODN STAT3 inhibitors, T40214 and T40231, on NSCLC bearing nude mice. NSCLC bearing nude mice were assigned to 5 groups, which were treated by vehicle, control ODN, T40214, T40231, and Paclitaxel, respectively. Tumors were measured, isolated and analyzed using Western blotting, immuno-histochemistry, RPA and TUNEL. Results show that GQ-ODN T40214 and T40231 significantly suppress the growth of NSCLC tumors in nude mice by selectively inhibiting the activation of Stat3 and its downstream proteins Bcl-2, Bcl-xL, Mcl-1, survivin, VEGF, Cyclin D1 and c-myc; thereby, promoting apoptosis and reducing angiogenesis and cell proliferation. These findings validate Stat3 as an important molecular target for NSCLC therapy and demonstrate the efficacy of GQ-ODN in inhibiting Stat3 phosphorylation.

Topics: Animals; Apoptosis; bcl-X Protein; Carcinoma, Non-Small-Cell Lung; Cell Proliferation; Cyclin D1; Immunohistochemistry; Inhibitor of Apoptosis Proteins; Lung Neoplasms; Mice; Mice, Nude; Microtubule-Associated Proteins; Myeloid Cell Leukemia Sequence 1 Protein; Neoplasm Proteins; Neovascularization, Pathologic; Oligodeoxyribonucleotides; Paclitaxel; Phosphorylation; Protein Conformation; Proto-Oncogene Proteins c-bcl-2; Proto-Oncogene Proteins c-myc; STAT3 Transcription Factor; Survivin; Vascular Endothelial Growth Factor A

Aberrant activation of the phosphatidylinositol 3-kinase (PI3K)-AKT/protein kinase B-signaling pathway has been associated with multiple human cancers, including thyroid cancer. Recently, we showed that, similar to human thyroid cancer, the PI3K-AKT pathway is overactivated in both the thyroid and metastatic lesions of a mouse model of follicular thyroid carcinoma (TRbeta(PV/PV) mice). This TRbeta(PV/PV) mouse harbors a knockin mutant thyroid hormone receptor beta gene (TRbetaPV mutant) that spontaneously develops thyroid cancer and distant metastasis similar to human follicular thyroid cancer. That the activation of the PI3K-AKT signaling contributes to thyroid carcinogenesis raised the possibility that this pathway could be a potential therapeutic target in follicular thyroid carcinoma. The present study tested this possibility by treating TRbeta(PV/PV) mice with LY294002 (LY), a potent and specific PI3K inhibitor, and evaluating the effect of LY on the spontaneous development of thyroid cancer. LY treatment inhibited the AKT-mammalian target of rapamycin (mTOR)-p70(S6K) signaling, and it decreased cyclin D1 and increased p27(Kip1) expression to inhibit thyroid tumor growth and reduce tumor cell proliferation. LY treatment increased caspase 3 and decreased phosphorylated-BAD to induce apoptosis. In addition, LY treatment reduced the AKT-matrix metalloproteinase 2 signaling to decrease cell motility to block metastatic spread of thyroid tumors. Thus, these altered signaling pathways converged effectively to prolong survival of TRbeta(PV/PV) mice treated with LY. No significant adverse effects were observed for wild-type mice treated similarly with LY. The present study provides the first preclinical evidence for the in vivo efficacy for LY in the treatment of follicular thyroid cancer.

Topics: Adenocarcinoma, Follicular; Animals; Apoptosis; Caspase 3; Cell Movement; Cell Proliferation; Chromones; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p27; Lung Neoplasms; Matrix Metalloproteinase 2; Mice; Mice, Mutant Strains; Morpholines; Neoplasm Invasiveness; Phosphoinositide-3 Kinase Inhibitors; Protein Kinases; Proto-Oncogene Proteins c-akt; Signal Transduction; Thyroid Hormone Receptors beta; Thyroid Neoplasms; TOR Serine-Threonine Kinases; Tumor Cells, Cultured

Carcinoid tumors are neuroendocrine malignancies that frequently metastasize and secrete hormones that cause debilitating symptoms in patients. In this study we report the effects of valproic acid (VPA), a drug long used for the treatment of epilepsy, on the growth and neuroendocrine phenotype of human carcinoid cancer cells. VPA treatment of gastrointestinal and pulmonary carcinoid cells resulted in a dose-dependent inhibition of cancer cell growth. Western blot analysis revealed degradation of cyclin D1 and an increase in cyclin-dependent kinases p21 and p27 with VPA treatment. Flow cytometry confirmed that the mechanism of VPA-induced growth inhibition is G(1) phase cell cycle arrest. Furthermore, VPA suppressed expression of the neuroendocrine tumor marker chromogranin A. In addition to these effects, VPA also increased levels of full-length Notch-1 and the active Notch-1 intracellular domain. Luciferase reporter assays incorporating the centromere-binding factor 1 (CBF-1) binding site and the achaete-scute complex-like 1 (ASCL-1) promoter confirmed the functional activity of VPA-induced Notch-1. Transfection of Notch-1 small-interfering RNA into carcinoid tumor cells blocked the effects of VPA on Notch-1 activation, ASCL-1 suppression, p21 induction, and cell growth inhibition. VPA also suppressed growth of carcinoid tumors in vivo in a mouse tumor xenograft experiment. These findings confirm the important role of Notch-1 in regulating the growth and neuroendocrine phenotype of carcinoid tumor cells. On the basis of this study, a clinical trial of VPA for patients with advanced carcinoid cancer will be conducted. Disclosure of potential conflicts of interest is found at the end of this article.

Topics: Animals; Antineoplastic Agents; Carcinoid Tumor; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Chromogranin A; Cyclin D1; Gastrointestinal Neoplasms; Genes, Reporter; Humans; Luciferases; Lung Neoplasms; Male; Mice; Mice, Nude; Phenotype; Receptor, Notch1; RNA Interference; Signal Transduction; Valproic Acid; Xenograft Model Antitumor Assays

The aim of this study was to investigate expression of cyclin D1, bcl-2, p53, Ki-67 and HER-2 proteins in 14 cases of non-small cell lung cancer and to establish their correlation to classical clinico-pathological findings, and alleged prognostic value to estimate biological potential of tumor. Retrospective pilot study of the surgically treated non-small cell lung cancer biopsy specimen, paraffin embedded, used immunohistochemical method to demonstrate expression of cyclin D1, bcl-2, p53, Ki-67 and HER-2. Protein quantification was performed by the semi-quantitative method. Achieved results were correlated with classical clinico-pathological parameters, like tumor size, histological type, differentiation level, presence of vascular invasion and metastasis in regional lymph nodes. Out of 14 cases of non-small cell lung cancer, squamous cell carcinoma was found in 7 patients, giant cell carcinoma in 3, adenocarcinoma in 2, and 1 case of pleomorphic and mucoepidermoid carcinoma. Expression of cyclin D1 was not found, while expression of HER-2 and bcl-2 protein was established in one cases each. p53 expression was noted in 8 cases (57,1%). Statistically positive significant correlation (p<0,05) was found among: presence of lymphovascular invasion to tumor tissue and appearance of nodal metastasis; proliferation Ki-67 index and level of tumor differentiation, i.e. size of tumor. Other investigated parameters showed no significant statistically dependence. p53 expression was not correlated to any of the investigated parameters what might imply the possibility that there is an independent pathway of this protein expression. Negative expression of bcl-2 protein points out to possibility that it is not included into process of tumor apoptosis, as well as that proteins cyclin D1 and HER-2 are not included into processes of the tumor genesis. Since the proliferative activity of the tumor, measured by the expression of Ki-67, is correlated to the gradus and size of the tumor mass, Ki-67 protein can be of a prognostic value to determine biological potential of non-small cell lung cancer.

Topics: Adult; Aged; Biopsy; Carcinoma, Non-Small-Cell Lung; Cyclin D1; Female; Gene Expression Regulation, Neoplastic; Humans; Ki-67 Antigen; Lung Neoplasms; Male; Middle Aged; Neoplasm Metastasis; Prognosis; Proto-Oncogene Proteins c-bcl-2; Receptor, ErbB-2; Tumor Suppressor Protein p53

To investigate the nuclear factor (NF)-kappaB activity in human non-small cell lung cancer (NSCLC) tissues and to explore the correlation between NF-kappaB activity and cell proliferation, between NF-kappaB activity and cell spontaneous apoptosis.. Thirty samples of non-small cell lung cancer tissues and 15 normal lung tissues were collected from May to October in 2006. NF-kappaB activation was determined by electrophoretic mobility shift assay (EMSA). CyclinD1 level was examined by RT-PCR and Western blot. Proliferation cell nuclear antigen (PCNA) protein was examined by immunohistochemical analysis. Spontaneous cell apoptosis was determined by the TUNEL method.. There was significant difference (F=78.96, P<0.01) in NF-kappaB activity among normal lung tissue group (24,826+/-3724), squamous-cell carcinoma tissue group (28,028+/-4204), and adenocarcinoma tissue group (35,425+/-5317). The NF-kappaB activity in the squamous-cell carcinoma group and the adenocarcinom tissue group was higher than that in the normal lung tissue group (all P<0.01); and the NF-kappaB activity is in the adenocarcinoma tissue group was higher compared with that in the squamous-cell carcinoma group (P<0.05). There was significant difference (F=62.43, P<0.01) in the cyclinD1 mRNA level among normal lung tissue group (2.04+/-0.24), the squamous-cell carcinoma group (2.91+/-0.37), and the adenocarcinoma group (4.13+/-0.36). There was significant difference (F=89.24, P<0.01) in cyclinD1 protein level among normal lung tissue group (0.31+/-0.06), the squamous-cell carcinoma group (0.43+/-0.07), and the adenocarcinoma group (0.58+/-0.08). There was significant difference (F=45.61, P<0.01) in PCNA protein level among the normal lung tissue group (0.32+/-0.09), the squamous-cell carcinoma group (0.42+/-0.10), and the adenocarcinima group (0.54+/-0.16). There was no significant difference (F=1.86, P>0.05) in apoptosis index among the normal lung tissue group (2.58%+/-0.39%), the squamous-cell carcinoma group (2.27%+/-0.34%), and the adenocarcinoma group (2.92%+/-0.59%). The NF-kappaB activity was positively correlated with cyclin D1 mRNA level, cyclin D1 protein level, and PCNA protein level in the squamous-cell carcinoma group (r=0.51, P<0.05, r=0.54, P<0.05, r=0.60, P<0.05), respectively; the NF-kappaB activity was also positively correlated with cyclinD1mRNA, cyclinD1protein level, and PCNA protein level in the adenocarcinoma group (r=0.60, P<0.05; r=0.64, P<0.05; r=0.68, P<0.05), respectively. The NF-kappaB activity in the squamous-cell group and the adenocarcinoma group was not related to cell apoptosis index.. NF-kappaB activity increased in NSCLC tissues. Abnormal NF-kappaB activation may be associated with cell proliferation, but do not affect spontaneous cell apoptosis in NSCLC tissues.

Topics: Adult; Aged; Aged, 80 and over; Apoptosis; Blotting, Western; Carcinoma, Non-Small-Cell Lung; Carcinoma, Squamous Cell; Cell Proliferation; Cyclin D1; Electrophoretic Mobility Shift Assay; Female; Humans; Lung; Lung Neoplasms; Male; Middle Aged; NF-kappa B; Proliferating Cell Nuclear Antigen; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger

Clinical chemoprevention trials of lung cancer have been somewhat disappointing and the development of highly effective chemopreventive agents is urgently needed. We previously showed that the organoselenium 1,4-phenylenebis(methylene)selenocyanate (p-XSC) is a potent chemopreventive agent in numerous preclinical animal models including a lung tumor model that employs carcinogens found in tobacco smoke. The goal of this study is to define molecular targets that will be highly promising in the design of future chemoprevention trials of non-small cell lung cancer (NSCLC), which is by far the most common type of lung cancer cases. In the present investigation, we showed that p-XSC at several doses (2.5, 5, 10 and 20 microM) including physiological levels (2.5-5.0 microM) of selenium is capable of inhibiting cell growth in a dose-dependent manner and inducing apoptosis in three NSCLC cells (NCI-H460, NCI-1299 and A549). To clarify the mechanism involved at the molecular level, we focused only on NCI-460 cells and examined the effects of p-XSC on markers that are known to be critical in the development of NSCLC. Using western blot analysis, we showed that p-XSC reduced the expression of cyclooxygenase-2 (COX-2) and phospholipase A2 (PLA2); although p-XSC inhibited both Akt and p-Akt but its effect was not significant. Using cDNA microarray approach (3800 genes per array) we found that p-XSC upregulates 22 genes by > or = 2-fold while downregulates 13 genes by < or = 0.5-fold; these altered genes include transcriptional factors, growth factors and those involved in xenobiotic metabolism as well as pro- and anti-apoptotic genes. Expression of selected genes was confirmed by RT-PCR; p-XSC reduced the levels of COX-2, PLA2, NF-kappaB and Cyclin D1 but enhanced the levels of glutathione peroxidase-5. Collectively, the results of this study showed that p-XSC alters several molecular markers in a manner that can account for its inhibitory effect of cell growth and induction of apoptosis; therefore, p-XSC may be considered a promising candidate for clinical chemoprevention of NSCLC.

Topics: Antigens, Human Platelet; Antineoplastic Agents; Apoptosis; Blotting, Western; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Proliferation; Chemoprevention; Cyclin D1; Cyclooxygenase 2; Dose-Response Relationship, Drug; Gene Expression; Humans; Lung Neoplasms; Membrane Proteins; NF-kappa B; Oligonucleotide Array Sequence Analysis; Oncogene Protein v-akt; Organoselenium Compounds; Reverse Transcriptase Polymerase Chain Reaction

AKT2, a serine/threonine kinase, is confirmed to be an oncogene. Abnormal expression and activation of AKT2 is observed in many kinds of tumor tissues. AKT2 is associated with the proliferation and invasion of cancers. This study was designed to investigate the expression of AKT2, Cyclin D1, and matrix metalloproteinase-9 (MMP-9) in human non-small cell lung cancer (NSCLC) tissue and their correlations to clinicopathologic features of NSCLC.. The expression of AKT2, Cyclin D1, and MMP-9 in 68 specimens of NSCLC, 38 specimens of corresponding adjacent tissues, and 14 specimens of no-cancerous lung tissue were assessed by immunohistochemistry; their correlations to clinicopathologic factors were analyzed using Chi-square test.. The positive rates of AKT2, Cyclin D1, and MMP-9 were significantly higher in NSCLC than in adjacent tissues and no-cancerous lung tissues (91.2% vs. 3.8%, 76.5% vs. 0%, 72.1% vs. 13.5%, P<0.05). The expression of AKT2 wasn't correlated to age, sex, histologic subtype and differentiation, and TNM stage of NSCLC patients (P>0.05), but was correlated to lymph node metastasis (P<0.05). The expression of Cyclin D1 and MMP-9 was correlated to lymph node metastasis and differentiation of squamous cell carcinoma (P<0.05); the expression of MMP-9 was correlated to TNM stage. The expression of AKT2 was positively correlated to the expression of Cyclin D1 and MMP-9 (P<0.05).. AKT2, Cyclin D1, and MMP-9 is related to development of lung cancer. The overexpression of Cyclin D1 and MMP-9 may relate to AKT2 regulation.

Topics: Adolescent; Adult; Aged; Carcinoma, Non-Small-Cell Lung; Cell Differentiation; Cyclin D1; Female; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Lymph Nodes; Lymphatic Metastasis; Male; Matrix Metalloproteinase 9; Middle Aged; Neoplasm Staging; Proto-Oncogene Proteins c-akt

The cyclin D1 (CCND1) A870G gene polymorphism is linked to the outcome in patients with resectable non-small cell lung cancer (NSCLC). Here, we investigated the impact of this polymorphism on smoking-induced cancer risk and clinical outcome in patients with NSCLC stages I-IV.. CCND1 A870G genotype was determined by polymerase chain reaction (PCR) and restriction fragment length polymorphism analysis (RFLP) of DNA extracted from blood. The study included 244 NSCLC patients and 187 healthy control subjects.. Patient characteristics were: 70% male, 77% smokers, 43% adenocarcinoma, and 27% squamous cell carcinoma. Eighty-one percent of the patients had stages III-IV disease. Median age at diagnosis was 60 years and median survival was 13 months. Genotype frequencies of patients and controls both conformed to the Hardy Weinberg equilibrium. The GG genotype significantly correlated with a history of heavy smoking (>or=40 py, P=0.02), and patients with this genotype had a significantly higher cigarette consumption than patients with AA/AG genotypes (P=0.007). The GG genotype also significantly correlated with tumor response or stabilization after a platinum-based first-line chemotherapy (P=0.04). Survival analysis revealed no significant differences among the genotypes.. Evidence was obtained that the CCND1 A870G gene polymorphism modulates smoking-induced lung cancer risk. Further studies are required to explore the underlying molecular mechanisms and to test the value of this gene polymorphism as a predictor for platinum-sensitivity in NSCLC patients.

Topics: Adult; Aged; Aged, 80 and over; Antineoplastic Combined Chemotherapy Protocols; Carcinoma, Non-Small-Cell Lung; Case-Control Studies; Cisplatin; Cyclin D1; Female; Genetic Predisposition to Disease; Genotype; Humans; Lung Neoplasms; Male; Middle Aged; Neoplasm Staging; Polymerase Chain Reaction; Polymorphism, Genetic; Polymorphism, Restriction Fragment Length; Smoking; Statistics, Nonparametric; Survival Analysis

Mutations in Ki-ras occur in approximately 30-50% of patients with adenocarcinoma (AC) of the lung. We previously reported the development of a bitransgenic mouse model that expressed the human Ki-ras(G12C) allele in a lung-specific, tetracycline-inducible manner and gave rise to benign lung tumors. In the current study, these benign tumors, which represent relatively early lesions in neoplastic progression, were analyzed for molecular alterations secondary to mutant Ki-ras expression to determine the gene(s) that contribute to adenoma (AD) development. Tumors were removed following doxycycline (DOX) treatment for 9 and 12 mo and examined for alterations in cell-cycle regulatory genes. Quantification of mRNA expression for cyclin D1, retinoblastoma, p16(Ink4a), p19(Arf), and survivin was carried out by real-time PCR. All of the tumors examined exhibited a mean reduction of approximately fivefold for the retinoblastoma gene (P < 0.02). Increased expression of both p19(Arf) and survivin were detected in a majority of the tumors examined (P < 0.01 and 0.001, respectively), but no change in cyclin D1 RNA expression was observed. A subset of the lung tumors (8/28) displayed reduced levels of p16(Ink4a) expression (P = 0.02). Immunohistochemical analysis confirmed the upregulation of p19(Arf) and survivin in all 10 of the lung tumors examined. However, increased staining for cyclin D1 was observed in the tumor tissue. In addition, increased levels of activated p53 were found in lung tumor tissues stained with an anti-phospho-p53 antibody, while an absence of staining was observed with an anti-phospho-pRb antibody in both normal control and tumor tissue. Analysis of the methylation status of p16(Ink4a) by methylation-specific PCR (MSP) demonstrated that seven of eight tumors exhibiting decreased expression of p16(Ink4a) had at least partial methylation of the promoter region. Single stranded conformational polymorphism (SSCP) analysis demonstrated that neither exons 1 or 2 of p16(Ink4a) nor exons 5-8 of p53 exhibited mutations. These data thus identify alterations in specific genes and pathways that combine with the mutation in Ki-ras to promote the formation of benign lung tumors and suggest potential targets for the development of novel chemotherapeutic and chemopreventive agents during the early stages of lung tumor progression.

Topics: Animals; Base Sequence; Cell Cycle; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p16; Disease Models, Animal; DNA Primers; DNA, Neoplasm; Genes, ras; Humans; Lung Neoplasms; Mice; Mice, Transgenic; Polymorphism, Single Nucleotide; ras Proteins; Retinoblastoma Protein

A well-known histone deacetylase inhibitor, trichostatin A, was applied to non-small-cell lung cancer cells to determine whether inhibition of histone deacetylase leads to the production of proteins that either arrest tumor cell growth or lead to tumor cell death.. Trichostatin A (0.01 to 1.0 micromol/L) was applied to one normal lung fibroblast and four non-small-cell lung cancer lines, and its effect was determined by flow cytometry, annexin-V staining, immunoprecipitation, and Western blot analysis.. Trichostatin A demonstrated tenfold greater growth inhibition in all four non-small-cell lung cancer lines compared with normal controls, with a concentration producing 50% inhibition ranging from 0.01 to 0.04 micromol/L for the tumor cell lines and 0.7 micromol/L for the normal lung fibroblast line. Trichostatin A treatment reduced the percentage of cells in S phase (10% to 23%) and increased G1 populations (10% to 40%) as determined by flow cytometry. Both annexin-V binding assay and upregulation of the protein, gelsolin (threefold to tenfold), demonstrated that the tumor cells were apoptotic, whereas normal cells were predominantly in cell cycle arrest. Trichostatin A increased histone H4 acetylation and expression of p21 twofold to 15-fold without significant effect on p16, p27, CDK2, and cyclin D1.. Collectively, these data suggest that inhibition of histone deacetylation may provide a valuable approach for lung cancer treatment. We evaluated trichostatin A as a potential candidate for anticancer therapy in non-small-cell lung cancer.

Topics: Antineoplastic Agents; Apoptosis; Carcinoma, Non-Small-Cell Lung; Cell Cycle; Cell Line; Cell Line, Tumor; Cell Survival; Cyclin D1; Enzyme Inhibitors; Gelsolin; Histone Deacetylase Inhibitors; Histones; Humans; Hydroxamic Acids; Lung; Lung Neoplasms

The ability of glucocorticoids (GCs) to regulate cell proliferation plays an important role in their therapeutic use. The canonical Wnt pathway, which promotes the proliferation of many cancers and differentiated tissues, is an emerging target for the actions of GCs, albeit existing links between these signaling pathways are indirect. By screening known Wnt target genes for their ability to respond differently to GCs in cells whose proliferation is either positively or negatively regulated by GCs, we identified c-myc, c-jun, and cyclin D1, which encode rate-limiting factors for G(1) progression of the cell cycle. Here we show that in U2OS/GR cells, which are growth-arrested by GCs, the glucocorticoid receptor (GR) represses cyclin D1 via Tcf-beta-catenin, the transcriptional effector of the canonical Wnt pathway. We demonstrate that GR can bind beta-catenin in vitro, suggesting that GC and Wnt signaling pathways are linked directly through their effectors. Down-regulation of beta-catenin by RNA interference impeded the expression of cyclin D1 but not of c-myc or c-jun and had no significant effect on the proliferation of U2OS/GR cells. Although these results revealed that beta-catenin and cyclin D1 are not essential for the regulation of U2OS/GR cell proliferation, considering the importance of the Wnt pathway for proliferation and differentiation of other cells, the repression of Tcf-beta-catenin activity by GR could open new possibilities for tissue-selective GC therapies.

Topics: Animals; beta Catenin; Cell Division; Cell Line, Tumor; Cyclin D1; Fibroblasts; Gene Expression; Haplorhini; Humans; In Vitro Techniques; Kidney; Luciferases; Lung Neoplasms; Osteosarcoma; Promoter Regions, Genetic; Rats; Receptors, Glucocorticoid; RNA, Small Interfering; Signal Transduction; TCF Transcription Factors; Wnt Proteins

Zanthoxyli Fructus belongs to the family of oranges and is used as a seasoning in Asian countries including Japan. This study found that a water extract of Zanthoxyli Fructus possessed anti-tumor activity against a wide variety of cancer cells including those from prostate (LNCaP, DU145, PC-3), breast (MCF-7, T47D, MDA-MB231), lung (NCI-H460, -H520), as well as leukemia (HL-60, NB4, Jurkat) in vitro, as measured by the trypan blue exclusion test. Importantly, Zanthoxyli Fructus slowed the proliferation of LNCaP, DU145, and MDA-MB231 cells present as xenografts in BALB/c nude mice without adverse effects. Further studies explored the molecular mechanism by which Zanthoxyli Fructus inhibited the proliferation of androgen-dependent human prostate cancer LNCaP cells because Zanthoxyli Fructus possessed the strongest anti-tumor activity against these cells. Zanthoxyli Fructus blocked androgen receptor (AR) signaling in conjunction with down-regulation of nuclear levels of AR and induced apoptosis of these cells, as measured by the reporter assay, Western blot analysis, and TUNEL assay, respectively. As expected, Zanthoxyli Fructus also decreased the level of the AR-target molecule, prostate-specific antigen in these cells. Furthermore, Zanthoxyli Fructus inhibited AKT kinase and down-regulated levels of cyclin D1 protein, as measured by the AKT kinase assay with GSK-3alpha/beta as a substrate and Western blot analysis, respectively. Taken together, Zanthoxyli Fructus might be useful as an adjunctive therapeutic agent for the treatment of individuals with a variety of cancer types.

Topics: Androgen Receptor Antagonists; Animals; Apoptosis; Breast Neoplasms; Cell Growth Processes; Cyclin D1; Down-Regulation; HL-60 Cells; Humans; Jurkat Cells; Lung Neoplasms; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasms, Hormone-Dependent; Phosphorylation; Plant Extracts; Promoter Regions, Genetic; Prostate-Specific Antigen; Prostatic Neoplasms; Proto-Oncogene Proteins c-akt; Receptors, Androgen; Signal Transduction; Transfection; Zanthoxylum

Based on in vitro studies, Rho guanine nucleotide exchange factors (RhoGEFs) are key regulators of mitogenic and transforming pathways. At least 1 family member, PDZ-RhoGEF, also integrates signaling between monomeric Rho G proteins and heterotrimeric G proteins through a so-called regulator of G-protein signaling (RGS) domain. Recently, the authors reported that 3 single-nucleotide polymorphisms (SNPs) in 2 members of the RGS family were associated with significant reductions in the risk of cancer.. For the current report, the authors studied the risk of lung cancer associated with a nonsynonymous SNP (rs868188; Ser1416Gly) in PDZ-RhoGEF in a large lung cancer case-control study of 2260 Caucasians and 369 Mexican Americans.. Compared with individuals who had the wild-type genotype (AA), Mexican Americans with the variant genotypes (AG and GG) had a significantly reduced risk for lung cancer (odds ratio [OR], 0.57; 95% confidence interval [95%CI], 0.34-0.94). The protective effect appeared to be more evident in younger individuals (OR, 0.42; 95%CI, 0.20-0.91), men (OR, 0.36; 95%CI, 0.18-0.71), and ever smokers (OR, 0.50; 95%CI, 0.29-0.88). A joint effect was observed between Ser1416Gly and polymorphisms in 2 cell-cycle control genes: p53 (intron 3) and cyclin D1 (CCND1). Tallying the variant alleles of the 4 RGS gene SNPs, a gene-dosage effect was apparent. Compared with individuals who had < 3 variant alleles, patients with > or = 3 variant alleles had a 51% reduction in lung cancer risk (OR, 0.49; 95%CI, 0.28-0.88).. To the authors' knowledge, this is the first epidemiological study to link PDZ-RhoGEF polymorphisms with cancer risk. The results suggest that there are interactions between RGS2, RGS6, and PDZ-RhoGEF and validate this family of proteins as key regulators of tumorigenesis.

Topics: Aged; Alleles; Case-Control Studies; Cell Transformation, Neoplastic; Cyclin D1; DNA, Neoplasm; Female; Genotype; Glycine; GTP-Binding Proteins; Guanine Nucleotide Exchange Factors; Humans; Lung Neoplasms; Male; Mexican Americans; Middle Aged; Odds Ratio; Polymorphism, Single Nucleotide; Rho Guanine Nucleotide Exchange Factors; Risk Factors; Serine; Signal Transduction; Tumor Suppressor Protein p53

The effect and mechanism of the ciglitazone on lung cancer cells A549 growth in vitro and in vivo were studied. Various concentrations of ciglitazone were added to the cultured A549 line, and the proliferation and differentiation of A549 cells were examined by MTT and cytometry analysis. A549 cells (1 x 10(6)/mouse) were inoculated subcutaneously into 20 nude mice, which were randomly divided into two groups: the control group, the ciglitazone treated group. The weights of subcutaneous tumors were measured. The expression of cyclin D1 and P21 in the lung was detected by immohistochemistry and Western blot respectively. The results showed that the proliferation of A549 was inhibited significantly by ciglitazone in a dose- and time-dependent manner. There were more cells arrested in G1 /G0 phase and the expression of PPARgamma was markedly up-regulated in ciglitazone-treated group. Direct injection of ciglitazone into A549-induced tumors could suppress tumor growth in nude mice and the growth inhibitory rate was 36%. The expression of cyclin D1 was decreased and P21 increased significantly in ciglitazone-treated group as compared with control group. It was concluded that ciglitazone could inhibit A549 proliferation dose-dependently and time-dependently and induce differentiation, which might be related to the modulation of cell cycle interfered by PPARgamma.

Topics: Animals; Cell Proliferation; Cyclin D1; Humans; Lung Neoplasms; Mice; Mice, Nude; PPAR gamma; Random Allocation; Thiazolidinediones; Tumor Cells, Cultured

To predict a malignant grade of lung cancer by fluorodeoxyglucose positron emission tomography (FDG-PET) scanning, we investigated the correlation between FDG uptake and pathological tumor stage, proliferative activities determined by Ki-67 and cyclin D1, and an alteration of p53, in clinical stage (c-stage) IA lung adenocarcinomas.. FDG-PET was performed for 71 patients with c-stage IA lung adenocarcinomas. FDG uptake was measured by a contrast ratio (CR) between the tumor and contralateral lung. Ki-67, cyclin D1 and p53 staining scores were examined by immunohistochemistry.. The lesions with ground-glass opacity were found in 26 patients, and solid lesions in 45 by computed tomography. The pathological tumor stages (p-stage) were stage IA in 59 and more advanced stages in 12. The latter had significantly higher CR value than the former (P < 0.001). Patients with CR > or = 0.55 could be predicted to be at advanced tumor stages, with a sensitivity of 0.83 and a specificity of 0.82. The CR and staining scores of Ki-67 were significantly correlated with each other (P < 0.0001), and both the values were significantly higher in advanced tumor stages than in p-stage IA, and were also significantly higher in tumors with intratumoral lymphatic, vascular and pleural involvements than in those without such features (P < 0.05-0.0001).. In c-stage IA lung adenocarcinomas, the FDG uptake can predict p-stage and tumor proliferative activity determined by Ki-67. For c-stage IA lung adenocarcinomas showing CR > or = 0.55, mediastinoscopy or neoadjuvant chemotherapy is indicated.

Topics: Adenocarcinoma; Adult; Aged; Aged, 80 and over; Cell Proliferation; Cyclin D1; Female; Fluorodeoxyglucose F18; Genes, Tumor Suppressor; Humans; Ki-67 Antigen; Lung Neoplasms; Male; Middle Aged; Neoplasm Staging; Positron-Emission Tomography; Predictive Value of Tests; Radiopharmaceuticals; Tumor Suppressor Protein p53

We previously identified DnaJ-like heat shock protein (HLJ1) as a gene associated with tumor invasion. Here, we investigated the clinical significance of HLJ1 expression in non-small-cell lung cancer (NSCLC) patients and its role in cancer progression.. We induced HLJ1 overexpression or knockdown in human lung adenocarcinoma CL1-5 cells and analyzed cell proliferation, anchorage-independent growth, in vivo tumorigenesis, cell motility, invasion, and cell cycle progression. Expression of genes that act downstream of HLJ1 was examined by DNA microarray analysis, pathway analysis, and western blotting. We measured HLJ1 expression in tumors and adjacent normal tissues of 71 NSCLC patients by quantitative reverse transcription-polymerase chain reaction. Associations between HLJ1 expression and disease-free and overall survival were determined using the log-rank test and multivariable Cox proportional hazards regression analysis. Validation was performed in an independent cohort of 56 NSCLC patients. Loss of heterozygosity (LOH) mapping of the HLJ1 locus was analyzed in 48 paired microdissected NSCLC tumors. All statistical tests were two-sided.. HLJ1 expression inhibited lung cancer cell proliferation, anchorage-independent growth, tumorigenesis, cell motility, and invasion, and slowed cell cycle progression through a novel STAT1/P21(WAF1) pathway that is independent of P53 and interferon. HLJ1 expression was lower in tumors than in adjacent normal tissue in 55 of 71 patients studied. NSCLC patients with high HLJI expressing tumors had reduced cancer recurrence (hazard ratio [HR] = 0.47; 95% confidence interval [CI] = 0.23 to 0.93; P = .03) and longer overall survival (HR = 0.38; 95% CI = 0.16 to 0.89; P = .03) than those with low-expressing tumors. Validation in the independent patient cohort confirmed the association between HLJ1 expression and patient outcome. LOH mapping revealed high frequencies (66.7% and 70.8%) of allelic loss and microsatellite instability (87.5% and 95.2%) of the HLJ1 locus at chromosome 1p31.1.. HLJ1 is a novel tumor suppressor in NSCLC, and high HLJ1 expression is associated with reduced cancer recurrence and prolonged survival of NSCLC patients.

Topics: Biomarkers, Tumor; Blotting, Northern; Blotting, Western; Carcinoma, Non-Small-Cell Lung; Cell Cycle; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Disease Progression; Disease-Free Survival; Flow Cytometry; Gene Expression Regulation, Neoplastic; HSP40 Heat-Shock Proteins; Humans; Loss of Heterozygosity; Lung Neoplasms; Microsatellite Repeats; Neoplasm Recurrence, Local; Odds Ratio; Oligonucleotide Array Sequence Analysis; Reproducibility of Results; Reverse Transcriptase Polymerase Chain Reaction; RNA, Small Interfering; STAT1 Transcription Factor; Survival Analysis; Transfection

Silibinin, a flavanone from milk thistle, inhibits the growth of tumors in several rodent models. We examined the effects of dietary silibinin on the growth, progression, and angiogenesis of urethane-induced lung tumors in mice.. A/J mice (15 per group) were injected with urethane (1 mg/g body weight) or saline alone and fed normal diets for 2 weeks, after which they were fed diets containing different doses of silibinin (0%-1% [wt/wt] silibinin) for 18 or 27 weeks. Immunohistochemistry and Western blot analysis were used to examine angiogenesis and enzymatic markers of inflammation, proliferation, and apoptosis. All statistical tests were two-sided.. Urethane-injected mice exposed to silibinin had statistically significantly lower lung tumor multiplicities than urethane-injected mice fed the control diet lacking silibinin (i.e., control mice). Mice that received urethane and 1% (wt/wt) dietary silibinin for 18 weeks had 93% fewer large (i.e., 1.5-2.5-mm-diameter) lung tumors than control mice (mean number of tumors/mouse: 27 in the urethane group versus 2 in the urethane + 1% silibinin group, difference = 25 tumors/mouse, 95% confidence interval [CI] = 13 to 37 tumors/mouse, P = .005). Lung tumors of silibinin-fed mice had 41%-74% fewer cells positive for the cell proliferation markers proliferating cell nuclear antigen and cyclin D1 than lung tumors of control mice. Tumor microvessel density was reduced by up to 89% with silibinin treatment (e.g., 56 microvessels/400x field in tumors from control mice versus 6 microvessels/400x field in tumors from urethane + 1% silibinin-treated mice [difference = 50 microvessels/400x field, 95% CI = 46 to 54 microvessels/400x field; P<.001]). Silibinin decreased lung tumor expression of vascular endothelial growth factor (VEGF) and of inducible nitric oxide synthase and cyclooxygenase-2, two enzymes that promote lung tumor growth and progression by inducing VEGF expression.. Silibinin inhibits lung tumor angiogenesis in an animal model and merits investigation as a chemopreventive agent for suppressing lung cancer progression.

Topics: Animals; Antineoplastic Agents; Apoptosis; Biomarkers, Tumor; Blotting, Western; Carcinogens; Cell Proliferation; Cyclin D1; Cyclooxygenase 2; Disease Models, Animal; Fibroblast Growth Factor 2; Immunohistochemistry; Inflammation; Lung Neoplasms; Mice; Microcirculation; Neovascularization, Pathologic; Nitric Oxide Synthase Type II; Platelet Endothelial Cell Adhesion Molecule-1; Proliferating Cell Nuclear Antigen; Silybin; Silymarin; Urethane; Vascular Endothelial Growth Factor A

Human lung cancer cell lines are widely used to test anticancer drugs. These in-vitro tests, however, preclude the detection of responses to paracrine factors from surrounding stroma. We have cocultured pulmonary fibroblasts CCD-19Lu, from a healthy donor, or HLF-A, from a patient with epidermoid carcinoma of the lung, with two human pulmonary adenocarcinoma cell lines to test the hypothesis that the fibroblasts stimulate the growth of the tumor cells. Both fibroblast cell lines significantly increased the proliferation of the pulmonary adenocarcinoma cell lines in 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide assays, with HLF-A fibroblasts yielding the most pronounced responses. The proliferation of the pulmonary adenocarcinoma cell lines in coculture with fibroblasts was blocked by antibodies against the transforming growth factor-alpha and amphiregulin. In addition, reverse transcription-polymerase chain reaction showed expression of mRNA for amphiregulin and transforming growth factor-alpha in all cell lines, whereas mRNA for the epidermal growth factor was detected only in pulmonary adenocarcinoma cell lines. Western blot analysis revealed that medium containing growth factors released by each fibroblast cell line activated extracellular signal-regulated kinase 1/2 in the both tested pulmonary adenocarcinoma cell lines, but activated Akt kinase only in A549 cells. Assessment of protein levels for cyclin D1 and cyclin E by Western blots demonstrated pronounced increases of both proteins in each pulmonary adenocarcinoma cell line, whereas protein levels for cyclin-dependent kinase inhibitor p21 remained unchanged. Immunocytochemical analysis showed positive immunoreactivity for P-extracellular signal-regulated kinase 1/2, cyclin D1 and cyclin E in pulmonary adenocarcinoma cells cocultured with fibroblasts or exposed to fibroblast-conditioned media. Our data suggest that the growth of pulmonary adenocarcinoma is stimulated by amphiregulin and transforming growth factor-alpha released from pulmonary fibroblasts. This may contribute to the disappointing clinical responses to anticancer drugs, which have shown promise in tests with lung cancer cell lines.

Topics: Adenocarcinoma; Amphiregulin; Animals; Antibodies, Blocking; Cell Line, Tumor; Cell Proliferation; Coculture Techniques; Culture Media, Conditioned; Cyclin D1; Cyclin E; EGF Family of Proteins; Enzyme Activation; Epidermal Growth Factor; Extracellular Signal-Regulated MAP Kinases; Fibroblasts; Glycoproteins; Immunohistochemistry; Intercellular Signaling Peptides and Proteins; Lung; Lung Neoplasms; Mice; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tetrazolium Salts; Thiazoles; Transforming Growth Factor alpha

Cyclin D1 is involved in normal regulation of the cell cycle and plays an important role in the transition from G1 to S phase of the cell cycle. The CCND1 gene has a G-->A polymorphism in exon 4 that increases the frequency of alternate splicing. We analyzed the potential role of CCND1 gene polymorphisms in lung cancer patients (n = 151) and in a matched control population (n = 151). DNA was isolated from blood samples, and exon 4 of CCND1 was amplified by polymerase chain reaction. After digestion with MspI, common CCND1 polymorphic alleles were analyzed by means of agarose gel electrophoresis. The data obtained were analyzed using multiple logistic regression. After adjustment for age, sex, and smoking status, the AG genotype was associated with an increased risk for overall lung cancer (odds ratio OR = 1.7, 95% confidence interval CI = 0.92-3.14). No association was found between AA genotype and risk of lung cancer. In smokers, the combined AG+AA genotypes of CCND1 were found to be significant (OR = 1.9, 95% CI = 1.03-3.71, P = 0.03). No positive association was found between CCND1 genotypes in nonsmokers. The results suggest that the CCND1 A870G gene polymorphisms may increase the risk of lung cancer in smokers from north India.

Topics: Aged; Cyclin D1; Disease Susceptibility; Female; Genotype; Humans; India; Lung Neoplasms; Male; Middle Aged; Polymorphism, Genetic; Smoking

Tobacco smoking is a major risk factor for lung cancer causing, among other effects, oxidative stress and lipid peroxidation. Malondialdehyde (MDA)-DNA adducts can be induced by direct DNA oxidation and by lipid peroxidation. We measured the relationship between bronchial MDA-DNA adducts and tobacco smoking, cancer status, and selected polymorphisms in 43 subjects undergoing a bronchoscopic examination for diagnostic purposes. MDA-DNA adducts were higher in current smokers than in never smokers (frequency ratio (FR) = 1.51, 95% confidence interval (CI) 1.01-2.26). MDA-DNA adducts were also increased in lung cancer cases with respect to controls, but only in smokers (FR = 1.70, 95% CI 1.16-2.51). Subjects with GA and AA cyclin D1 (CCND1) genotypes showed higher levels of MDA-DNA adducts than those with the wild-type genotype (FR = 1.51 (1.04-2.20) and 1.45 (1.02-2.07)). Lung cancer cases with levels of MDA-DNA adducts over the median showed a worse, but not statistically significant, survival, after adjusting for age, gender, and packyears (hazard ratio = 2.48, 95% CI 0.65-9.44). Our findings reinforce the role of smoking in lung carcinogenesis through oxidative stress. Subjects who carry at least one variant allele of the CCND1 gene could accumulate DNA damage for altered cell-cycle control and reduced DNA repair proficiency.

Topics: Aged; Bronchi; Cyclin D1; DNA Adducts; DNA Damage; DNA Repair; Female; Genotype; Humans; Lung Neoplasms; Male; Malondialdehyde; Middle Aged; Polymorphism, Genetic; Risk Factors; Smoking

Desmoid tumors arising in the lung and pleura are extremely rare and can resemble other, more common neoplasms native to these sites. Alterations of the adenomatous polyposis coli/beta-catenin pathway have been detected in sporadic desmoid tumors and have been associated with nuclear accumulation of beta-catenin and overexpression of cyclin D1.. To analyze the expression of beta-catenin and cyclin D1 in desmoid tumors and solitary fibrous tumors (SFTs), and to compare the utilities of these substances for distinguishing between these entities with those of other, more commonly used stains.. Formalin-fixed, paraffin-embedded sections of 4 desmoid tumors (1 pulmonary, 1 pleural, 2 pleural/chest wall), and 5 benign and 6 malignant SFTs of the pleura were immunostained for beta-catenin, cyclin D1, ALK1, CD34, vimentin, desmin, smooth muscle actin, muscle-specific actin, S100, and pancytokeratin. Staining intensity and the percentage of stained tumor cells were assessed semiquantitatively.. Diffuse moderate or strong nuclear staining for beta-catenin was found in all desmoid tumors, 4 of 5 benign SFTs, and 2 of 6 malignant SFTs. All cases except 1 benign SFT showed concurrent cytoplasmic staining. Nuclear and cytoplasmic cyclin D1 staining was increased in all groups. The best distinction between desmoid tumors and SFTs was provided by CD34 (desmoid tumors, 0/4; SFTs, 8/11) and smooth muscle actin (desmoid tumors, 4/4; SFTs, 0/11).. Our findings suggest that alterations in the adenomatous polyposis coli/beta-catenin pathway and cyclin D1 dysregulation may contribute to the pathogenesis of pleuropulmonary desmoid tumors and SFTs. CD34 and smooth muscle actin stains are particularly useful for differentiating between pleuropulmonary desmoid tumors and SFTs.

Topics: Actins; Adult; Aged; Antigens, CD34; beta Catenin; Cell Nucleus; Child, Preschool; Cyclin D1; Cytoplasm; Diagnosis, Differential; Female; Fibromatosis, Aggressive; Humans; Immunohistochemistry; Leiomyoma; Lung Neoplasms; Male; Middle Aged; Muscle, Smooth; Pleural Neoplasms; Staining and Labeling

Arrest defective 1 (ARD1), an acetyltransferase, is essential for the yeast life cycle. Although its human homologue (hARD1) has been identified, its biological functions in human cells remain unclear. In the present study, we examined the biological function of hARD1. In H1299 and A549 lung cancer cells, hARD1-silencing RNA inhibited cell proliferation and induced G(1) arrest. Cyclin D1 was also found to be down-regulated in these growth-arrested cells, and the ectopic expression of cyclin D1 rescued cell growth. hARD1 knockdown repressed the promoter activity of the cyclin D1 gene, which inhibited the transcription of cyclin D1. Moreover, hARD1 knockdown reduced the binding of beta-catenin/TCF4 transcription factor to cyclin D1 promoter and repressed its transcriptional activity. Inversely, hARD1 expression increased the transcriptional activity of beta-catenin. Both endogenous and ectopically expressed hARD1 was coimmunoprecipitated with beta-catenin. hARD1 knockdown did not affect beta-catenin expression or degradation but noticeably reduced acetylated beta-catenin. The beta-catenin binding and acetylation by hARD1 were observed in vitro. Therefore, it is suggested that hARD1 participates in proliferation of lung cancer cells via the activation of beta-catenin.

Topics: Acetylation; Acetyltransferases; beta Catenin; Carcinoma, Non-Small-Cell Lung; Cell Growth Processes; Cell Line, Tumor; Cyclin D1; G1 Phase; Humans; Lung Neoplasms; N-Terminal Acetyltransferase A; N-Terminal Acetyltransferase E; Promoter Regions, Genetic; Protein Binding; RNA, Small Interfering; TCF Transcription Factors; Transcription Factor 7-Like 2 Protein

Many studies have highlighted the aberrant expression and prognostic significance of individual proteins in either the Rb (particularly cyclin D1, p16INK4A, and pRb) or the p53 (p53 and p21Waf1) pathways in non-small cell lung cancer. We hypothesize that cumulative abnormalities within each and between these pathways would have significant prognostic potential regarding survival.. Our study population consisted of 106 consecutive surgically resected cases of predominantly early-stage non-small cell lung cancer from the National Cancer Institute-Mayo Clinic series, and assessment of proteins involved both immunohistochemical (cyclin D1, p21Waf1, pRb, p16INK4A, and p53) and mutational analysis (p53) in relationship to staging and survival.. Cyclin D1 overexpression was noted in 48% of the tumors, p16INK4A negative in 53%, pRb negative in 17%, p53 immunopositive in 50%, p53 mutation frequency in 48%, and p21(Waf1) overexpression in 47%, none with prognostic significance. Cyclin D1 overexpression in pRb-negative tumors revealed a significantly worse prognosis with a mean survival of 2.3 years (P = 0.004). A simultaneous p53 mutation dramatically reduced the mean survival time to 0.9 years (P = 0.007). Cyclin D1 overexpression with either a p53 mutation or a p53 overexpression was also associated with a significantly poorer prognosis (P = 0.0033 and 0.0063, respectively).. Some cumulative abnormalities in the Rb and p53 pathways (e.g., cyclin D1 overexpression and p53 mutations) significantly cooperate to predict a poor prognosis; however, the complexity of the cell cycle protein interaction in any given tumor warrants caution in interpreting survival results when specific protein abnormalities are taken in isolation.

Topics: Aged; Carcinoma, Non-Small-Cell Lung; Cell Cycle; Cyclin D1; DNA Mutational Analysis; Female; Gene Expression Regulation, Neoplastic; Heterozygote; Humans; Immunohistochemistry; Lung Neoplasms; Male; Middle Aged; Mutation; Prognosis; Protein Binding; Retinoblastoma Protein; Time Factors; Treatment Outcome; Tumor Suppressor Protein p53

DNA repair enzyme genetic polymorphisms have been postulated to increase the risk of certain cancers in the presence of tobacco carcinogen exposures. The XPD protein is an important component of the TFIIH transcription factor complex. XPD genetic polymorphisms resulting in amino acids substitutions may lead to alterations in TFIIH helicase activity, resulting in repair and transcription defects. Cyclin D1 is a key regulatory protein for the transition of cells from the G(1)-S cell cycle phase. The CCND1 G870A polymorphism has been reported to enhance alternate splicing of a stable mRNA variant, which may result in the bypass of the G(1)/S cell cycle checkpoint. In this study, XPD G23591A (Asp312Asn) and A35931C (Lys751Gln) polymorphisms and the CCND1 G870A splice variant frequencies were determined in 273 upper aero-digestive tract cancer cases and 269 controls. The XPD Asp312Asn variant frequency was significantly different among cases and controls and conferred an odds ratio (OR) of 1.3 (95% CI 1.0-1.8). However, individuals with the CCND1 G870A and XPD Lys751Gln variants had higher age adjusted ORs of 3.2 (95% CI 2.2-4.6) and 2.2 (95% CI 1.5-3.2), respectively. Furthermore, a significant gene-gene interaction was observed among cases with at least two variant alleles for both CCND1 and XPD genes [OR 7.09 (95% CI 4.03-12.5)]. Smokers with a combination of at least one variant allele of both CCND1 and XPD genes also had an elevated risk as compared to nonsmokers. This is the first study to suggest an associative interaction between XPD and CCND1 genetic polymorphisms, tobacco exposure, and cancer risk.

Topics: Alternative Splicing; Carcinoma, Squamous Cell; Cyclin D1; DNA Helicases; DNA Primers; DNA-Binding Proteins; Exons; Female; Genetic Variation; Head and Neck Neoplasms; Humans; Lung Neoplasms; Male; Middle Aged; Polymorphism, Single Nucleotide; Reference Values; Smoking; Transcription Factors; Xeroderma Pigmentosum Group D Protein

The aim of this study was to assess the impact of cyclin D1 overexpression (considered separately or jointly with previously assessed p53 and pRb statuses) on survival in a group of 111 surgically treated non-small cell lung cancer patients (NSCLC).. Cyclin D1 accumulation was assessed immunohistochemically, with the use of monoclonal antibody (DCS-6, DakoCytomation) and the alkaline phosphatase-anti-alkaline phosphatase (APAAP) technique.. Overexpression of cyclin D1 was found in 55 samples (49%), whereas the altered phenotypes cyclin D1+/p53+ or cyclin D1+/pRb- were found in 23 (22%) and 9 samples (9%), respectively. Statistical analysis was performed for different cut-off values and the only significant differences were found if samples with some expression of each protein were considered positive. There was no relationship between cyclin D1 overexpression and major clinicopathological factors, including p53 expression; however, there was a direct correlation between cyclin D1 and pRb protein expression (p=0.007). Cyclin D1 accumulation did not influence patients' survival. Of all possible cyclin D1/p53, cyclin D1/pRb and cyclin D1/p53/pRb phenotypes, patients with cyclin D1-/p53+ phenotype had shortened overall survival compared to other patients (p=0.027, HR=1.8). In the multivariate analysis, the only variable associated with shortened overall and disease-free survival was the stage of disease (p<0.001).. These results suggest the lack of prognostic value of cyclin D1 overexpression in NSCLC patients.

Topics: Biomarkers, Tumor; Carcinoma, Non-Small-Cell Lung; Cyclin D1; Female; Humans; Immunoenzyme Techniques; Lung Neoplasms; Male; Middle Aged; Prognosis; Retinoblastoma Protein; Survival Rate; Tumor Suppressor Protein p53

To investigate the apoptosis inducing effect of tea polyphenols (TPP) on human lung cancer cell (LCC) and its associative mechanism.. The apoptosis inducing effect of TPP on LCC in vitro, and its influence on expression of the related gene were determined by MTT assay, laser scanning confocal microscopy and flow cytometry.. TPP in different concentration (50,100,200 and 400 microg/ml) had dose-dependent inhibitory effect on LCC, the inhibitory rate was 28.69+/-1.27% ,46. 19+/-1.79% ,64.61+/-1.29%, 75.90+/-1.96%, respectively. The inhibited LCC were blocked in (G0/G1 phase, and could not transferred to S and G2/ M phase of cell cycle. Meanwhile, TPP could induce apoptosis of LCC, the apoptotic rate being 4.76+/-0.11 %, 5.78+/-0.38 %, 10.06+/-0.67 %, 24.44+/-0.44 %, respectively. Morphologic changes of cells were seen in laser scanning confocal microscopy observation. Compared to the control group, intracellular Ca2+ concentration, Annexin V expression, phospatase and tensin homologe deleted on chromosome ten (PTEN) protein and expression gradually increased, while Cyclin D1 protein expression gradually decreased in the TPP treated groups along with the increasing of TPS concentration.. TTP can induce LCC apoptosis, the mechanism is related to the change of intracellular Ca2+ concentration, PTEN protein and Cyclin D1 protein expression.

Topics: Annexin A5; Antineoplastic Agents, Phytogenic; Apoptosis; Cyclin D1; Dose-Response Relationship, Drug; Flavonoids; Humans; Lung Neoplasms; Phenols; Phosphoric Monoester Hydrolases; Polyphenols; PTEN Phosphohydrolase; Tea; Tumor Cells, Cultured; Tumor Suppressor Proteins

Cigarette smoke contains several carcinogens known to initiate and promote tumorigenesis as well as metastasis. Nicotine is one of the major components of the cigarette smoke and the 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a tobacco-specific carcinogen. Here, we demonstrated that NNK stimulated cell proliferation in normal human bronchial epithelial cells (NHBE) and small airway epithelial cells (SAEC). Cells exposed to NNK resulted in an increase in the level of cyclin D1 protein (as early as 3-6 h). Increased phosphorylation of the Rb Ser(795) was detected at 6-15 h after NNK treatment and thereby promoted cells entering into the S phase (at 15-21 h). The increased cyclin D1 protein level was induced through activation of the transcription factor, nuclear factor kB (NFkappaB), in the NHBE cells. Treatment of the NHBE cells with PD98059, an ERK1/2 (extracellular signal-regulated protein kinase)-specific inhibitor, specifically suppressed the NNK-induced IkappaBalpha phosphorylation at position 32 of the serine residue, suggesting that the ERK1/2 kinase was involved in the IkappaBalpha phosphorylation induced by NFkappaB activation. To determine whether the NNK-induced NFkappaB activation and cyclin D1 induction were also observed in vivo, A/J mice were treated with NNK (9.1 mg) for 20 weeks and the results showed a significant induction of cyclin D1 and NFkappaB translocation determined by immunoblotting analyses. We further demonstrated that the nicotine acetylcholine receptor (nAchR), which contains the alpha3-subunit, was the major target mediating NNK-induced cyclin D1 expression in the NHBE cells. In summary, our findings demonstrate for the first time that NNK could stimulate normal human bronchial cell proliferation through activation of the NFkappaB, which in turn up-regulated the cyclin D1 expression.

Topics: Bronchi; Carcinogens; Cell Proliferation; Cells, Cultured; Cyclin D1; Dose-Response Relationship, Drug; Epithelial Cells; Humans; Lung Neoplasms; Mitogen-Activated Protein Kinase 3; NF-kappa B; Nitrosamines; Phosphorylation; Receptors, Nicotinic; Retinoblastoma Protein; Up-Regulation

Experimental evidence suggests that lung cancer development and progression can be linked to an increased proliferation rate.. To evaluate the immunohistochemical expression of seven components of the cell cycle machinery in a series of well characterised non-small cell lung cancer (NSCLC) specimens (n = 105).. Multivariate analysis revealed that simultaneous loss of expression of three of these factors--cyclin D1, the cyclin dependent kinase inhibitor p16, and the tumour suppressor retinoblastoma protein Rb2/p130--correlated with survival, confirming the hypothesis that the cyclin D1-p16-retinoblastoma tumour suppressor pathway is inactivated in most lung cancer samples.. These results suggest that loss of control of cell cycle checkpoints is a common occurrence in lung cancer and support the idea that functional cooperation between different cell cycle regulatory proteins constitutes another level of regulation in cell growth control and tumour suppression.

Topics: Aged; Aged, 80 and over; Biomarkers, Tumor; Carcinoma, Non-Small-Cell Lung; Cell Cycle Proteins; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p16; Female; Humans; Immunoenzyme Techniques; Lung Neoplasms; Male; Middle Aged; Neoplasm Proteins; Neoplasm Staging; Prognosis; Proteins; Retinoblastoma-Like Protein p130; Survival Analysis

Somatic mutations in the kinase domain of the epidermal growth factor receptor (EGFR), including L858R and exon 19 deletions, underlie responsiveness to gefitinib and erlotinib in non-small cell lung cancer (NSCLC). Acquired resistance to these tyrosine kinase inhibitors is in some cases mediated by a second mutation, T790M. Ansamycin antibiotics, such as geldanamycin, potently inhibit heat shock protein 90 (Hsp90), promoting ubiquitin-mediated degradation of oncogenic kinases that require the chaperone for proper conformational folding. Here, we show that L858R and deletion mutant EGFR proteins found in NSCLC interact with the chaperone and are sensitive to degradation following Hsp90 inhibition. In NIH/3T3 cells expressing either wild-type or mutant EGFR, diminution of expression of both L858R and EGFR delL747-S752, P753S occurred following exposure to 50 nmol/L geldanamycin over 24 hours, whereas partial diminution of wild-type EGFR required a minimum of 200 nmol/L drug. In time course experiments, mutant EGFR expression was depleted after only 4 hours of exposure to 1 micromol/L geldanamycin, whereas diminution of wild-type EGFR was less substantial and seen only following 12 hours. Similarly, EGFR proteins in NSCLC cell lines harboring EGFR mutations, including NCI-H1650, NCI-H3255, and NCI-H1975, were also more sensitive to geldanamycin-induced degradation compared with the protein in wild-type cells. Exposure of EGFR-mutant cell lines to geldanamycin induced marked depletion of phospho-Akt and cyclin D1 as well as apoptosis. These data suggest mutational activation of EGFR is associated with dependence on Hsp90 for stability and that Hsp90 inhibition may represent a novel strategy for the treatment of EGFR-mutant NSCLC.

Topics: Animals; Antibiotics, Antineoplastic; Apoptosis; Benzoquinones; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cyclin D1; Cysteine Proteinase Inhibitors; ErbB Receptors; Gene Deletion; HSP90 Heat-Shock Proteins; Humans; Lactams, Macrocyclic; Lung Neoplasms; Mice; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; NIH 3T3 Cells; Phosphorylation; Protein Serine-Threonine Kinases; Protein Structure, Tertiary; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Quinones

D-type cyclins (cyclins D1, D2, and D3) promote G1-S progression and are aberrantly expressed in cancer. We reported previously that all-trans-retinoic acid chemo-prevented carcinogenic transformation of human bronchial epithelial (HBE) cells through proteasomal degradation of cyclin D1. Retinoic acid is shown here to activate distinct mechanisms to regulate different D-type cyclins in HBE cells. Retinoic acid increased cyclin D2, decreased cyclin D3 and had no effect on cyclin D1 mRNA expression. Retinoic acid decreased cyclin D1 and cyclin D3 protein expression. Repression of cyclin D3 protein preceded that of cyclin D3 mRNA. Proteasomal inhibition prevented the early cyclin D3 degradation by retinoic acid. Threonine 286 (T286) mutation of cyclin D1 stabilized cyclin D1, but a homologous mutation of cyclin D3 affecting threonine 283 did not affect cyclin D3 stability, despite retinoic acid treatment. Lithium chloride and SB216763, both glycogen synthase kinase 3 (GSK3) inhibitors, inhibited retinoic acid repression of cyclin D1, but not cyclin D3 proteins. Notably, phospho-T286 cyclin D1 expression was inhibited by lithium chloride, implicating GSK3 in these effects. Expression of cyclin D1 and cyclin D3 was deregulated in retinoic acid-resistant HBE cells, directly implicating these species in retinoic acid response. D-type cyclins were independently targeted using small interfering RNAs. Repression of each D-type cyclin suppressed HBE growth. Repression of all D-type cyclins cooperatively suppressed HBE growth. Thus, retinoic acid repressed cyclin D1 and cyclin D3 through distinct mechanisms. GSK3 plays a key role in retinoid regulation of cyclin D1. Taken together, these findings highlight these cyclins as molecular pharmacologic targets for cancer chemoprevention.

Topics: Bronchi; Cell Line; Cell Transformation, Neoplastic; Cyclin D1; Cyclin D2; Cyclin D3; Cyclins; Epithelial Cells; Glycogen Synthase Kinase 3; Humans; Lung Neoplasms; RNA, Messenger; RNA, Small Interfering; Transfection; Tretinoin

Overwhelming evidence has demonstrated tobacco smoke (TS) is causally associated with various types of cancers, especially lung cancer. Sustained epithelial cell hyperplasia and squamous metaplasia are considered as preneoplastic lesions during the formation of lung cancer. The cellular and molecular mechanisms leading to lung cancer due to TS are not clear. Mitogen-activated protein kinases (MAPK)/activator protein-1 (AP-1) can be activated by various stimuli and play a critical role in the control of cell proliferation and differentiation. To date, information on the response of the MAPK/AP-1 pathway during hyperplasia and squamous metaplasia induced by TS is lacking. We therefore investigated the effects of TS on the development of epithelial hyperplasia and squamous metaplasia, regulation of MAPK/AP-1 activation, and expression of AP-1-regulated cell cycle proteins and differentiation markers in the lungs of rats. Exposure of rats to TS (30 mg/m(3) or 80 mg/m(3), 6 h/day, 3 days/week for 14 weeks) dramatically induced cell proliferation and squamous metaplasia in a dose-dependent manner, effects that paralleled the activation of AP-1-DNA binding activity. Phosphorylated ERK1/2, JNK, p38 and ERK5 were significantly increased by exposure to TS, indicating the activation of these MAPK pathways. Expression of Jun and Fos proteins were differentially regulated by TS. TS upregulated the expression of AP-1-dependent cell cycle proteins including cyclin D1 and proliferating cell nuclear antigen (PCNA). Among the AP-1-dependent cell differentiation markers, keratin 5 and 14 were upregulated, while loricrin, filaggrin and involucrin were downregulated following TS exposure. These findings suggest the important role of MAPK/AP-1 pathway in TS-induced pathogenesis, thus providing new insights into the molecular mechanisms of TS-associated lung diseases including lung cancers.

Topics: Animals; Cell Proliferation; Cyclin D1; Enzyme Activation; Filaggrin Proteins; Hyperplasia; Intermediate Filament Proteins; JNK Mitogen-Activated Protein Kinases; Keratins; Lung Neoplasms; Male; Membrane Proteins; Metaplasia; Mitogen-Activated Protein Kinases; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Proliferating Cell Nuclear Antigen; Protein Precursors; Proto-Oncogene Proteins c-fos; Proto-Oncogene Proteins c-jun; Rats; Rats, Inbred WKY; Signal Transduction; Smoking; Transcription Factor AP-1

In many human lung adenocarcinoma cell lines, a pathway involving epidermal growth factor receptor (EGFR), ErbB2 and ErbB3 receptors, phosphatidyl inositol 3-kinase (PI3K), Akt, glycogen synthase kinase 3-beta (GSK3-beta), and cyclin D1 controls cell growth, survival, and invasiveness. We have investigated this pathway in paired transformed/nontransformed cell lines from murine peripheral lung epithelium, E9/E10 and A5/C10. The E9 and A5 carcinoma lines expressed ErbB3 and transforming growth factor-alpha (TGF-alpha) and responded to TGF-alpha stimulation with protein complex formation including the p85 regulatory subunit of PI3K, activation of Akt, phosphorylation of GSK3-beta, and increased cyclin D1 protein and the cell cycle. ErbB3 and TGF-alpha were not detected in the nontransformed E10 and C10 cell lines. Nevertheless, exposure of E10 or C10 cells to TGF-alpha activated PI3K and Akt and increased cyclin D1 and cell growth. The effector pathway from the EGFR to PI3K in these nontransformed cells included the adaptor Grb2, the docking protein Gab1, and the phosphatase Shp2. Gab1 was highly expressed in E10 and C10 cells but not in the malignant E9 and A5 sister lines. Complexes of EGFR/Grb2/Gab1/Shp2 after TGF-alpha stimulation were prominent only in E10 and C10 cells. Thus, alternate pathways downstream of EGFR regulate mitosis in these paired malignant versus nontransformed lung cell lines.

Topics: Adaptor Proteins, Signal Transducing; Adenocarcinoma; Animals; Cell Line, Tumor; Cyclin D1; Enzyme Activation; Epithelial Cells; ErbB Receptors; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Humans; Intracellular Signaling Peptides and Proteins; Lung Neoplasms; Mice; Phosphatidylinositol 3-Kinases; Phosphoproteins; Protein Tyrosine Phosphatase, Non-Receptor Type 11; Protein Tyrosine Phosphatases; Proto-Oncogene Proteins c-akt; Receptor, ErbB-2; Receptor, ErbB-3; Transforming Growth Factor alpha

[6]-Gingerol, a pungent ingredient of ginger (Zingiber officinale Roscoe, Zingiberaceae), has anti-bacterial, anti-inflammatory, and anti-tumor-promoting activities. Here, we describe its novel anti-angiogenic activity in vitro and in vivo. In vitro, [6]-gingerol inhibited both the VEGF- and bFGF-induced proliferation of human endothelial cells and caused cell cycle arrest in the G1 phase. It also blocked capillary-like tube formation by endothelial cells in response to VEGF, and strongly inhibited sprouting of endothelial cells in the rat aorta and formation of new blood vessel in the mouse cornea in response to VEGF. Moreover, i.p. administration, without reaching tumor cytotoxic blood levels, to mice receiving i.v. injection of B16F10 melanoma cells, reduced the number of lung metastasis, with preservation of apparently healthy behavior. Taken together, these results demonstrate that [6]-gingerol inhibits angiogenesis and may be useful in the treatment of tumors and other angiogenesis-dependent diseases.

Topics: Animals; Aorta; Blotting, Western; Catechols; Cell Cycle; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cells, Cultured; Collagen; Cornea; Cyclin D1; DNA; Dose-Response Relationship, Drug; Drug Combinations; Electrophoresis, Polyacrylamide Gel; Endothelium, Vascular; Fatty Alcohols; Fibroblast Growth Factor 2; G1 Phase; Humans; In Vitro Techniques; Laminin; Lung Neoplasms; Male; Mice; Mice, Inbred C57BL; Models, Chemical; Mutagens; Neoplasm Metastasis; Neoplasm Transplantation; Neoplasms, Experimental; Neovascularization, Pathologic; NIH 3T3 Cells; Plant Extracts; Proteoglycans; Rats; Rats, Sprague-Dawley; Umbilical Veins; Vascular Endothelial Growth Factor A; Zingiber officinale

N-(4-hydroxyphenyl) retinamide [4-HPR], a synthetic retinoid, has been shown to inhibit tumor cell growth, invasion, and metastasis by a mechanism that is not fully understood. Because the nuclear factor-kappaB (NF-kappaB) has also been shown to regulate proliferation, invasion, and metastasis of tumor cells, we postulated that 4-HPR modulates the activity of NF-kappaB. To test this postulate, we examined the effect of this retinoid on NF-kappaB and NF-kappaB-regulated gene products. We found that 4-HPR potentiated the apoptosis induced by tumor necrosis factor (TNF) and chemotherapeutic agents, suppressed TNF-induced invasion, and inhibited RANKL-induced osteoclastogenesis, all of which are known to require NF-kappaB activation. We found that 4-HPR suppressed both inducible and constitutive NF-kappaB activation without interfering with the direct DNA binding of NF-kappaB. 4-HPR was found to be synergistic with Velcade, a proteasome inhibitor. Further studies showed that 4-HPR blocked the phosphorylation and degradation of IkappaBalpha through the inhibition of activation of IkappaBalpha kinase (IKK), and this led to suppression of the phosphorylation and nuclear translocation of p65. 4-HPR also inhibited TNF-induced Akt activation linked with IKK activation. NF-kappaB-dependent reporter gene expression was also suppressed by 4-HPR, as was NF-kappaB reporter activity induced by TNFR1, TRADD, TRAF2, NIK, and IKK but not that induced by p65 transfection. The expression of NF-kappaB-regulated gene products involved in antiapoptosis (IAP1, Bfl-1/A1, Bcl-2, cFLIP, and TRAF1), proliferation (cyclin D1 and c-Myc), and angiogenesis (vascular endothelial growth factor, cyclooxygenase-2, and matrix metalloproteinase-9) were also down-regulated by 4-HPR. This correlated with potentiation of apoptosis induced by TNF and chemotherapeutic agents.

Topics: Antineoplastic Agents; Apoptosis; Boronic Acids; Bortezomib; Carcinoma, Small Cell; Carrier Proteins; Cell Growth Processes; Cyclin D1; Cyclooxygenase 2; Down-Regulation; Drug Synergism; Enzyme Activation; Fenretinide; Genes, myc; Humans; I-kappa B Kinase; I-kappa B Proteins; Lung Neoplasms; Membrane Glycoproteins; NF-kappa B; NF-KappaB Inhibitor alpha; Oncogene Protein v-akt; Osteoclasts; Phosphorylation; Promoter Regions, Genetic; Proto-Oncogene Proteins c-myc; Pyrazines; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Transcription Factor RelA; Tumor Necrosis Factor-alpha; Vascular Endothelial Growth Factor A

The development and progression of epithelial cancers are the result of an imbalance in signals promoting and inhibiting cellular proliferation and apoptosis. The aim of this study is to evaluate the expression of cell-cycle and apoptosis regulators and correlate them with clinical outcome in the most frequent carcinomas, in order to establish common prognostic biomarkers independent of cancer origin. Using tissue microarrays (TMAs), we have analysed the immuno-expression of Ki-67, Bcl-2, Bax, cyclin D1, cyclin D3, CDK1, CDK2, CDK6, p16, p21, and p27 in a series of 205 carcinomas of the large bowel, breast, lung and prostate (80, 73, 37 and 15 cases, respectively). By univariate analysis, positivity for p27, p16 and Bcl-2 was associated with better overall survival (P<0.0135, P<0.0442 and P<0.0001, respectively). The risk of mortality was 2.3-fold greater in patients without Bcl-2 expression. TMA immunohistochemical analysis identified a subset of epithelial cancers with overlapping alterations in cell-cycle checkpoints, apoptosis regulators and tumour suppressor pathways. We found that in most common epithelial cancers, regardless of origin, Bcl-2 appears to be the key biological factor influencing clinical behaviour.

Topics: Adult; Analysis of Variance; bcl-2-Associated X Protein; Biomarkers, Tumor; Breast Neoplasms; Cell Cycle Proteins; Cyclin D1; Cyclin D3; Cyclin-Dependent Kinase Inhibitor p16; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase Inhibitor p27; Cyclin-Dependent Kinases; Cyclins; Female; Humans; Immunohistochemistry; Intestinal Neoplasms; Ki-67 Antigen; Lung Neoplasms; Male; Middle Aged; Neoplasms; Prognosis; Prostatic Neoplasms; Proto-Oncogene Proteins c-bcl-2; Survival Analysis; Tissue Array Analysis

To investigate the relationship between expression of cell cycle-related protein cyclin D1, p27kip1 and the pathogenesis of bronchioloalveolar carcinoma (BAC) and the value of prediction of prognosis.. Cyclin D1 and p27kip1 protein were detected by immunohistochemical En Vision method in 43 BACs.. The positivity of cyclin D1 in BAC was 65.1% (28/43), which was significantly higher than that in normal pulmonary tissue (0/13), P<0.01. No statistically significant association was found between cyclin D1 expression data and sex, age, tobacco-use history, histologic subtype (mucinous vs nonmucinous), stromal fibrosis, lymph node metastasis, clinical stage or postoperative survival period (P>0.05), while cyclin D1 expression was found to be negatively correlated with tumor size (P<0.05). The positivity of p27kip1 in BACs was 51.2% (22/43), significantly lower than that in normal pulmonary tissue (12/13), P<0.01. p27kip1 expression level was not associated with sex, age, tobacco-use history, tumor size or histologic subtype (P>0.05), but was negatively correlated with stromal fibrosis, lymph node metastasis and clinical stage (P<0.05); and positively associated with postoperative survival period (P<0.01). The survival rate of p27kip1 positive group was significantly higher than that of p27kip1 negative group (P<0.01). No statistically significant correlation was found between cyclin D1 and p27kip1 expression.. Increased cyclin D1 expression and decreased p27kip1 expression are related to the pathogenesis of BAC; decreased p27kip1 expression is associated with metastasis progression; immunodetection of p27kip1 is useful for assessment of prognosis.

Topics: Adenocarcinoma, Bronchiolo-Alveolar; Adult; Age Distribution; Aged; Biomarkers, Tumor; Cell Cycle Proteins; China; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p27; Female; Humans; Lung Neoplasms; Male; Middle Aged; Prognosis; Reproducibility of Results; Retrospective Studies; Risk Assessment; Risk Factors; Sensitivity and Specificity; Sex Distribution; Survival Analysis; Tumor Suppressor Proteins

Flavopiridol is the potent inhibitor of cdks sharing its function with endogenous cdk inhibitors, and causes arrest at both the G1 and G2 phases of the cell cycle resulting in apoptosis in various tumor cell lines. Cyclin-dependent kinase inhibitor p16INK4a induces cell cycle arrest in G1 or G2 or both, and is inactivated in many malignant tumors. In this study, we focused on the effects of flavopiridol on chemically-induced rat lung adenocarcinoma, osteosarcoma and malignant fibrous histiocytoma (MFH) cell lines showing different pattern of p16INK4a status. The data demonstrated that flavopiridol inhibited cellular growth in a dose- and time-dependent manner, inducing apoptosis within 24 h in all cell lines at a concentration of 300 nM. The growth inhibition rate was the greatest for lung adenocarcinoma cells, lacking p16INK4a expression associated with methylation-mediated gene silencing; 83% at a concentration of 300 nM for 72-h treatment; while the growth of osteosarcoma and MFH cells, both expressing p16INK4a, were inhibited at similar levels; 54-61% for osteosarcoma and 61-64% for MFH cell lines. Then, we further investigated the influence of p16INK4a induction upon the effect of flavopiridol in p16INK4a-deficient lung adenocarcinoma cells. 5-aza 2'-deoxycytidine (5-Aza-CdR) induced p16INK4a expression and inhibited cellular growth in lung adenocarcinoma at a similar level to that with flavopiridol treatment. After the induction of p16INK4a expression by 5-Aza-CdR, the growth inhibition rates of flavopiridol in the p16INK4a-induced lung adenocarcinoma cells could not achieve comparable inhibition to that in the p16INK4a-deficient cells; the efficacy was reduced compared to original p16INK4a-deficient cells at each concentration of 50, 100 and 500 nM for 72-h treatment. These data indicate that flavopiridol shows cell type specific inhibition and possibly acts in a more compensatory manner for endogenous p16INK4a function in tumor cells having the aberrations of p16INK4a gene.

Topics: Adenocarcinoma; Animals; Apoptosis; Bone Neoplasms; Cell Division; Cell Line, Tumor; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase Inhibitor p16; Cyclin-Dependent Kinases; DNA Methylation; Flavonoids; Gene Expression Regulation, Neoplastic; Histiocytoma, Benign Fibrous; Lung Neoplasms; Osteosarcoma; Piperidines; Promoter Regions, Genetic; Proto-Oncogene Proteins; Rats; RNA, Messenger

The relation between expression of cell cycle-regulator molecules and apoptosis was examined in surgical specimens and cultured human lung carcinoma cell lines. Immunohistochemical analysis for 133 cases revealed 2 types of staining pattern. The first group consisted of 95 cases (71.4%) characterized by apoptotic cells showing intensely positive staining for cdk4 and cyclin D1 but negative for other proteins (type A). In the second group (type B), comprising 38 cases (28.6%), apoptotic cells exhibited intense positive staining for any cyclins and cdks. Most of the latter cases had lost expression of Rb protein. When tumor cells retrieved from paraffin-embedded tissue were examined by flow cytometry, higher proportions of cells expressing only cdk4 or cyclin D1 in type A cases and of cells expressing any cyclin or cdk in type B cases showed a subdiploid DNA content. In survival analysis using the LI of apoptotic cells and cyclin/cdk-positive cells, the high-apoptosis/high-cyclin D1 group showed the poorest prognosis. Furthermore, forced overexpression of only cdk4 or cyclin D1 induced apoptosis in cultured cells with normal Rb protein, whereas overexpression of any cyclin or cdk induced apoptosis in cells defective for Rb protein. In conclusion, upregulation of cdk4/cyclin D1 may be a primary and critical factor in induction of apoptosis in human lung carcinomas in vivo. Moreover, inactivation of Rb protein renders cells more prone to apoptosis by abnormal expression of any cell-cycle protein.

Topics: Apoptosis; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinases; DNA, Neoplasm; Flow Cytometry; Humans; Immunohistochemistry; Lung Neoplasms; Prognosis; Proto-Oncogene Proteins

To study the effects of 9-cis-retinoic acid (9-cis-RA) on cell cycle and expression of cyclin D1 and cdk4 in lung cancer cells.. 9-cis-RA (1 x 10(-6) mol.L-1) was used to treat lung cancer cells for 24 h; Flow cytometry (FCM) was used to detect the percent of G0/G1 phase and S phase cells of three groups including blank control, DMSO control and 9-cis-RA groups; RT-PCR was used to analyze the expression changes of cyclin D1 and cdk4 before and after treatment with 9-cis-RA in lung cancer cells.. The percent of G0/G1 phase cells of 9-cis-RA groups was significantly higher than that of the control groups (P < 0.01 or P < 0.05) and the percent of S phase cells of 9-cis-RA groups was lower than that of the control groups (P < 0.01 or P < 0.05); the expression of cyclin D1 of PG, SPC-A1 and L78 cells was decreased (P < 0.01) and the expression of cdk4 of PG, A549 and L78 cells was also decreased (P < 0.01) after treatment with 9-cis-RA.. Most of the proliferation and the expression of cyclin D1 and cdk4 of PG, A549, SPC-A1 and L78 were inhibited by 9-cis-RA.

Topics: Adenocarcinoma; Alitretinoin; Antineoplastic Agents; Carcinoma, Squamous Cell; Cell Division; Cell Line, Tumor; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinases; G1 Phase; Humans; Lung Neoplasms; Proto-Oncogene Proteins; Resting Phase, Cell Cycle; S Phase; Tretinoin

Topics: Antineoplastic Agents; Apoptosis; Chromans; Cyclin D1; Humans; Lung Neoplasms; Prostaglandin D2; Proto-Oncogene Proteins p21(ras); Receptors, Cytoplasmic and Nuclear; RNA, Messenger; Thiazolidinediones; Transcription Factors; Troglitazone

A total of 111 pulmonary neuroendocrine tumors comprising 13 typical carcinoids, five atypical carcinoids, 44 large-cell neuroendocrine carcinomas and 49 small-cell carcinomas were immunohistochemically studied for dysregulated cyclin B1 expression and disruption of the Rb/p16/cyclin D1 pathway (Rb pathway), and the results were correlated with tumor proliferation activity and clinical outcome. Overexpression of cyclins B1 and D1, respectively, was detected in no and 15% typical carcinoids, 20 and 20% atypical carcinoids, 84 and 32% large-cell neuroendocrine carcinomas, 84 and 10% small-cell carcinomas. Loss of Rb and p16 expression, respectively, was observed in no and 14% typical carcinoids, no and 40% atypical carcinoids, 49 and 18% large-cell neuroendocrine carcinomas, 84 and 8% small-cell carcinomas. In summary, 29% typical carcinoids, 20% atypical carcinoids, 78% large-cell neuroendocrine carcinomas and 93% small-cell carcinomas had Rb pathway aberrations. Rb pathway aberration was mostly attributed to Rb loss in small-cell carcinomas, while p16 loss and/or cyclin D1 overexpression besides Rb loss also played an important role in large-cell neuroendocrine carcinomas, while cyclin D1 overexpression was the only cause of Rb pathway aberration in carcinoid tumors. Thus, both cyclin B1-associated G2/M arrest and Rb-mediated G1 arrest are consistently compromised in high-grade large-cell neuroendocrine carcinoma and small-cell carcinoma, but are generally intact or occasionally altered in carcinoid tumor; the mechanisms involved in Rb pathway aberration among the tumor categories are different, reflecting a genetic divergence among the individual tumor categories. Cyclin B1 expression closely correlated with the Ki-67 labeling index either in the individual tumor categories or overall tumors (P < 0.0001, r = 0.742), suggesting that cyclin B1 is one of the key factors regulating cell proliferation in pulmonary neuroendocrine tumors. Neither cyclins B1 and D1, Rb, p16, nor Ki-67 correlated with patient survival in individual tumor categories, suggesting that the prognostic significance of these factors is tumor-type specific.

Topics: Biomarkers, Tumor; Carcinoid Tumor; Carcinoma, Large Cell; Carcinoma, Small Cell; Cyclin B; Cyclin B1; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p16; Humans; Immunohistochemistry; Ki-67 Antigen; Lung Neoplasms; Neuroendocrine Tumors; Retinoblastoma Protein; Signal Transduction; Survival Analysis

A(3) adenosine receptor (A(3)AR) activation was shown to inhibit the growth of various tumor cells via the down-regulation of nuclear factor kappaB and cyclin D1. To additionally elucidate whether A(3)AR is a specific target, a survey of its expression in tumor versus adjacent normal cells was conducted.. A(3)AR mRNA expression in various tumor tissues was tested in paraffin-embedded slides using reverse transcription-PCR analysis. A comparison with A(3)AR expression in the relevant adjacent normal tissue or regional lymph node metastasis was performed. In addition, A(3)AR protein expression was studied in fresh tumors and was correlated with that of the adjacent normal tissue.. Reverse transcription-PCR analysis of colon and breast carcinoma tissues showed higher A(3)AR expression in the tumor versus adjacent non-neoplastic tissue or normal tissue. Additional analysis revealed that the lymph node metastasis expressed even more A(3)AR mRNA than the primary tumor tissue. Protein analysis of A(3)AR expression in fresh tumors derived from colon (n = 40) or breast (n = 17) revealed that 61% and 78% had higher A(3)AR expression in the tumor versus normal adjacent tissue, respectively. The high A(3)AR expression level in the tumor tissues was associated with elevated nuclear factor kappaB and cyclin D1 levels. High A(3)AR mRNA expression was also demonstrated in other solid tumor types.. Primary and metastatic tumor tissues highly express A(3)AR indicating that high receptor expression is a characteristic of solid tumors. These findings and our previous data suggest A(3)AR as a potential target for tumor growth inhibition.

Topics: Blotting, Western; Breast Neoplasms; Carcinoma, Ductal, Breast; Cell Line, Tumor; Colonic Neoplasms; Cyclin D1; Down-Regulation; Humans; Lung Neoplasms; Lymphatic Metastasis; Melanoma; Neoplasm Metastasis; Neoplasms; NF-kappa B; Receptor, Adenosine A3; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger

Cigarette smoke (CS) has been linked to cardiovascular, pulmonary, and malignant diseases. CS-associated malignancies including cancers of the larynx, oral cavity, and pharynx, esophagus, pancreas, kidney, bladder, and lung; all are known to overexpress the nuclear factor-kappaB (NF-kappaB)-regulated gene products cyclin D1, cyclooxygenase (COX)-2, and matrix metalloprotease-9. Whether the COX-2 inhibitor, celecoxib, approved for the treatment of colon carcinogenesis and rheumatoid arthritis, affects CS-induced NF-kappaB activation is not known, although the role of NF-kappaB in regulation of apoptosis, angiogenesis, carcinogenesis, and inflammation is established. In our study, in which we examined DNA binding of NF-kappaB in human lung adenocarcinoma H1299 cells, we found that cigarette smoke condensate (CSC)-induced NF-kappaB activation was persistent up to 24 h, and celecoxib suppressed CSC-induced NF-kappaB activation. Celecoxib was effective even when administered 12 h after CSC treatment. This effect, however, was not cell type-specific. The activation of inhibitory subunit of NF-kappaB kinase (IkappaB), as examined by immunocomplex kinase assay, IkappaB phosphorylation, and IkappaB degradation was also inhibited. Celecoxib also abrogated CSC-induced p65 phosphorylation and nuclear translocation and NF-kappaB-dependent reporter gene expression. CSC-induced NF-kappaB reporter activity induced by NF-kappaB inducing kinase and IkappaB alpha kinase but not that activated by p65 was also blocked by celecoxib. CSC induced the expression of NF-kappaB-regulated proteins, COX-2, cyclin D1, and matrix metalloproteinase-9, and celecoxib abolished the induction of all three. The COX-2 promoter that is regulated by NF-kappaB was activated by CSC, and celecoxib suppressed its activation. Overall, our results suggest that chemopreventive effects of celecoxib may in part be mediated through suppression of NF-kappaB and NF-kappaB-regulated gene expression, which may contribute to its ability to suppress inflammation, proliferation, and angiogenesis.

Topics: Carcinoma, Non-Small-Cell Lung; Celecoxib; Cyclin D1; Cyclooxygenase 2; Cyclooxygenase 2 Inhibitors; Cyclooxygenase Inhibitors; DNA, Neoplasm; Enzyme Activation; Gene Expression Regulation, Neoplastic; Genes, Reporter; Humans; I-kappa B Kinase; Isoenzymes; Lung Neoplasms; Matrix Metalloproteinase 9; Matrix Metalloproteinase Inhibitors; Membrane Proteins; NF-kappa B; Phosphorylation; Prostaglandin-Endoperoxide Synthases; Protein Serine-Threonine Kinases; Pyrazoles; Smoke; Smoking; Sulfonamides

The cyclooxygenase 2 (COX-2) inhibitor celecoxib (also called celebrex), approved for the treatment of colon carcinogenesis, rheumatoid arthritis, and other inflammatory diseases, has been shown to induce apoptosis and inhibit angiogenesis. Because NF-kappa B plays a major role in regulation of apoptosis, angiogenesis, carcinogenesis, and inflammation, we postulated that celecoxib modulates NF-kappa B. In the present study, we investigated the effect of this drug on the activation of NF-kappa B by a wide variety of agents. We found that celecoxib suppressed NF-kappa B activation induced by various carcinogens, including TNF, phorbol ester, okadaic acid, LPS, and IL-1 beta. Celecoxib inhibited TNF-induced I kappa B alpha kinase activation, leading to suppression of I kappa B alpha phosphorylation and degradation. Celecoxib suppressed both inducible and constitutive NF-kappa B without cell type specificity. Celecoxib also suppressed p65 phosphorylation and nuclear translocation. Akt activation, which is required for TNF-induced NF-kappa B activation, was also suppressed by this drug. Celecoxib also inhibited the TNF-induced interaction of Akt with I kappa B alpha kinase (IKK). Celecoxib abrogated the NF-kappa B-dependent reporter gene expression activated by TNF, TNF receptor, TNF receptor-associated death domain, TNF receptor-associated factor 2, NF-kappa B-inducing kinase, and IKK, but not that activated by p65. The COX-2 promoter, which is regulated by NF-kappa B, was also inhibited by celecoxib, and this inhibition correlated with suppression of TNF-induced COX-2 expression. Besides NF-kappa B, celecoxib also suppressed TNF-induced JNK, p38 MAPK, and ERK activation. Thus, overall, our results indicate that celecoxib inhibits NF-kappa B activation through inhibition of IKK and Akt activation, leading to down-regulation of synthesis of COX-2 and other genes needed for inflammation, proliferation, and carcinogenesis.

Topics: Carcinogens; Carcinoma, Non-Small-Cell Lung; Celecoxib; Cell Line, Tumor; Cyclin D1; Cyclooxygenase Inhibitors; Enzyme Activation; Genes, Reporter; Humans; I-kappa B Kinase; I-kappa B Proteins; Interleukin-1; Lipopolysaccharides; Lung Neoplasms; MAP Kinase Signaling System; Matrix Metalloproteinase Inhibitors; Neoplasm Proteins; NF-kappa B; NF-KappaB Inhibitor alpha; Okadaic Acid; Phosphorylation; Promoter Regions, Genetic; Protein Processing, Post-Translational; Protein Serine-Threonine Kinases; Proteins; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Pyrazoles; Receptors, Tumor Necrosis Factor; Sulfonamides; Tetradecanoylphorbol Acetate; TNF Receptor-Associated Factor 1; TNF Receptor-Associated Factor 2; Transcription Factor RelA; Tumor Necrosis Factor-alpha

The purpose of this study was to investigate the impact of survivin, cyclin D1, integrin beta1, and vascular endothelial growth factor (VEGF) in tumor on survival of patients with small adenocarcinoma of the lung. Seventy-two patients with pathologic stage I resected tumors <2 cm in diameter were entered into the study. Each patient underwent curative surgical resection for lung cancer between July 1992 and November 1999. The resected tumors were subjected to immunostaining for each gene. Thirty-five, 26, 6, and 16 patients had tumors with >10% survivin-, >20% cyclin D1-, >10% integrin beta1-, and >10% VEGF-positive cells, respectively. When the survival of 72 patients was compared according to each gene expression, the overall survival of patients with positive expression of survivin, cyclin D1, and integrin [beta]1 was significantly worse than that of individuals whose tumors had negative expression of each gene. By multivariate analysis controlling for each gene expression, no gene expression was an independent marker of poor prognosis, however, the overall survival of the complex gene expression (2 or more gene-positive) group (n = 35) was significantly worse than that of 0 or 1 gene-positive group (n = 37; log-rank test, P = 0.0011; Wilcoxon test, P = 0.0011). When the association between survival and pathologic factors, including lymphatic invasion, venous invasion, type of bronchioalveolar carcinoma, and complex gene positive expression was analyzed, only complex gene-positive expression was found to be a significant independent factor (hazard ratio = 0.085, P = 0.0299). It can be concluded that multiple increased expression of oncogene is a poor prognostic factor in patients with small adenocarcinoma of the lung.

Topics: Adenocarcinoma; Adult; Aged; Aged, 80 and over; Biomarkers; Cyclin D1; Female; Gene Expression; Humans; Immunohistochemistry; Inhibitor of Apoptosis Proteins; Integrin beta1; Lung Neoplasms; Male; Microtubule-Associated Proteins; Middle Aged; Neoplasm Proteins; Prognosis; Survival Analysis; Survivin; Vascular Endothelial Growth Factor A

Integrin-linked kinase (ILK), bound to the cytoplasmic tails of integrin beta1, beta2, and beta3, is thought to signal through AKT and glycogen synthase kinase-3beta (GSK-3beta) for survival and proliferation regulation. To determine the role of ILK in the cellular radiation response, stably transfected A549 lung cancer cells overexpressing either wild-type (ILK-wk) or hyperactive ILK (ILK-hk) were studied for survival, signaling, proliferation, and examined in immunofluorescence and adhesion assays. Strong radiosensitization was observed in ILK-hk in contrast to ILK-wk mutants and empty vector controls. ILK small interfering RNA transfections showed radioresistance similar to irradiation on fibronectin. AKT, GSK-3beta-cyclin D1, mitogen-activated protein kinase kinase 1/2-mitogen-activated protein kinase, and c-Jun NH2-terminal kinase signaling was dysregulated in irradiated ILK-hk mutants. Immunofluorescence stainings of ILK-hk cells indicated disturbed ILK and paxillin membrane localization with concomitant decrease in focal adhesions. Profound ILK-hk-dependent changes in morphology were characterized by spindle-like cell shape, cell size reduction, increased cell protrusions, strong formation of membranous f-actin rings, and significantly reduced adhesion to matrix proteins. Additionally, ILK-wk and ILK-hk overexpression impaired beta1-integrin clustering and protein Tyr-phosphorylation. Taken together, the data provide evidence that ILK signaling modulates the cellular radiation response involving diverse signaling pathways and through changes in f-actin-based processes such as focal adhesion formation, cell adhesion, and spreading. Identification of ILK and its signaling partners as potential targets for tumor radiosensitization might promote innovative anticancer strategies by providing insight into the mechanism of cell adhesion-mediated radioresistance, oncogenic transformation, and tumor growth and spread.

Topics: Actins; Cell Line, Tumor; Cyclin D1; Cytoskeletal Proteins; Extracellular Matrix Proteins; Focal Adhesions; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Humans; Integrin beta1; Lung Neoplasms; MAP Kinase Signaling System; Paxillin; Phosphoproteins; Phosphorylation; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Radiation Tolerance; Retinoblastoma Protein; Subcellular Fractions; Transfection; Tyrosine

Caveolin-1 (Cav-1) is the principal structural component of caveolae membrane domains in non-muscle cells, including mammary epithelia. There is now clear evidence that caveolin-1 influences the development of human cancers. For example, a dominant-negative mutation (P132L) in the Cav-1 gene has been detected in up to 16% of human breast cancer samples. However, the exact functional role of caveolin-1 remains controversial. Mechanistically, in cultured cell models, Cav-1 is known to function as a negative regulator of the Rasp42/44 MAP kinase cascade and as a transcriptional repressor of cyclin D1 gene expression, possibly explaining its in vitro transformation suppressor activity. Genetic validation of this hypothesis at the in vivo and whole organismal level has been prevented by the lack of a Cav-1 (-/-)-null mouse model. Here, we examined the role of caveolin-1 in mammary tumorigenesis and lung metastasis using a molecular genetic approach. We interbred a well characterized transgenic mouse model of breast cancer, MMTV-PyMT (mouse mammary tumor virus-polyoma middle T antigen), with Cav-1 (-/-)-null mice. Then, we followed the onset and progression of mammary tumors and lung metastases in female mice over a 14-week period. Interestingly, PyMT/Cav-1 (-/-) mice showed an accelerated onset of mammary tumors, with increased multiplicity and tumor burden ( approximately 2-fold). No significant differences were detected between PyMT/Cav-1 (+/+) and PyMT/Cav-1 (+/-) mice, indicating that complete loss of caveolin-1 is required to accelerate both tumorigenesis and metastasis. Molecularly, mammary tumor samples derived from PyMT/Cav-1 (-/-) mice showed ERK-1/2 hyperactivation, cyclin D1 up-regulation, and Rb hyperphosphorylation, consistent with dys-regulated cell proliferation. PyMT/Cav-1 (-/-) mice also developed markedly advanced metastatic lung disease. Conversely, recombinant expression of Cav-1 in a highly metastatic PyMT mammary carcinoma-derived cell line, namely Met-1 cells, suppressed lung metastasis by approximately 4.5-fold. In vitro, these Cav-1-expressing Met-1 cells (Met-1/Cav-1) demonstrated a approximately 4.8-fold reduction in invasion through Matrigel-coated membranes. Interestingly, delivery of a cell permeable peptide encoding the caveolin-1 scaffolding domain (residues 82-101) into Met-1 cells was sufficient to inhibit invasion. Coincident with this decreased invasive index, Met-1/Cav-1 cells exhibited marked reductions in MMP-9 and MMP-2

Topics: Animals; Caveolin 1; Caveolins; Cell Line, Tumor; Cell Membrane; Cell Movement; Cells, Cultured; Collagen; Cyclin D1; Disease Progression; Drug Combinations; Electrophoresis, Polyacrylamide Gel; Enzyme Activation; Female; Gene Expression Regulation, Neoplastic; Immunoblotting; Inhibitory Concentration 50; Laminin; Lung Neoplasms; Male; Mammary Neoplasms, Animal; Mammary Tumor Virus, Mouse; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Mice; Mice, Transgenic; Microscopy, Fluorescence; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mutation; Neoplasm Invasiveness; Neoplasm Metastasis; Peptides; Phosphorylation; Proteoglycans; Recombinant Proteins; Retinoblastoma Protein; Retroviridae; Time Factors; Up-Regulation

Many studies showed that hypoxia inducible factor-1 alpha (HIF-1alpha)was an essential component for hypoxia- induced cell cycle arrest, but the definite mechanism and the degree of HIF-1alpha affecting cell cycle arrest were unknown yet. This study was to explore the probable mechanism of hypoxia-induced tumor cell cycle arrest.. Human lung adenocarcinoma cell line A549 were divided into 3 groups: 12-h hypoxia group, 24-h hypoxia group, and control group. The hypoxia groups were exposed to hypoxic conditions (37degrees C, 5% CO2, and 2.0% O2) for 12 h, and 24 h, respectively, while control group was exposed to normal oxygen conditions (37 degrees C, 5% CO2, and 21% O2) for 24 h. Flow cytometry was used to measure the distribution of cell cycles and the expression of cyclin D1. The expression of HIF-1alpha, and p53 was detected using immunohistochemistry.. (1) The ratio of G(0)/G(1) in 12-h hypoxia group was (70.20+/-3.33)%, and in 24-h hypoxia group was (82.85+/-1.75)%, significantly higher than that in control group [(50.36+/-4.09) %] (F=202.34, P< 0.01).(2) There was significant difference in cyclin D1 expression among 12-h hypoxia group [(80.22+/-1.55)%], 24-h hypoxia group [(73.65+/-2.10)%], and control group [(90.35+/-2.68)%] (F=100.45, P< 0.01). (3) HIF-1alpha expression in 12-hypoxia group, 24-hypoxia group, and control group was 0.16 +/- 0.02, 0.26 +/- 0.05, and 0.01 +/- 0.00, respectively, with significant difference among the 3 groups (F=105.28, P< 0.01). (4) In hypoxia groups, cyclin D1 expression was negatively correlated with G(0)/G(1) arrest (r=-0.91, P< 0.01), HIF-1alpha expression was positively correlated with p53 expression (r=0.84, P< 0.01), and negatively correlated with cyclin D1 expression (r=-0.90, P< 0.01), and p53 expression was negatively correlated with cyclin D1 expression (r=-0.78,P< 0.01).. Hypoxia can cause G(0)/G(1) cell cycle arrest in human lung adenocarcinoma cell line A549. HIF-1alpha-p53-cyclin D1 pathway might play an important role in the hypoxia-induced G(0)/G(1) cell cycle arrest of human lung adenocarcinoma cell line A549.

Topics: Adenocarcinoma; Cell Hypoxia; Cell Line, Tumor; Cyclin D1; G1 Phase; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Lung Neoplasms; Resting Phase, Cell Cycle; Transcription Factors; Tumor Suppressor Protein p53

Asbestos exposure causes activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) in lung epithelial cells, the targets of asbestos-associated lung carcinomas. The functional significance of ERK1/2 activation in pulmonary epithelial and mesothelial cells is unclear. Using serum-stimulated mouse alveolar type II epithelial cells as a model for cell cycle reentry, we show that the duration of phospho-ERK1/2 in the nucleus determines cell fate in response to crocidolite asbestos. In response to 10% serum, a proliferative stimulus, phosphorylated ERK1/2 initially accumulated in the nucleus, and reduction of nuclear phospho-ERK1/2 after 2 to 4 hours was followed by expression of cyclin D1 and S-phase entry. Low levels of asbestos (<0.5 microg/cm2) promoted S-phase entry in low (2%) serum through an epidermal growth factor receptor-dependent pathway but did not promote cell cycle progression or induce apoptosis in the presence of high (10%) serum-containing medium. Higher levels of asbestos (1.0 to 5.0 microg/cm2) prolonged the localization of phospho-ERK1/2 in the nucleus in the presence of high serum, impeded S-phase entry, and induced apoptosis in a dose-dependent manner. Immunofluorescence microscopy indicated that the duration of signaling by phospho-ERK1/2 in the nucleus was predictive of cell fate at any concentration of asbestos. After 8 hours of exposure, cells with nuclear phospho-ERK1/2 also were positive for nuclear localization of apoptosis-inducing factor (AIF), an early event in apoptosis. In contrast, asbestos-exposed cells that displayed cytoplasmic phospho-ERK1/2 at 8 hours expressed cyclin D1 and proceeded to S phase. Our studies show that prolonged localization of phospho-ERK1/2 in the nucleus is incompatible with expression of cyclin D1 and is predictive of asbestos-associated cell death by AIF, thereby providing an approach for determining cell fate in asbestos-induced tumorigenesis.

Topics: Animals; Apoptosis; Apoptosis Inducing Factor; Asbestos, Crocidolite; Cell Cycle; Cell Division; Cell Nucleus; Cyclin D1; Flavoproteins; Lung Neoplasms; MAP Kinase Signaling System; Membrane Proteins; Mice; Mitochondria; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Phosphorylation; Pulmonary Alveoli

Recently, the authors identified molecular signatures and pathways associated with nonsmall cell lung carcinoma histology and lung development. They hypothesized that genetic classifiers of histology would provide insight into lung tumorigenesis and would be associated with clinical outcome when evaluated in a broader set of specimens.. Associations between patient survival and immunostaining for 11 representative histologic classifiers (epidermal growth factor receptor [EGFR], CDK4, syndecan-1, singed-like, TTF-1, keratin 5, HDAC2, docking protein 1, integrin alpha3, P63, and cyclin D1) were examined using a tissue microarray constructed from nonsmall cell lung carcinoma specimens.. Sixty-three tumors were examined, including 43 adenocarcinomas, 11 large cell carcinomas, and 9 squamous cell carcinomas. Sixty-three percent of tumors were clinical Stage I lesions, and 37% were Stage II-III lesions. In a multivariate analysis that controlled for age, gender, and race, syndecan-1 expression was found to be associated with a significant reduction in the risk of death (hazard ratio, 0.31 [95% confidence interval, 0.18-0.87]; P < 0.05). Multivariate analysis also indicated that EGFR expression was associated with a significant reduced risk of death.. The authors demonstrated that expression of either of the nonsmall cell lung carcinoma subtype classifiers syndecan-1 and EGFR was associated with a 30% reduction in the risk of death, with this reduction being independent of histology and other confounders. The results of the current study suggest that loss of expression of these histologic classifiers is associated with biologic aggressiveness in lung tumors and with poor outcome for patients with such tumors. If their significance can be validated prospectively, these biomarkers may be used to guide therapeutic planning for patients with nonsmall cell lung carcinoma.

Topics: Adenocarcinoma; Aged; Carcinoma, Large Cell; Carcinoma, Non-Small-Cell Lung; Carcinoma, Squamous Cell; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinases; ErbB Receptors; Female; Histone Deacetylase 2; Histone Deacetylases; Humans; Immunohistochemistry; Integrin alpha Chains; Keratin-5; Keratins; Lung Neoplasms; Male; Membrane Glycoproteins; Membrane Proteins; Multivariate Analysis; Nuclear Proteins; Proteoglycans; Proto-Oncogene Proteins; Repressor Proteins; Syndecan-1; Syndecans; Thyroid Nuclear Factor 1; Transcription Factors

To study the inhibitory effects of ciglitazone, a synthetic ligand of peroxisome proliferator-activated receptors (PPAR), on human lung cancer growth in vitro and in vivo and its mechanisms.. Human lung cancer A549 cells cultured in vitro were treated with different concentrations of ciglitazone. The proliferative activity and cell cycle of A549 cells were determined by MTT assay and flow cytometry. Expression of PPARgamma protein was detected by Western blot. A549 cells (1 x 10(6) cells/nude mouse) were inoculated subcutaneously into nude mice, which were randomly divided into two groups, 10 in each: control group (group A) and ciglitazone treated group (group B). When the tumors grew to a size with diameter around 1 cm, ciglitazone 100 microl (100 micromol/L) was intratumorally injected every other day in group B mice. A total of 15 injections were given. Mice in group A were similarly treated with normal saline. One month later, tumors were excised and weighed. Expression of cyclin D1 and p21 protein were detected by immunohistochemistry and Western blot.. Growth of A549 cells was significantly inhibited in group B in a dose-dependent and time-dependent fashion as compared with that in group A. Most of the ciglitazone-treated cells arrested in G(1)/G(0) phase and the expression of PPARgamma protein was markedly up-regulated. The tumor weights in group A was (2.79 +/- 0.33) g and that in group B was (1.51 +/- 0.40) g, with an inhibition rate of 47.0%. The expression level of cyclin D1 in group A was significantly higher than that in group B, while the expression level of p21 protein in group A was significantly lower than that in group B.. Ciglitazone can effectively inhibit the growth of human lung cancer A549 and induce its differentiation by cell cycle arrest via PPARgamma activation.

Topics: Animals; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Dose-Response Relationship, Drug; Female; G1 Phase; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Mice; Mice, Inbred BALB C; Mice, Nude; PPAR gamma; Random Allocation; Resting Phase, Cell Cycle; Thiazolidinediones; Time Factors

Topics: Alleles; Cyclin D1; DNA Mutational Analysis; DNA Primers; Drosophila Proteins; Genotype; Homeodomain Proteins; Homozygote; Humans; Lung; Lung Neoplasms; Membrane Proteins; Polymerase Chain Reaction; Polymorphism, Restriction Fragment Length; Receptors, G-Protein-Coupled; Substrate Specificity

The occurrence and development of tumors are controled by oncogene, antioncogene and tumor metastasis-related gene. Cyclin D1 is the expressive product of CCND1(a kind of oncogen). Vascular endothelial growth factor (VEGF) regulates the growth of neoplastic angiogenesis, which plays a role in the invasion and metastasis of tumor. The objective of this study was to detect the expression of Cyclin D1 and VEGF in non-small cell lung cancer (NSCLC), and to explore their association with the prognosis of NSCLC.. Immunohistochemistry was used to detect the expression of Cyclin D1 and VEGF in pathological tissue sections of 55 cases of NSCLC and 10 cases of non-malignant tissue from lung lesions. The relationship between the expression of Cyclin D1 and VEGF in 55 NSCLC sections and the age, sex, smoke, size of tumor, histopathological type, differentiation, stage, lymph node metastasis and survival time were analyzed statistically.. The expression rates of Cyclin D1 and VEGF in the 55 NSCLC tissue sections were 61.82% and 74.55%, respectively. In 10 cases of non-malignant tissue sections, cyclin D1 expression was not detected and VEGF expression (+/-) was only in 2 cases. The expression of Cyclin D1 and VEGF showed: (1) there was no significant difference among age, sex, histopathological type, stage, differentiation, tumor size and smoking level; (2) there were significant differences between the patients with and without lymph node metastasis; (3) there were significant differences of survival time between positive and negative expression groups. It meant Cyclin D1 and VEGF would be the poor prognostic factors.. The expression of Cyclin D1 and VEGF occurred in more than 60% cases of NSCLC, which may play a role in the biologic behavior and prognosis of NSCLC.

Topics: Adult; Aged; Carcinoma, Non-Small-Cell Lung; Cyclin D1; Female; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Lymphatic Metastasis; Male; Middle Aged; Neoplasm Staging; Prognosis; Survival Rate; Vascular Endothelial Growth Factor A

Using laser capture microdissection (LCM), fluorescent microsatellite analysis and immunohistochemical analysis, we have constructed a detailed topographical molecular map of the entire bronchial tree surrounding a primary bronchial squamous carcinoma in order to establish the relationship between the molecular damage within the airway and that in the tumour itself. Allelic imbalance was analysed using markers on chromosomes 3, 9, 13 and 17. In addition, immunohistochemical analysis for p53 and cyclin D1 expression was performed. Analysis revealed allelic imbalance at several loci at the tumour site but also in 83% of the histologically normal airway specimens of the upper and lower lobes. The fractional allele loss (FAL) value was statistically higher (0.75+/-0.13) in the tumour site than in the distal site of the upper (0.42+/-0.09) and lower lobes (0.31+/-0.08). Immunohistochemical analysis revealed overexpression of p53 and cyclin D1 protein within histologically normal bronchial epithelium, thus confirming previous reports for their early involvement in lung tumour development. This is to date the largest in-depth study of allelic imbalance using LCM in a single individual. The patterns of allele-specific imbalance observed support a clonal or oligoclonal expansion model of outgrowths throughout the lung. The widespread incidence of genetic changes in the whole of lung most likely represents smoking-induced alterations and emphasize the complexity of the field cancerization concept. Our findings point to the need for in-depth studies of the whole bronchial tree tissue surrounding lung carcinomas, in order to identify the genetic changes that differentiate preneoplastic and neoplastic stages in lung carcinogenesis.

Topics: Aged; Alleles; Allelic Imbalance; Bronchi; Bronchial Neoplasms; Carcinoma, Non-Small-Cell Lung; Carcinoma, Squamous Cell; Cell Transformation, Neoplastic; Clone Cells; Cyclin D1; Disease Progression; DNA, Neoplasm; Epithelial Cells; Gene Expression Regulation, Neoplastic; Genes, p53; Humans; Lasers; Lung; Lung Neoplasms; Male; Microsatellite Repeats; Neoplasm Proteins; Neoplastic Stem Cells; Proliferating Cell Nuclear Antigen; Tumor Suppressor Protein p53

Rb protein in its hypophosphorylated form acts as a cell cycle regulator for G1 arrest. Both cyclin D1 overexpression and P16(INK4) loss of protein produce persistent hyperphosphorylation of Rb with resultant evasion of cell cycle arrest. To better establish the mechanisms of loss of Rb function in neuroendocrine lung tumors, we performed an immunohistochemical analysis of the P16(INK4)/cyclin D1/Rb pathway in the spectrum of neuroendocrine tumors, including 34 typical carcinoids (TCs), 25 atypical carcinoids (ACs), 42 large cell neuroendocrine carcinomas (LCNECs), and 79 small cell lung carcinomas (SCLCs). Absence of Rb expression was not observed in TCs but was seen in 21% of ACs, 68% of LCNECs, and 87% of SCLCs. P16 was expressed in 91% of TCs, 77% of ACs, 78% of LCNECs, and 93% of SCLCs. Cyclin D1 was overexpressed in 6% of TCs, 20% of ACs, 9.5% of LCNECs, and 1.3% of SCLCs. There was an inverse relationship between Rb and P16 in high-grade tumors (P < 0.001) and a direct relationship between cyclin D1 and Rb (P < 0.001) in all tumors, demonstrating that P16 and cyclin D1 act exclusively on the Rb pathway for cell cycle regulation. Overall, the Rb pathway (Rb/P16(INK4)/cyclin D1) was altered more frequently in ACs than in TCs (P = 0.001) and more frequently in LCNECs than in ACs (P = 0.001). Although Rb-negative tumors had shorter survival in the overall group (P < 0.001) as a result of lack of Rb in most SCLCs, cyclin D1 overexpression and P16 loss did not influence survival in any individual category. We conclude that Rb pathway of G1 arrest is consistently compromised in high-grade neuroendocrine lung tumors (92%), primarily through loss of Rb protein, and is intact in low-grade TCs. In ACs an intermediate level of alterations (59%) is seen, consistent with their less-aggressive behavior compared with high-grade tumors. The specific profile of the Rb pathway parameters might provide specific therapeutic targets in neuroendocrine lung tumors.

Topics: Adult; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p16; Female; G1 Phase; Humans; Immunohistochemistry; Lung Neoplasms; Male; Middle Aged; Neuroendocrine Tumors; Prognosis; Retinoblastoma Protein; Survival Rate

Heterogeneous nuclear ribonucleoprotein B1 (hnRNP B1), an RNA binding protein, is a useful marker for early detection of lung squamous cell carcinoma because it is overexpressed in the early stages of lung cancer, including bronchial dysplasia, a premalignant lesion of lung squamous cell carcinoma. In the case of adenocarcinoma, we investigated the utility of hnRNP B1 for both detection of early adenocarcinoma and discrimination of non-invasive lesion, atypical adenomatous hyperplasia (AAH) from adenocarcinoma. hnRNP B1, cyclin D1, p16, and Ki-67 were analyzed in lung adenocarcinoma tissues and divided into early and overt adenocarcinoma and AAH, using immunohistochemistry. The intensity of these molecular markers was compared among three groups and also analyzed for 4 patients who showed both adenocarcinoma and AAH. Thirty-six of 54 (67%) adenocarcinoma patients showed positive staining of hnRNP B1: 14/20 (70%) early adenocarcinoma and 22/34 (65%) overt adenocarcinoma. In contrast, overexpression of hnRNP B1 in non-invasive lesion, AAH was observed in only 9% (1/11). Overexpression of cyclin D1 and decrease of p16 were frequently observed in both adenocarcinoma and AAH. These results suggest that hnRNP B1 would be a candidate of molecular marker for detection of early lung adenocarcinoma. In addition, combined analysis of hnRNP B1 and cell cycle-related genes, such as cyclin D1 and p16, might aid in discrimination of AAH from early adenocarcinoma.

Topics: Adenocarcinoma; Aged; Biomarkers, Tumor; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p16; Disease Progression; Female; Heterogeneous-Nuclear Ribonucleoprotein Group A-B; Humans; Hyperplasia; Immunoenzyme Techniques; Ki-67 Antigen; Lung; Lung Neoplasms; Male; Middle Aged; Neoplasm Proteins; Precancerous Conditions

Topics: Alleles; Carcinoma, Non-Small-Cell Lung; Cyclin D1; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Respiratory Mucosa; Smoking

Cyclin D1 is overexpressed in almost 60% of resectable non-small-cell lung cancer (NSCLC). In the absence of cyclin D1 gene amplification, overexpression is characterized by allelic imbalanced transcript levels.. The aims were to study cyclin D1 expression by immunohistochemistry and allelic balance of transcripts in tumor-free bronchial epithelia from patients with resectable NSCLC by using monoclonal antibodies (48 patients and 288 sites), microdissection/reverse transcriptase polymerase chain reaction/restriction fragment length polymorphism analyses (24 patients and 144 sites). Derived data were related to patient characteristics-in particular, smoking habits.. In 167 (58%) of 288 sites, cyclin D1 was overexpressed, with cytoplasmic and nuclear sublocalization in 53% and 7% of all sites, respectively. Nuclear overexpression was more frequent in premalignant versus normal or hyperplastic epithelia (55% v 3%; P <.0001). Allele-specific expression imbalances were found in 69 (48%) of 144 sites; in particular, those in which cyclin D1 was overexpressed (P =.004). In 14 (58%) of 24 patients, balanced or imbalanced transcript ratios and degree of expression were consistent at all sites for the same patient, whereas in another 10 patients, transcript balances and cyclin D1 expression patterns varied across the sites. Nuclear cyclin D1 expression in at least one site (14 of 48 patients) was linked to heavy smoking (> 40 pack-years; P =.02) and shorter overall survival (P =.01).. Allele-specific, probably damage-driven, deregulation of the cyclin D1 gene may precede and perhaps facilitate the spread of preneoplastic clones across the bronchial epithelial surface in a significant number of patients. Cyclin D1 expression at multiple bronchial sites may identify a subgroup of heavy-smoking patients with poor outcome.

Topics: Adult; Aged; Aged, 80 and over; Alleles; Carcinoma, Non-Small-Cell Lung; Cyclin D1; Female; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Male; Middle Aged; Polymorphism, Restriction Fragment Length; Proportional Hazards Models; Prospective Studies; Respiratory Mucosa; Smoking; Statistics, Nonparametric; Survival Rate; Switzerland

Bronchial carcinogenesis is a multistep process characterized by accumulation of genetic and molecular abnormalities, which precedes and accompanies the preinvasive lesions known as dysplasia and carcinoma in situ (CIS). We hypothesized that the level of accumulated molecular abnormalities in dysplasia assessed by immunohistochemical markers might reflect the severity of the carcinogenic process, thus allowing for risk assessment in smokers.. We performed a prospective analysis of bronchial biopsies in 48 former smokers who had at least one area of metaplasia. Twenty-two of the patients had a previous history of lung cancer. Eighty bronchial lesions were recorded at baseline, including 31 metaplasia, 12 mild dysplasia, 9 moderate dysplasia, 9 severe dysplasia, and 19 CISs. Forty-one percent of the patients had multiple preinvasive lesions. Immunohistochemical analysis of P53, cyclin D1, cyclin E, Bax, and Bcl2 was performed. Aberrant expression of one of these proteins as compared with normal bronchi was recorded as one molecular alteration.. After 18 months, 17 patients were diagnosed with lung cancer. No isolated parameter, including dysplastic grade or any isolated molecular alteration, was significantly associated with cancer occurrence at 18 months follow-up, using a logistic regression statistical analysis. In contrast, considering CIS and cancer as end point, more than two immunohistochemical abnormalities were associated with cancer or CIS occurrence (P = 0.02).. We concluded that the cumulative index of immunohistochemical abnormalities in a random dysplasia is associated with CIS or lung cancer in the cancerization field of symptomatic smokers, independently of the histopathological grade of dysplasia. This set of histopathological biomarkers might be useful in risk assessment and provide intermediate end points for chemopreventive trials.

Topics: Adult; Aged; Aged, 80 and over; bcl-2-Associated X Protein; Biomarkers; Bronchi; Bronchial Neoplasms; Carcinoma in Situ; Cyclin D1; Cyclin E; Cyclin-Dependent Kinase Inhibitor p16; Female; Follow-Up Studies; Humans; Immunohistochemistry; Lung Neoplasms; Male; Middle Aged; Multivariate Analysis; Precancerous Conditions; Prospective Studies; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Risk Assessment; Tumor Suppressor Protein p53

Cigarette smoke (CS) is a major cause of a variety of malignancies including cancers of the larynx, oral cavity and pharynx, esophagus, pancreas, kidney, bladder and lung. The signal transduction pathway that mediates the effects of CS is not well understood but nuclear factor-kappa B (NF-kappaB) is probably involved. The gas phase of CS contains free radicals such as superoxide radicals, hydroxyl radicals and hydrogen peroxide, which potentially can activate NF-kappaB. Benzo[a]pyrene, another potent carcinogen of CS, can also activate NF-kappaB, but by an as yet unknown mechanism. Various other agents that activate NF-kappaB are either tumor initiators or tumor promoters, and NF-kappaB activation can block apoptosis, promote proliferation and mediate tumorigenesis. Therefore, NF-kappaB is an ideal target for preventing CS-induced lung carcinogenesis. Thus, agents that abrogate NF-kappaB activation have the potential to suppress lung carcinogenesis. Because curcumin, a diferuloylmethane, is anticarcinogenic, we investigated the effect of this phytochemical on CS-induced NF-kappaB activation and NF-kappaB-regulated gene expression in human non-small cell lung carcinoma cells. Exposure of cells to CS induced persistent activation of NF-kappaB, and pre-treatment with curcumin abolished the CS-induced DNA-binding of NF-kappaB, IkappaBalpha kinase activation, IkBalpha phosphorylation and degradation, p65 nuclear translocation and CS-induced NF-kappaB-dependent reporter gene expression. The inhibition of NF-kappaB activation correlated with suppression of CS-induced NF-kappaB-dependent cyclin D1, cyclooxygenase-2 and matrix metalloproteinase-9 expression. Overall our results indicate that CS-induced NF-kappaB activation and NF-kappaB-regulated gene expression in human non-small cell lung carcinoma cells is suppressed by curcumin through suppression of IkappaBalpha kinase.

Topics: Antineoplastic Agents; Blotting, Western; Carcinoma, Non-Small-Cell Lung; Cells, Cultured; Curcumin; Cyclin D1; Cyclooxygenase 2; DNA Primers; Down-Regulation; Epithelial Cells; Humans; I-kappa B Kinase; Isoenzymes; Luciferases; Lung Neoplasms; Matrix Metalloproteinase 9; Membrane Proteins; NF-kappa B; Phosphorylation; Prostaglandin-Endoperoxide Synthases; Protein Serine-Threonine Kinases; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Smoking; Transfection

RASSF1A is a recently identified 3p21.3 tumor suppressor gene. The high frequency of epigenetic inactivation of this gene in a wide range of human sporadic cancers including non-small cell lung cancer (NSCLC) and neuroblastoma suggests that RASSF1A inactivation is important for tumor development. Although little is known about the function of RASSF1A, preliminary data suggests that it may have multiple functions. To gain insight into RASSF1A functions in an unbiased manner, we have characterized the expression profile of a lung cancer cell line (A549) transfected with RASSF1A. Initially we demonstrated that transient expression of RASSF1A into the NSCLC cell line A549 induced G(1) cell cycle arrest, as measured by propidium iodide staining. Furthermore, annexin-V staining showed that RASSF1A-expressing cells had an increased sensitivity to staurosporine-induced apoptosis. We then screened a cDNA microarray containing more than 6000 probes to identify genes differentially regulated by RASSF1A. Sixty-six genes showed at least a 2-fold change in expression. Among these were many genes with relevance to tumorigenesis involved in transcription, cytoskeleton, signaling, cell cycle, cell adhesion, and apoptosis. For 22 genes we confirmed the microarray results by real-time RT-PCR and/or Northern blotting. In silico, we were able to confirm the majority of these genes in other NSCLC cell lines using published data on gene expression profiles. Furthermore, we confirmed 10 genes at the RNA level in two neuroblastoma cell lines, indicating that these RASSF1A target genes have relevance in non-lung cell backgrounds. Protein analysis of six genes (ETS2, Cyclin D3, CDH2, DAPK1, TXN, and CTSL) showed that the changes induced by RASSF1A at the RNA level correlated with changes in protein expression in both non-small cell lung cancer and neuroblastoma cell lines. Finally, we have used a transient assay to demonstrate the induction of CDH2 and TGM2 by RASSF1A in NSCLC cell lines. We have identified several novel targets for RASSF1A tumor suppressor gene both at the RNA and the protein levels in two different cellular backgrounds. The identified targets are involved in diverse cellular processes; this should help toward understanding mechanisms that contribute to RASSF1A biological activity.

Topics: Apoptosis; Carcinoma, Non-Small-Cell Lung; Cyclin D1; Down-Regulation; G1 Phase; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Genes, Tumor Suppressor; Humans; Lung Neoplasms; Neoplasm Proteins; Neuroblastoma; Oligonucleotide Array Sequence Analysis; Reverse Transcriptase Polymerase Chain Reaction; Staurosporine; Transfection; Tumor Cells, Cultured; Tumor Suppressor Proteins

Recent study indicated that the components of Toona sinensis Roemor have potent anti-inflammatory and analgesic effects. These components have also been reported to inhibit the growth of boils in vivo. In this study, we investigated the effect of crude extract from the leaves of Toona sinensis Roemor on the proliferation of A549 lung cancer cells. We found that the extract effectively blocked cell cycle progression by inhibiting the expression of cyclin D1 and E in A549 cells. Additionally, incubation of the extract led to activation of caspase-3-like proteases and apoptotic cell death. Conversely, the extract did not show any significant cytotoxic effect on primarily cultured human foreskin fibroblasts or MRC-5 human lung fibroblasts. Therefore, antiproliferative action of the extract is specific for tumor cells. Our results suggest that the components of Toona sinensis Roemor have potent anticancer effects in vitro and identification of the useful components in the extract may lead to the development of a novel class of anticancer drugs.

Topics: Adenocarcinoma; Antineoplastic Agents; Blotting, Western; Cell Cycle; Cyclin D1; Cyclin E; Drug Screening Assays, Antitumor; Fibroblasts; Flow Cytometry; Humans; Lung Neoplasms; Meliaceae; Phytotherapy; Plant Extracts; Plant Leaves; Tumor Cells, Cultured

Topics: Animals; Annexin A5; Antineoplastic Combined Chemotherapy Protocols; Cyclin D1; Genes, bcl-1; Genes, Reporter; Humans; Luciferases; Lung Neoplasms; Lymphoma; Mice; Mice, Inbred BALB C; Organotechnetium Compounds; Rabbits; Radionuclide Imaging; Radiopharmaceuticals; Recombinant Fusion Proteins; Time Factors

Tetrandrine, isolated from the root of Stephania tetrandra, has been used in Chinese medicine for the treatment of silicosis and arthritis, and it also has anti-tumor/growth activities. However, the signaling pathways of tetrandrine-induced growth arrest and apoptosis in cancer cells remain unclear. We investigated the molecular mechanisms of tetrandrine-induced apoptosis and growth arrest in human lung carcinoma cells. Upon treatment with tetrandrine, a time-dependent inhibition of cell growth was observed and cells developed many of the hallmark features of apoptosis. Flow cytometry analysis confirmed that tetrandrine increased populations of both apoptotic sub-G1 and G1 phase. Tetrandrine-induced growth inhibition was associated with induction of Cdk inhibitor p21, inhibition of cyclin D1 and activation of caspase-3. Tetrandrine also affected the expression patterns of cytoskeletons including distribution of F-actin and expression level of microtubule. These results suggest that tetrandrine merits further investigation as a cell cycle blocker as well as a cancer chemopreventive agent.

Topics: Actins; Alkaloids; Antineoplastic Agents, Phytogenic; Apoptosis; Benzylisoquinolines; Caspase 3; Caspases; Cell Cycle; Cell Division; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Drugs, Chinese Herbal; Flow Cytometry; Growth Inhibitors; Humans; Immunoblotting; Lung Neoplasms; Microtubules; Tumor Cells, Cultured

There is a need to identify lung cancer prevention mechanisms. All-trans-retinoic acid (RA) was reported previously to inhibit N-nitrosamine-4-(methylnitrosamino)-1-(3 pyridyl)-1-butanone (NNK) carcinogenic transformation of BEAS-2B human bronchial epithelial cells (J. Langenfeld et al., Oncogene, 13: 1983-1990, 1996). This study was undertaken to identify pathways targeted during this chemoprevention.. Because epidermal growth factor receptor (EGFR) overexpression is frequent in non-small cell lung cancers (NSCLC) and bronchial preneoplasia, BEAS-2B cells, carcinogen-transformed BEAS-2B(NNK) cells, and retinoid chemoprevented BEAS-2B(NNK RA) cells were each examined for EGFR expression. Whether RA treatment regulated directly EGFR expression or reporter plasmid activity was studied. RA effects on epidermal growth factor (EGF) induction of EGFR-phosphotyrosine levels, cyclin D1 expression and mitogenesis were examined in BEAS-2B cells.. Findings reveal that NNK-mediated transformation of BEAS-2B cells increased EGFR expression. RA treatment repressed EGFR expression and reporter plasmid activity in these cells. This treatment reduced EGF-dependent mitogenesis as well as EGFR-associated phosphotyrosine levels and cyclin D1 expression. These findings extend prior work by highlighting EGFR as a chemoprevention target in the lung. Notably, RA treatment prevented transformation as well as outgrowth of EGFR overexpressing bronchial epithelial cells, despite NNK exposure. After acute NNK exposure, p53-induced species that appear after DNA damage or oxidative stress were evident before an observed increase in EGFR expression.. These findings indicate how effective chemoprevention prevents carcinogenic transformation of bronchial epithelial cells when repair of genomic damage does not select against EGFR overexpressing cells. This implicates EGFR as a chemoprevention target in the carcinogen-exposed bronchial epithelium.

Topics: Antineoplastic Agents; Blotting, Western; Carcinogens; Cell Transformation, Neoplastic; Cyclin D1; DNA Primers; ErbB Receptors; Gene Expression Regulation; Humans; Lung Neoplasms; Mitosis; Nitrosamines; Phosphotyrosine; Reverse Transcriptase Polymerase Chain Reaction; Transfection; Tretinoin

Lovastatin, the drug used for the treatment of hypercholesterolemia, has previously been reported to exert antitumor activity in experimental murine models. Butyrate and butyric acid derivatives are well known to induce differentiation and apoptosis of tumour cells and also have recently gained acceptance as potential anticancer agents. In this study, we examined the antitumor effects of the combination of lovastatin and butyrate or its prodrug tributyrin in vitro and in vivo against a murine Lewis lung carcinoma (3LL). This combination therapy showed synergistic antitumor activity against 3LL cells in vitro. These effects were at least in part due to apoptosis induction that occurred after 12 hr of incubation with lovastatin and butyrate and was preceded by changes in cell cycle distribution of treated cells and expression of p21, p53 and cyclin D1. Remarkably, a systemic treatment of syngeneic mice inoculated with 3LL cells with both drugs resulted in significant tumour growth retardation.

Topics: Animals; Antineoplastic Agents; Blotting, Western; Carcinoma, Lewis Lung; Cell Cycle; Cell Survival; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Drug Synergism; Drug Therapy, Combination; Female; Flow Cytometry; Formazans; Lovastatin; Lung Neoplasms; Male; Mice; Mice, Inbred C57BL; Mice, Inbred DBA; Tetrazolium Salts; Triglycerides; Tumor Suppressor Protein p53

Male strain A mice were fed a diet containing chemopreventive agents. After 1 and 3 weeks on the diets, lung nuclear fractions were examined for expression of cyclin D1/2 with western blot analysis. In animals fed a diet containing a mixture of myoinositol and dexamethasone, a treatment found previously to be effective in preventing the development of tobacco smoke-induced lung tumors in A/J mice, cyclin D1/2 expression was reduced to 30-40% of control levels. A similar decrease in cyclin D1/2 expression was found when animals were fed either myoinositol or dexamethasone alone. Paradoxically, tobacco smoke by itself had a similar effect on cyclin D1/2 expression. On the other hand, several agents that had been previously found not to be effective against tobacco smoke carcinogenesis [phenethyl isothiocyanate, 1,4-phenylenebis(methylene)selenoisocyanate, N-acetylcysteine, acetylsalicylic acid, D-limonene and beta carotene] did not decrease cyclin D1/2 expression after 1 or 3 weeks of feeding. It was concluded that expression of cyclin D1/2 might be a potentially useful marker in the identification of chemopreventive agents for tobacco smoke and could be of some help in the evaluation of their effects.

Topics: Acetylcysteine; Animals; Anticarcinogenic Agents; Aspirin; Blotting, Western; Cell Nucleus; Cyclin D1; Cyclin D2; Cyclins; Cyclohexenes; Dexamethasone; Free Radical Scavengers; Immunohistochemistry; Inositol; Isothiocyanates; Limonene; Lung; Lung Neoplasms; Male; Mice; Nicotiana; Organoselenium Compounds; Tea; Terpenes; Time Factors

Pathways involving p53 and pRb tumor suppressor genes are frequently deregulated during lung carcinogenesis. Through its location at the interface of these pathways, Mdm2 can modulate the function of both p53 and pRb genes. We have examined here the pattern of expression of Mdm2 in a series of 192 human lung carcinomas of all histological types using both immunohistochemical and Western blot analyses and four distinct antibodies mapping different epitopes onto the Mdm2 protein. Using Immunohistochemistry (IHC), Mdm2 was overexpressed as compared to normal lung in 31% (60 out of 192) of all tumors analysed, whatever their histological types. Western blotting was performed on 28 out of the 192 tumoral samples. Overexpression of p85/90, p74/76 and p57 Mdm2 isoforms was detected in 18% (5 out of 28), 25% (7 out of 28) and 39% (11 out of 28) of the cases respectively. Overall, overexpression of at least one isoform was observed in 14 out of 28 (50%) lung tumors and concomittant overexpression of at least two isoforms in 7 out of 28 (25%) cases. A good concordance (82%) was observed between immunohistochemical and Western blot data. Interestingly, a highly significant inverse relationship was detected between p14(ARF) loss and Mdm2 overexpression either in NSCLC (P=0.0089) or in NE lung tumors (P<0.0001). Furthermore, a Mdm2/p14(ARF) >1 ratio was correlated with a high grade phenotype among NE tumors overexpressing Mdm2 (P=0.0021). Taken together, these data strongly suggest that p14(ARF)and Mdm2 act on common pathway(s) to regulate p53 and/or pRb-dependent or independent functions and that the Mdm2 : p14(ARF) ratio might act as a rheostat in modulating the activity of both proteins.

Topics: Biomarkers, Tumor; Blotting, Western; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p16; DNA, Neoplasm; Female; Humans; Immunoenzyme Techniques; Lung Neoplasms; Male; Neoplasm Proteins; Nuclear Proteins; Prognosis; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-mdm2; Retinoblastoma Protein; Survival Rate; Tumor Cells, Cultured; Tumor Suppressor Protein p14ARF; Tumor Suppressor Protein p53

The relationships between overexpression of cyclin D1 or cyclin E and clinicopathological factors were investigated in 157 patients with non-small cell lung cancers (NSCLCs) using immunohistochemical analysis. Fifty-eight cases of NSCLCs (58/157, 37%) showed the overexpression of cyclin D1, and 64 cases (64/157, 41%) were positive for cyclin E. Cyclin E and cyclin D1 were infrequently concurrently overexpressed (17/157, 10.8%). Overexpression of cyclin E was more frequently observed in squamous cell carcinoma (29/57, 51%) compared with that in adenocarcinoma (28/86, 33%) (P<0.05). In addition, overexpression of cyclin E was more frequently observed in poorly or moderately differentiated NSCLCs (52/103, 50%) than in well-differentiated ones (12/54, 22%) regardless of their histological types (P<0.01). On the contrary, there was no statistically significant relationship between cyclin D1 overexpression and histological types or grade of tumor differentiation. These findings suggest that expression of cyclin E was frequently independent of that of cyclin D1 and played some roles in the grade of tumor differentiation in NSCLCs.

Topics: Adult; Aged; Aged, 80 and over; Carcinoma, Non-Small-Cell Lung; Cell Transformation, Neoplastic; Cyclin D1; Cyclin E; Female; Humans; Lung Neoplasms; Male; Middle Aged

Activation of Akt by the phosphatidylinositol 3'-OH kinase (PI3K) results in the inhibition of proapoptotic signals and the promotion of survival signals (L. P. Kane et al., Curr. Biol. 9:601-604, 1999; G. J. Kops et al., Nature 398:630-634, 1999). Evidence supporting the importance of the PI3K/Akt signaling pathway in tumorigenesis stems from experiments with transgenic mice bearing polyomavirus middle T antigen under the control of the mouse mammary tumor virus long terminal repeat promoter. Mammary epithelium-specific expression of polyomavirus middle T antigen results in the rapid development of multifocal metastatic mammary tumors, whereas transgenic mice expressing a mutant middle T antigen decoupled from the phosphatidylinositol 3'-OH kinase (MTY315/322F) develop extensive mammary gland hyperplasias that are highly apoptotic. To directly assess the role of Akt in mammary epithelial development and tumorigenesis, we generated transgenic mice expressing constitutively active Akt (HAPKB308D473D or Akt-DD). Although expression of Akt-DD interferes with normal mammary gland involution, tumors were not observed in these strains. However, coexpression of Akt-DD with MTY315/322F resulted in a dramatic acceleration of mammary tumorigenesis correlated with reduced apoptotic cell death. Furthermore, coexpression of Akt-DD with MTY315/322F resulted in phosphorylation of the FKHR forkhead transcription factor and translational upregulation of cyclin D1 levels. Importantly, we did not observe an associated restoration of wild-type metastasis levels in the bitransgenic strain. Taken together these observations indicate that activation of Akt can contribute to tumor progression by providing an important cell survival signal but does not promote metastatic progression.

Topics: Animals; Antigens, Polyomavirus Transforming; Cell Survival; Cyclin D1; DNA-Binding Proteins; Enzyme Activation; Epithelium; Female; Forkhead Box Protein O1; Forkhead Transcription Factors; Hyperplasia; I-kappa B Proteins; Lung Neoplasms; Male; Mammary Glands, Animal; Mammary Neoplasms, Experimental; Mice; Mice, Transgenic; Phosphatidylinositol 3-Kinases; Phosphorylation; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Signal Transduction; Transcription Factors

Primary pulmonary osteosarcoma is an extremely rare malignancy. To date, only 12 cases have been reported, with a high mortality rate. The authors report on a newly diagnosed patient and describe investigations that were performed using immunohistochemistry and comparative genomic hybridization (CGH).. The clinical course of a woman age 37 years is presented. Along with routine histologic examination, immunohistochemistry was used to demonstrate differentiation-associated proteins, oncoproteins, and other markers; CGH analysis for genomic alterations; and histochemistry to demonstrate alkaline phosphatase activity.. Immunohistochemical analysis showed varying expression patterns using antibodies against a panel of tumor markers. Most notable was high overexpression of BCL-2 and cyclin D. CGH analysis showed that this neoplasm contained a much higher level of genetic aberrations compared with skeletal osteosarcoma.. This tumor exhibited features common to skeletal osteosarcomas but also had some unique features. Genome analysis suggests that this tumor has several genetic aberrations in common with extraskeletal osteosarcoma. The novel regions of instability identified within the tumor genome may contribute toward the unique tumor phenotype and relative chemoresistance.

Topics: Adult; Biomarkers; Bone Neoplasms; Chromosome Aberrations; Combined Modality Therapy; Cyclin D1; Female; Humans; Immunohistochemistry; Lung Neoplasms; Nucleic Acid Hybridization; Osteosarcoma; Phenotype; Proto-Oncogene Proteins c-bcl-2

To elucidate the mechanism of action of sodium butyrate (NaB), we examined its effect on the expression of some cell cycle-related proteins (cyclins D1 and E, p16(ink4), p21(waf1), p27(kip1)) in 2 human non-small cell lung cancer cell lines (NCI-460 and NCI-H23) characterized by wild- type and mutant TP53, respectively. The growth of both cell lines was inhibited in a dose-dependent manner and this process was accompanied by a modulation of cell cycle-related proteins. In NCI-H460, the p27(kip1) and p16(ink4) protein levels were markedly increased following NaB treatment, whereas p21(waf1) was only slightly elevated, with a peak at 2 mM NaB, and p53 was unaffected by any concentration. By contrast, in NCI-H23, a marked increase in p21(waf1) protein was paralleled by decreased p53 levels, whereas all the other investigated proteins remained stable. The results suggest that NaB blocks the growth of both cell lines by induction of cyclin-dependent kinase inhibitors (in particular, p21(waf1) in NCI-H23 and p27(kip1) and p16(ink4) in NCI-H460) through a p53-dependent or p53-independent mechanism, and open up interesting perspectives for the use of NaB as an alternative or additional strategy in the treatment of non-small cell lung carcinoma.

Topics: Blotting, Western; Butyrates; Carcinoma, Non-Small-Cell Lung; Cell Cycle; Cell Cycle Proteins; Cell Division; Cyclin D1; Cyclin E; Cyclin-Dependent Kinase Inhibitor p16; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase Inhibitor p27; Cyclins; Dose-Response Relationship, Drug; Flow Cytometry; Genes, p53; Humans; Lung Neoplasms; Microtubule-Associated Proteins; Mutation; Sodium; Tumor Cells, Cultured; Tumor Suppressor Protein p53; Tumor Suppressor Proteins

Loss of the G1 checkpoint appears to be extremely common among virtually all neoplasms. A variety of genetic and epigenetic mechanisms have been demonstrated to play significant roles in this process. In a consecutive series of early stage non-small cell lung cancer (NSCLC), we have established the loss of expression of the G1 Cdk inhibitors p15INK4b) and p16INK4a by DNA methylation is very common (37%), and methylation of p16INK4a is tightly correlated with loss of expression of p16INK4a protein (P = 0.0018). Furthermore, methylation of p15INK4b and p16INK4a appear inversely correlated, although methylation of p15INK4b is an infrequent event in this cohort (4%). Methylation was detected in all stages of NSCLC equally, and did not correlate with survival in these patients. Evidence for methylation was more frequent in squamous cell carcinomas in comparison to other tumor histologies (P = 0.0156). In addition, over-expression of cyclin D1 was found to be tightly restricted (P = 0.0032) to those tumors that had retained wild-type expression of pRB, and did not correlate with methylation or expression of p16INK4a gene product. Although loss of p16INK4a function remains tightly correlated with pRB expression, loss of other regulatory elements in NSCLC such as p53 mutation and cyclin D1 over-expression appear independent of loss of the p16INK4a gene product.

Topics: Aged; Amino Acid Sequence; Base Sequence; Carcinoma, Non-Small-Cell Lung; Carcinoma, Squamous Cell; Carrier Proteins; Cell Cycle; Cell Cycle Proteins; Cohort Studies; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p15; Cyclin-Dependent Kinase Inhibitor p16; Cyclin-Dependent Kinases; DNA Methylation; Gene Expression Regulation, Neoplastic; Genes, p53; Genes, ras; Genes, Tumor Suppressor; Humans; Lung Neoplasms; Male; Middle Aged; Mutation; Neoplasm Staging; Retinoblastoma Protein; RNA Splicing; Tumor Suppressor Proteins

A large number of biological factors that seem to have important prognostic significance have been identified in non-small cell lung cancers (NSCLCs). In the present study, we have characterized expression of cyclin D1 and cyclin E in a cohort of 217 resected NSCLCs from a single institution by immunohistochemistry to analyze their expression in relation to the growth fraction determined by Ki-67 and to prognosis, and then we have constructed a risk-stratification model of cancer death by multiple biological factors in p-stage I NSCLCs. The cyclin E labeling index (LI) was significantly associated with the Ki-67 LI (r = 0.45; P < 0.001). Tumors having high-level cyclin E expression (cyclin E LI > or =30%) showed a significantly higher Ki-67 LI than tumors having low-level cyclin E expression (cyclin E LI <30%; P < 0.001), whereas positive or negative cyclin D1 expression was not associated with the Ki-67 LI (P = 0.1). Cyclin E expression was a significant and independent unfavorable prognostic factor (hazards ratio = 2.09; P = 0.03), as reported previously (Clin. Cancer Res., 6: 11-16, 2000), whereas cyclin D1 expression was not. These findings indicate different roles of cyclin D1 and cyclin E in cell proliferation and in the prognosis of NSCLCs. Furthermore, we stratified this cohort of p-stage I NSCLCs into different survival groups by using biological factors, including cyclin E, Ki-67, and ras p21, which previously we have found to be independent prognostic factors among 10 factors studied in p-stage I NSCLCs. Four groups of patients with markedly different survivals were identified with 5-year survival rates that ranged from 96% for patients with no factors altered to 41% for patients with all three factors altered (P < 0.001). This combination of biological factors was a significant and independent prognostic factor (hazards ratio = 7.94; P = 0.001).

Topics: Aged; Carcinoma, Non-Small-Cell Lung; Cell Division; Cohort Studies; Cyclin D1; Cyclin E; Female; G1 Phase; Humans; Immunohistochemistry; Ki-67 Antigen; Lung Neoplasms; Male; Middle Aged; Prognosis; Proportional Hazards Models; Proto-Oncogene Proteins p21(ras); Risk Factors; Survival Rate

Molecular analysis of microdissected tissue samples is used for analyzing tissue heterogeneity of histological specimens. We have developed a rapid one-step microdissection technique, which was applied for the selective procurement of tissue areas down to a minimum of 10 cell profiles. The special features of our microdissection system consist of an ultrasonically oscillating needle and a piezo-driven micropipette. The validity of this technique is demonstrated in human lung large-cell carcinoma by real-time quantitative reverse transcriptase-polymerase chain reaction assays of vimentin, cyclin D1, and carcinoembryonic antigen after linear RNA amplification. mRNA expression values of microdissected samples scattered around those of bulk tumor tissue and showed differential mRNA expression between samples of tumor parenchyma and supportive stromal cells for vimentin and carcinoembryonic antigen as confirmed by immunohistochemistry. In conclusion, this procedure requires simple equipment, is easily performed, and delivers microdissected tissue samples of oligocellular clusters suitable for further molecular analysis.

Topics: Carcinoembryonic Antigen; Carcinoma, Large Cell; Colon; Cyclin D1; Dissection; Humans; Immunohistochemistry; Lung Neoplasms; Needles; Reverse Transcriptase Polymerase Chain Reaction; RNA, Neoplasm; Transcription, Genetic; Ultrasonics; Vimentin

Bronchioloalveolar (BA) carcinoma of the lung is considered to have a better prognosis than that of common adenocarcinomas of the lung. However, a minor component of the BA pattern is common in many lung adenocarcinomas and the criteria for designating an adenocarcinoma as BA are not well defined. We assessed the clinicopathologic features of 238 cases of lung adenocarcinoma with a partial or predominant BA pattern. Tumors were classified as BA if more than 75% of the tumor had a BA growth pattern. In other words, the tumor grew along pre-existing lung structures without invasion or destruction of parenchyma. Tumors with 50% to 75% BA pattern were considered mixed and tumors with less than 50% BA pattern were designated as solid/acinar (S/A). Fixed, paraffin-embedded tissue sections of each neoplasm were also assessed using immunohistochemical methods with a panel of antibodies specific for p53, retinoblastoma protein, p16, cyclin D1, and cyclin E, and the results were correlated with clinical and pathologic parameters. Our results show that the 5-year survival rate of patients with BA and mixed tumors, 63% and 60%, respectively, was significantly better than that of patients with S/A tumors (P =.026). Patients with BA tumors were more frequently women (55.9%) compared with patients with mixed (48.3%) and S/A (43.8%) tumors. Bronchioloalveolar and mixed tumors were similarly associated with tobacco use, 88.2% and 85%, respectively; slightly less than S/A tumors (93.8%). Clinical and pathologic parameters did not correlate with immunohistochemical results. In conclusion, patients with BA or mixed tumors have similar 5-year survival, better than that of patients with S/A tumors, suggesting that adenocarcinomas can be designated as BA when at least 50% of the tumor has a BA pattern.

Topics: Adenocarcinoma, Bronchiolo-Alveolar; Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Cyclin D1; Cyclin E; Cyclin-Dependent Kinase Inhibitor p16; Female; Fluorescent Antibody Technique, Indirect; Humans; Immunohistochemistry; Lung Neoplasms; Lymph Nodes; Lymphatic Metastasis; Male; Middle Aged; Prognosis; Retinoblastoma Protein; Survival Rate; Tumor Suppressor Protein p53

Coumarin in vivo has antitumor activity in various types of cancer. In vitro, coumarin and 7-hydroxycoumarin, its major biotransformation product in humans, inhibit the proliferation of several human tumor cell lines. The molecular mechanisms of these effects are unknown. To gain information about these mechanisms, we studied the effects of coumarin and 7-hydroxycoumarin in the human lung adenocarcinoma cell line A-427 on the inhibition of: (i) cell proliferation; (ii) cell cycle progression; and (iii) expression of cyclins D1, E and A. The inhibitory concentrations 50 (IC(50)) of both compounds were estimated by cytostatic assays of tetrazolium (MTT) reduction. The effects on cell cycle progression were assayed with propidium iodide and BrdU using DNA histograms and multiparametric flow cytometry. The percentages of cells expressing cyclins D1, E, and A were estimated by means of bivariate flow cytometry using propidium iodide, and FITC-conjugated monoclonal antibodies for each cyclin. The IC(50) (+/-S.E.M. n=3) of 7-hydroxycoumarin and coumarin at 72 h exposure, were 100+/-4.8 and 257+/-8.8 microg/ml, respectively. 7-Hydroxycoumarin at the concentration of 160 microg/ml (1 mM), inhibited the G(1)/S transition of the cell cycle, an action consistent with the cytostatic effect. No significant decreases of cyclins E and A were observed. In contrast, cyclin D1 significantly decreased, which appears to indicate an action of 7-hydroxycoumarin in early events of phase G(1). However, messenger RNA of cyclin D1, assayed by RT-PCR, did not change. This suggests a posttranscriptional effect. The effects of coumarin were not significant. Cyclin D1 is overexpressed in many types of cancer, and its inhibition has been proposed as a pharmacological and therapeutic target for novel antitumor agents. Knowledge of the decrease of cyclin D1 by 7-hydroxycoumarin may lead to its use in cancer therapy, as well as to the development of more active compounds.

Topics: Adenocarcinoma; Antineoplastic Agents; Biotransformation; Cell Cycle; Cell Division; Coumarins; Cyclin D1; Dose-Response Relationship, Drug; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tumor Cells, Cultured; Umbelliferones

The association of the immunohistochemical expressions of cyclin D1, p16 and the retinoblastoma gene product (pRB) with the prognoses of 106 patients with non-small cell lung cancer (NSCLC) at stages I and II after a complete resection was investigated. We used antibodies recognizing nuclear and cytoplasmic cyclin D1, p16 and pRB. In 106 tumors, the positive rates of cyclin D1, p16 and pRB were 46, 54 and 48%, respectively. Cyclin D1-positive (cyclin D1(+)) patients had significantly poorer survival prognoses than cyclin D1-negative (cyclin D1(-)) patients (log-rank test, P=0.0002; Wilcoxon test, P=0.0005), whereas p16-positive (p16(+)) patients had significantly better prognoses than p16-negative (p16(-)) patients (log-rank test, P=0.0063; Wilcoxon test, P=0.0044). The survival period was over 65% for patients with cyclin D1(-)/p16(+) (n=34) at 120 months after surgery, whereas patients with cyclin D1(+)/p16(-) patients (n=22) had a 50% survival period at 49 months. The cumulative survival rate of cyclin D1(+)/p16(-) patients was significantly lower than that of cyclin D1(-)/p16(+) patients (log-rank test, P=0.0004; Wilcoxon test, P=0.0002). The pRB did not influence significantly the survival rate. Our results indicate that cyclin D1 and p16, especially a combination of cyclin D1 and p16, are very useful to predict the prognosis of patient with NSCLC after curative resection independent of pathological stages I and II.

Topics: Aged; Carcinoma, Non-Small-Cell Lung; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p16; Female; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Lung Neoplasms; Male; Middle Aged; Prognosis; Retinoblastoma Protein; Survival Analysis

p21 (p21WAF1/CIP1) is involved in cell cycle regulation, as an inhibitor of cyclin dependent kinases (CDK2, CDK4 and CDK6). However, subsequent in vitro studies have suggested that p21 may influence this process by an additional mechanism, in particular through the regulation of cyclin D1 subcellular localisation. This study of primary resectable non-small cell lung cancer (NSCLC) was designed to examine p21 functions in association with the expression of cyclin D1 (including its subcellular localisation), p16INK4a and pRb. p21 expression was examined in 50 NSCLC (stage I-IIIA) and in several normal lung samples all of which had previously been studied for cyclin D1 (DNA, RT-PCR, immunostaining), p16INK4a (DNA, RT-PCR, immunostaining), and pRb (immunostaining). As assessed by immunoblotting and immunostaining, p21 was expressed at low levels in normal lung tissue with immunoreactivity seen in a small number of bronchial epithelial cells only. In NSCLC, p21 expression (> or =10% of positive cells) was observed in 42% (21/50) of cases. High p21 expression was associated with well differentiated tumours (p = 0.01) and cyclin D1 nuclear staining (p = 0.02). Furthermore, we found an inverse correlation with p16INK4a (p = 0.004) and a direct correlation with pRb expression (p = 0.02). Risk of relapse was associated with p16INK4a and p21 status with no relapse in patients with normal p16INK4a and p21. Our results confirm that a large number of NSCLC have a low level of p21 expression. The associations of p21 and nuclear cyclin D1, pRb, p16INK4a support the relevance of pathways linked to lung carcinogenesis that involve p21 but may act in addition to direct CDK inhibition.

Topics: Aged; Carcinoma, Non-Small-Cell Lung; Carrier Proteins; Cell Cycle; Cell Nucleus; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p16; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Enzyme Inhibitors; Female; Genes, Tumor Suppressor; Humans; Lung; Lung Neoplasms; Male; Middle Aged; Prognosis; Retinoblastoma Protein

Pulmonary large-cell neuroendocrine carcinoma (LCNEC) is a newly proposed clinicopathologic entity; a few cases of LCNEC have been reported in other sites, such as the uterine cervix and the thymus. In the salivary glands, LCNEC is extremely rare and is not recognized as a specific entity in the World Health Organization classification. We retrospectively reviewed from our files 1675 cases of surgically resected primary parotid gland tumors and found 2 cases of LCNEC that fulfilled the criteria of pulmonary LCNEC. These cases occurred in 72- and 73-year-old men who had short histories of enlarging parotid gland tumors. The tumors were composed of large cells that exhibited organoid, solid, trabecular, and rosette-like growth patterns with a high mitotic rate and a conspicuous tendency for necrosis. The tumor cells were polygonal and characterized by a moderate nuclear:cytoplasmic ratio, coarse chromatin, and conspicuous nucleoli. Immunohistochemical examination revealed that the tumor cells were positive for six general neuroendocrine markers, cytokeratin, p53, bcl-2, epidermal growth factor receptor, and cyclin D1. Markedly reduced expressions of p21Waf1 and p27Kip1 were also noticed. The Ki-67 labeling index was more than 50% in both cases. One case showed loss of heterozygosity at TP53 accompanied by a p53 gene point mutation. Loss of heterozygosity at chromosome 9p21 was detected in both cases; one was accompanied by a p16 gene silent point mutation. Both patients died of the disease, with recurrence 5 months and 4 years after surgery, respectively. These findings indicate that LCNEC is a rare but distinct salivary gland tumor with highly aggressive biologic behavior. Multiple alterations of cell cycle regulators and tumor suppressor genes may play an important role in presenting the biologic characteristics of this rare parotid gland tumor.

Topics: Aged; Base Sequence; Carcinoma, Large Cell; Carcinoma, Neuroendocrine; Carcinoma, Non-Small-Cell Lung; Cyclin D1; Diagnosis, Differential; DNA Mutational Analysis; DNA, Neoplasm; ErbB Receptors; Humans; Keratins; Ki-67 Antigen; Loss of Heterozygosity; Lung Neoplasms; Male; Microscopy, Electron; Parotid Neoplasms; Point Mutation; Proto-Oncogene Proteins c-bcl-2; Tumor Suppressor Protein p53

Inflammatory myofibroblastic tumor (IMT) is composed of myofibroblasts, plasma cells, and lymphocytes. Cytokines are possibly involved in its pathogenesis. Human herpesvirus-8 (HHV-8) encodes cell cycle regulatory and signaling proteins. A combination of nested PCR with several negative controls and Southern blot methods showed the presence of HHV-8 DNA in seven cases of IMT. Additionally, strong expression was demonstrated by in situ hybridization in many tumoral nuclei. Most of the myofibroblasts in all of the cases were immunoreactive for human IL-6 and cyclin D1. These cytokines probably have a paracrine action and may sustain myofibroblastic growth. HHV-8 could play an essential role in triggering IMT development by a local reactivation of viral lytic replication. The relationship between HHV-8 and immunosuppression status as the only associated cause for tumorigenesis should be revised.

Topics: Adult; Aged; Cyclin D1; DNA, Viral; Female; Granuloma, Plasma Cell; Herpesvirus 8, Human; Humans; Interleukin-6; Leg; Lung Neoplasms; Lymph Nodes; Lymphatic Diseases; Male; Middle Aged; Soft Tissue Neoplasms

Gap junctional intercellular communication (GJIC) and connexin expression are frequently decreased in neoplasia and may contribute to defective growth control and loss of differentiated functions. GJIC, in E9 mouse lung carcinoma cells and WB-aB1 neoplastic rat liver epithelial cells, was elevated by forced expression of the gap junction proteins, connexin43 (Cx43) and connexin32 (Cx32), respectively. Transfection of Cx43 into E9 cells increased fluorescent dye-coupling in the transfected clones, E9-2 and E9-3, to levels comparable to the nontransformed sibling cell line, E10, from which E9 cells originated. Transduction of Cx32 into WB-aB1 cells also increased dye-coupling in the clone, WB-a/32-10, to a level that was comparable to the nontransformed sibling cell line, WB-F344. The cell cycle distribution was also affected as a result of forced connexin expression. The percentage of cells in G(1)-phase increased and the percentage in S-phase decreased in E9-2 and WB-a/32-10 cells as compared to E9 and WB-aB1 cells. Concomitantly, these cells exhibited changes in G(1)-phase cell cycle regulators. E9-2 and WB-a/32-10 cells expressed significantly less cyclin D1 and more p27(kip-1) protein than E9 and WB-aB1 cells. Other growth-related properties (expression of platelet-derived growth factor receptor-beta, epidermal growth factor receptor, protein kinase C-alpha, protein kinase A regulatory subunit-Ialpha, and production of nitric oxide in response to a cocktail of pro-inflammatory cytokines) were minimally altered or unaffected. Thus, enhancement of connexin expression and GJIC in neoplastic mouse lung and rat liver epithelial cells restored G(1) growth control. This was associated with decreased expression of cyclin D1 and increased expression of p27(kip-1), but not with changes in other growth-related functions.

Topics: Animals; Carcinoma; Cell Communication; Cell Cycle Proteins; Cell Division; Connexins; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p27; Cytokines; Diffusion; Fluorescent Dyes; G1 Phase; Gap Junction beta-1 Protein; Gap Junctions; Gene Expression Regulation, Neoplastic; Liver Neoplasms, Experimental; Lung Neoplasms; Mice; Microtubule-Associated Proteins; Neoplasm Proteins; Nitric Oxide; Protein Kinases; Rats; Receptors, Growth Factor; Recombinant Fusion Proteins; Transfection; Tumor Cells, Cultured; Tumor Suppressor Proteins

Ex-chromate workers are frequently afflicted with lung cancers, especially central-type squamous cell carcinomas (SCCs) of the lung. However, little is known about the molecular and cellular biologic characteristics of chromate-induced lung cancers. We investigated expression of cyclin D1, bcl-2, and p53 proteins in chromate-induced lung cancers by immunohistochemistry, compared with those in lung cancers from nonexposed individuals and those in individuals with pneumoconiosis. Of 19 chromate-induced lung cancers, 16 tumors were SCCs, including 11 central and 5 peripheral types. Eleven (69%) of 16 chromate SCCs showed cyclin D1 expression. In contrast, cyclin D1 expression was observed in only 3 (12%) of 26 SCCs from nonexposed individuals and 6 (16%) of 37 SCCs that developed in patients with pneumoconiosis, respectively. The frequency of cyclin D1 expression proved to be significantly higher in chromate-induced SCCs than in SCCs from nonexposed individuals and from those with pneumoconiosis (P < .001). When comparisons were extended to all histologic types of lung cancer, cyclin D1 expression was observed significantly more often in chromate-induced lung cancers than in lung cancers from nonexposed subjects and those from patients with pneumoconiosis (11 [58%] of 19 v 5 [10%] of 52, P < .001, and 7 [11%] of 63, P < .001, respectively). Frequencies of bcl-2 and p53 expression were not significantly different among lung cancers from ex-chromate workers, nonexposed individuals and those with pneumoconiosis. The current study suggests that cyclin D1 expression may be involved in the development of chromate-induced lung cancers, although its underlying mechanism remains to be determined.

Topics: Adenocarcinoma; Carcinoma, Large Cell; Carcinoma, Small Cell; Carcinoma, Squamous Cell; Chromates; Cyclin D1; Head and Neck Neoplasms; Humans; Immunohistochemistry; Lung Neoplasms; Occupational Exposure; Proto-Oncogene Proteins c-bcl-2; Smoking; Tumor Suppressor Protein p53

Uncontrolled cell proliferation is the hallmark of malignant tumours. Thus, the proliferative potential of tumour cells is an important prognostic factor. However, evaluation of the prognostic significance of the expression of proteins involved in regulation of cell proliferation remains controversial. In the present study, expression of Ki-67, PCNA and cyclin D1 was estimated in a group of 89 surgically resected non-small cell lung carcinomas using immunohistochemistry. The results were compared with expression of bcl-2 and p53 and with clinicopathological parameters including patients' survival. Ki-67 and PCNA were found to be moderately and highly expressed in 39% and 44% of the tumours, respectively. There was a strong correlation between Ki67 and PCNA expression. Forty five of 88 tumours (51%) showed overexpression of cyclin D1. Surprisingly, cyclin D1 was mainly localized in the cytoplasm and only a small group of tumours (9/88, 10%) showed nuclear staining as well. Bcl-2 and p53 expression was observed in 69% and 30% of the tumours, respectively. All these markers were found to be independent of clinicopathological parameters, except for Ki-67 and bcl-2 expression, which was associated with squamous cell carcinomas. It is concluded that none of the markers that were studied can be used as an independent prognostic factor, whereas the following combinations of markers may have favourable prognostic value: p53 positivity and low Ki-67 expression, p53 positivity and lack of cyclin D1 expression, bcl-2 positivity and low Ki-67 expression, and lack of cyclin D1 expression and low Ki-67 expression.

Topics: Adenocarcinoma; Biomarkers, Tumor; Bronchial Neoplasms; Carcinoma, Non-Small-Cell Lung; Cyclin D1; Humans; Immunohistochemistry; Ki-67 Antigen; Lung Neoplasms; Prognosis; Proliferating Cell Nuclear Antigen; Proto-Oncogene Proteins c-bcl-2; Time Factors; Tumor Suppressor Protein p53

Cyclins D1 (cD1) and E (cE) are G1 phase cyclins believed to participate in the pathogenesis of malignancy. Overexpression of cD1 has been reported to influence prognosis in squamous cell carcinomas (SCC) of the larynx, but was not significant in a limited study of non-small cell lung cancers (NSCLC). Altered expression of cE has been proposed as another potential prognostic marker in malignancy but its possible role in NSCLC has not been elucidated. In order to determine the prognostic value of cD1 and cE in NSCLC, paraffin-embedded sections of 467 NSCLC were immunostained with monoclonal antibody to cD1 (1:500, PharMingen, San Diego, CA) and 400 NSCLC with MA to cE (1:2500, PharMingen) using an enhanced sensitivity avidin-biotin complex technique. The number of tumor cells with nuclear and/or cytoplasmic immunopositivity was graded on a scale of: 0 = less than 1%, 1 = 1 to 10%, 2 = 10 to 25%, 3 = 25 to 50%, 4 = 50 to 75%, 5 = more than 75%. Results were correlated with survival by Kaplan-Meier survival plot using Stat-View software (Abacus Concepts, Berkeley, CA). Overall, 426 NSCLC with cD1 and 360 NSCLC with cE had adequate follow-up (median, 76 mo) for survival analysis. Both cyclins independently showed significance in prognosis of SCC but not other cell types. For cD1, absence of immunostaining was associated with worse prognosis than any immunopositivity for all stages of SCC (P = .025). For cE, Stage I and II SCC with less than 50% immunopositivity had a worse prognosis (P = .029). Of 70 Stage I and II SCC immunostained for both monoclonal antibodies, 55% of patients with tumors that demonstrated both absence of cD1 staining and cE immunopositivity in less than 50% of cells were dead at 5 years compared to 35% of patients with tumors that demonstrated positive staining with cD1 and cE immunopositivity in more than 50% of cells. These results strongly suggest cD1 and cE can independently predict prognosis in early stage SCC. Worse prognosis was associated with loss of expression, consistent with mechanisms other than overexpression of these cyclins in the progression of SCC.

Topics: Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Carcinoma, Non-Small-Cell Lung; Carcinoma, Squamous Cell; Cyclin D1; Cyclin E; Female; Humans; Immunoenzyme Techniques; Lung Neoplasms; Male; Middle Aged; Neoplasm Staging; Prognosis; Survival Analysis; Survival Rate

p16, an inhibitor of cell cycle machinery, is frequently inactivated in non-small cell carcinoma of the lung (NSCCL). To clarify the significance of p16 inactivation in the progression of lung adenocarcinoma, we immunohistochemically evaluated p16 protein status and Rb, p53 and cyclin D1 expression in 51 surgically resected adenocarcinomas that were less than 3 cm in diameter (median follow-up period: 52.5 months). Twenty-one of 51 adenocarcinomas showed negative immunostaining for p16. Twenty adenocarcinomas were also negative for Rb, while 31 and 13 were positive for p53 and cyclin D1, respectively. Loss of p16 expression was significantly correlated with scar grade, lymphatic permeation, lymph node metastasis and clinical stage. Rb protein expression was also inversely correlated with scar grade, pleural involvement and vascular invasion. When the cases were stratified according to the expression of both proteins, the Rb-/p16- subset (7/51) consisted of poorly differentiated adenocarcinoma with a higher grade of invasion. While Rb, p53 and cyclin D1 protein status showed no significant correlations with prognosis, p16 inactivation was significantly correlated with poor prognosis, and the prognosis of Rb-/p16- was the worst among the 4 subsets. Inactivation of p16 may play a role in accelerating scar formation and lymph node metastasis, and may contribute through these mechanisms to poor prognosis in patients with small-sized lung adenocarcinoma.

Topics: Adenocarcinoma; Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p16; Female; Humans; Immunohistochemistry; Lung Neoplasms; Male; Middle Aged; Prognosis; Retinoblastoma Protein; Survival Analysis; Tumor Suppressor Protein p53

Lung cancer results from a stepwise accumulation of genetic and molecular abnormalities with unknown temporal relationships to precursor bronchial lesions. In a search for biomarkers of malignant progression, we analyzed the expression of the tumor suppressor gene Rb and of the proteins regulating its phosphorylation and function in G1 arrest, p16INK4A and cyclin D1, in preinvasive bronchial lesions accompanying cancer in 75 patients, in comparison with similar lesions in 22 patients with no cancer history. Rb was constantly expressed in preinvasive lesions, including carcinoma in situ (CIS). In contrast, p16 expression was lost in moderate dysplasia (12%) and in CIS (30%) in patients with lung cancer. p16 loss occurred exclusively in patients who displayed loss of p16 expression in their related invasive carcinoma. Loss of p16 expression was not seen in nine patients with dysplasia but no cancer progression. Cyclin D1 overexpression was seen in hyperplasia and metaplasia (6%), mild dysplasia (17%), moderate dysplasia (46%), and CIS (38%) in patients with cancer but was lost in 5% of the patients during the process of invasion; it was also observed in patients with no cancer progression (14%). Our results indicate that Rb protein function can be invalidated before invasion through alteration of the Rb phosphorylation pathway, by p16 inhibition, and/or by cyclin D1 overexpression and suggest a role for p16 and cyclin D1 deregulation in progression of preinvasive bronchial lesions to invasive carcinoma.

Topics: Biomarkers, Tumor; Bronchi; Bronchial Neoplasms; Carcinoma; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p16; Follow-Up Studies; Humans; Lung Neoplasms; Neoplasm Invasiveness; Retinoblastoma Protein

Aberrations in the pathway composed of p16, cyclin D1/CDK4,6 and pRb (pRb pathway) which controls the transition from G1 to S phase occur frequently in various types of tumors. In the present study we analyzed immunohistochemically the expression of pRb, p16 and cyclin D1 in 1 12 primary non-small cell lung carcinomas (NSCLC). Loss of expression of pRb and p16 proteins was demonstrated in 15/112 cases and 64/112 cases, respectively. Inverse expression of pRb and p16 proteins was observed in 61 cases and was statistically correlated with advanced stage of the disease (p=0.03). Overexpression of cyclin D1 was detected in 34 cases and was more frequently observed in stage I than in stage III of the disease (p=0.02). Concomitant overexpression of cyclin D1 and lack of p16 was observed in 57% of cyclin D1-positive tumors. In summary, 82 of 112 analyzed cases showed an aberrant expression of at least one of the investigated proteins. These results indicate that although pRb protein expression is altered only in a small percentage of NSCLCs, the pRb pathway is disrupted very frequently in this type of tumor. There were no statistically significant correlations between changes in protein expression and histological type of tumor, gender, smoking habits and occupation of patients.

Topics: Adenocarcinoma; Adult; Aged; Carcinoma, Large Cell; Carcinoma, Non-Small-Cell Lung; Carcinoma, Squamous Cell; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p16; Female; Humans; Immunohistochemistry; Lung Neoplasms; Male; Middle Aged; Poland; Retinoblastoma Protein; Smoking

We developed an immunohistochemical assay specific for cyclin D1 and suitable for formalin-fixed and paraffin-embedded sections, to evaluate cyclin D1 expression in a group of 135 surgically resected lung-cancer patients for the purpose of investigating the prognostic role of this protein in lung cancer. In addition, we compared cyclin D1 expression with the expression of proliferating cell nuclear antigen (PCNA), considered to be a reliable index of the proliferation rate. We found cyclin D1 expressed in more than 60% of the neoplastic cells in 26.5% of our specimens. A total of 24.5% of the specimens showed cyclin D1 expression in a percentage of cells ranging from 30 to 60%; 36.7% of the specimens expressed cyclin D1 in less than 30% of the cells; and 12.2% of the specimens expressed cyclin D1 in less than 1% of the evaluated cells. Western blot analyses confirmed the specificity of this assay by correlating statistically in a highly significant fashion with the immunohistochemical results (P = 0.0003). Furthermore, we found a direct relationship between cyclin D1 and PCNA immunodetection (P = 0.0004), which correlated cyclin D1 overexpression with a higher tumor proliferation rate. When we analyzed our data statistically, cyclin D1 expression was found to be a negative prognostic marker (P < 0.00005) whose expression correlates with a shorter patient survival time.

Topics: Adenocarcinoma; Carcinoma, Small Cell; Carcinoma, Squamous Cell; Cyclin D1; Humans; Immunohistochemistry; Lung Neoplasms; Neoplasm Staging; Prognosis; Proliferating Cell Nuclear Antigen; Protein Biosynthesis; Survival Analysis; Transcription, Genetic

Cyclin D1 antisense (D1AS)-transfected lung epithelial cell lines were serum deprived and then analyzed for three hallmarks of apoptosis: appearance of single-strand DNA breaks, alteration of apoptosis-related protein expression, and induction of chromatin condensation. Single-strand DNA breaks appeared at significant levels 24 h after serum deprivation, whereas induction of chromatin condensation was observed after 72 h. The antioxidants dimethyl sulfoxide, ascorbate, and glutathione, as well as insulin-like growth factor-I, inhibited induction of DNA damage in this assay. Additionally, proliferating cell nuclear antigen expression is completely suppressed in the D1AS cells, indicating a mechanism to explain the reduced capacity for DNA repair. Increased expression of cyclin D1, which is a common lesion in lung cancer, may thus prevent induction of apoptosis in an oxidizing and growth factor-poor environment. Reducing cyclin D1 expression in lung cancer cells by expression of D1AS RNA disrupted these protective pathways.

Topics: Antioxidants; Apoptosis; Ascorbic Acid; Culture Media, Serum-Free; Cyclin D1; Dimethyl Sulfoxide; DNA Damage; DNA Repair; Gene Expression Regulation, Neoplastic; Glutathione; Humans; Insulin-Like Growth Factor I; Lung Neoplasms; Oligodeoxyribonucleotides, Antisense; Proliferating Cell Nuclear Antigen; Tumor Cells, Cultured

The objectives of this study were: (1) to determine, using immunohistochemistry, the level of expression of the cell cycle factors p53, p21 and cyclin D1 in a group of bronchioloalveolar carcinomas (BACs), and to compare these data to relevant published data for lung carcinoma; (2) to determine if higher expression rates for these factors in BAC were associated statistically with advanced clinical stage, greater tumour size, tobacco abuse, and/or BAC subtype; (3) to seek, using Fisher's exact t-test and paired data groups, any significant associations within the expression data for p53, p21 and cyclin D1.. A panel of monoclonal antibodies against p53, p21 and cyclin D1 was applied to 19 bronchioloalveolar carcinomas (17 surgical pathology cases and two autopsies) from the tissue archives of St. Louis University. These immunohistochemical stains were graded on a semiquantitative scale according to the prevalence of nuclear staining within the tumour (< 10% positive cells = 0, 10-25% = 1+, 25-50% = 2+, 50-75% = 3+ and 75-100% = 4+). Six of 19 (32%) of BACs showed 1+ or greater p53 positivity, six of 19 (32%) showed 1+ or greater nuclear cyclin D1 positivity, and nine of 19 (47%) of BACs showed 1+ or greater p21 nuclear positivity. A statistically significant correlation was found between p53 and cyclin D1 expression (P = 0.046, Fisher's exact t-test), but not between p53 and p21, or between p21 and cyclin D1. No statistically significant association was found between the cell cycle factor expression data and subtype of BAC (mucinous vs. nonmucinous), tumour diameter, clinical stage or tobacco-use history.. BACs show p53 immunostain positivity at a frequency similar to that published for p53 mutations in lung adenocarcinomas in general. Cyclin D1 and p21 nuclear expression characterizes a significant proportion of BACs, with cyclin D1 and p53 expression showing a statistically significant association. Aberrations in p53, p21, and cyclin D1 expression may be important in the development of a significant proportion of BACs.

Topics: Adenocarcinoma, Bronchiolo-Alveolar; Adult; Aged; Cell Cycle; Cell Nucleus; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Female; Genes, p53; Humans; Immunohistochemistry; Lung Neoplasms; Male; Middle Aged; Tumor Suppressor Protein p53

Induction of differentiation in a variety of model systems is accompanied by cell cycle exit and inhibition of Cdk2 kinase activity. We asked whether inhibition of Cdk2 activity is sufficient to allow differentiation to occur in a non-small cell lung cancer cell line. Treatment of NCI-H358 with flavopiridol, an inhibitor of multiple Cdk's, resulted in growth arrest and induction of mucinous differentiation. The onset of differentiation coincided temporally with loss of Cdk2 kinase activity. Western analysis revealed that flavopiridol treatment resulted in depletion of both cyclin E and D1, suggesting that loss of the regulatory subunits is at least partially responsible for the loss of Cdk kinase activity. Similarly, roscovitine, an inhibitor of Cdk's 1, 2, and 5, but not Cdk4, also induced differentiation in NCI-H358, although the resulting pattern of expression of cell cycle regulatory genes differed from the pattern obtained with flavopiridol. Furthermore, stable expression of an antisense Cdk2 construct in NCI-H358 also resulted in the appearance of a marker of mucinous differentiation. These results show that the inhibition of activity of cyclin dependent kinases, particularly Cdk2, by multiple different mechanisms is accompanied by differentiation. Thus, induction of differentiation is one potential mechanism of action for agents that down-regulate Cdk activity.

Topics: Carcinoma, Non-Small-Cell Lung; CDC2-CDC28 Kinases; Cell Differentiation; Cell Division; Cyclin D; Cyclin D1; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinases; Cyclins; Enzyme Inhibitors; Flavonoids; G1 Phase; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Neoplasm Proteins; Piperidines; Protein Serine-Threonine Kinases; Purines; Roscovitine; Signal Transduction; Transfection; Tumor Cells, Cultured

Cyclin D1, like p16INK4 (p16) and retinoblastoma (RB) proteins, participates in the cell cycle control at the G1-S transition. We have previously demonstrated altered p16 and RB protein status in non-small-cell lung cancers (NSCLCs) and their potential synergistic effect with altered p53 protein on proliferative activity (Kinoshita et al (1996) Cancer Res 56: 5557-5562). In the present study, cyclin D1 expression was studied by immunohistochemistry in the same cohort of 111 resected NSCLCs as in our previous study, and the amount of the cyclin D1 gene was analysed by Southern blot analysis in 29 NSCLCs. Cyclin D1 expression was analysed in relation to the status of p53, p16 and RB proteins, and proliferative activity determined by the Ki-67 index. It was also analysed in relation to survival of 77 patients with NSCLCs which were potentially curatively resected between 1990 and 1995. We found that: (1) cyclin D1 was expressed in 13 (11.7%) of 111 NSCLCs; (2) the cyclin D1 gene was neither significantly amplified nor rearranged; (3) cyclin D1 expression significantly correlated with altered p53 protein expression (P = 0.04), whereas it did not correlate with p16 and RB protein status; (4) proliferative activity tended to be higher in cyclin D1-positive (+) tumours than in cyclin D1-negative (-) tumours, although this difference was not statistically significant (P = 0.08); and (5) patients with cyclin D1+ tumours survived longer than patients with cyclin D1- tumours (5-year survival rates, 89% and 64% respectively, by the Kaplan-Meier method; P = 0.045 by the log-rank test), and cyclin D1 expression tended to be a favourable prognostic factor (P = 0.08 in univariate analysis). These findings suggest the involvement of cyclin D1 in the development and progression of NSCLCs, their proliferative activity and clinical outcome of NSCLC patients.

Topics: Aged; Biomarkers, Tumor; Carcinoma, Non-Small-Cell Lung; Cell Division; Cyclin D1; Disease Progression; Female; Gene Expression Regulation, Neoplastic; Genes, p53; Humans; Ki-67 Antigen; Lung Neoplasms; Male; Middle Aged; Prognosis; Tumor Suppressor Protein p53

Inactivation of the Rb pathway in non-small cell lung carcinoma (NSCLC) occurs mostly through inactivation of the cyclin-dependent kinase inhibitor p16(INK4A) and/or up-regulation of cyclin D1. In order to assess the frequency and the prognostic value of these abnormalities in NSCLC, immunohistochemical analysis of Rb, p16(INK4), and cyclin D1 has been performed on 168 cases of NSCLC including 77 squamous cell carcinomas, 43 adenocarcinomas, and 48 basaloid carcinomas. The reduced survival rate of basaloid carcinoma (stage I-II) compared with other histological types of NSCLC was confirmed (p = 0.008). Loss of protein expression of Rb and p16(INK4A) was observed in 12 per cent and 58 per cent of NSCLC cases respectively and cyclin D1 overexpression in 43 per cent. There was an inverse correlation between Rb and p16 expression ( p < 0.0001) and a direct correlation between Rb and cyclin D1 expression ( p = 0.0007). In univariate analysis, Rb-negative adenocarcinomas at stages I-II had a significantly shorter survival than Rb-positive cases ( p = 0.04) and stages I-II p16-positive cases had a shorter survival than p16-negative cases ( p = 0.02), which was more significant in basaloid carcinoma ( p = 0.003). p16 status retained its influence on survival in multivariate analysis at stage I-II for all cases ( p = 0.01) and for basaloid carcinoma ( p = 0.005). Cyclin D1 overexpression did not influence survival. Combined Rb/p16/cyclin D1 phenotypes in univariate analysis showed a shorter survival for Rb-negative/p16-positive/cyclin D1-negative tumours ( p = 0.002). These results, linked to previous data, indicate that the Rb pathway of G1 arrest is initially disrupted in the vast majority of NSCLCs (83 per cent), but could not confirm an unfavourable role for each individual event (p16(INK4A) loss or cyclin D1 up-regulation) in prognosis.

Topics: Adenocarcinoma; Biomarkers, Tumor; Carcinoma, Non-Small-Cell Lung; Carcinoma, Squamous Cell; Carcinoma, Transitional Cell; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p16; Humans; Immunoenzyme Techniques; Lung Neoplasms; Multivariate Analysis; Neoplasm Proteins; Prognosis; Retinoblastoma Protein; Survival Rate

Retinoids have demonstrated activity in the chemoprevention of aerodigestive tract cancer. Potentially contributing to their lung cancer chemopreventive effects, retinoids inhibit the growth of human bronchial epithelial (HBE) cells. We observed previously that all-trans retinoic acid (t-RA) arrests the growth of HBE cells in the G0 phase of the cell cycle through activation of retinoic acid receptor-dependent pathways, which enhances the association of E2F-4 with retinoblastoma protein family members, converting E2F into a transcriptional suppressor. In this study, we examined the mechanism by which t-RA blocks cell cycle progression in HBE cells and the possibility that this signaling event is blocked in non-small cell lung cancer (NSCLC) cells that are refractory to the growth inhibitory effects of t-RA. t-RA suppressed the expression and activity of cyclin D1, cyclin E, and cyclin-dependent kinases (CDK)-2 and CDK-4, increased expression of the CDK inhibitor p27, and shifted the retinoblastoma protein to a hypophosphorylated form. Posttranslational mechanisms contributed to the changes in CDK-2, CDK-4, and p27 levels, which, in the case of CDK-4, involved the ubiquitin-proteasome pathway. In contrast, despite retinoic acid receptor transcriptional activation, these signaling events did not occur in a NSCLC cell line that is refractory to growth inhibition by t-RA. These findings provide the first evidence that t-RA activates degradation of CDK-4 through the ubiquitin-proteasome pathway, a novel mechanism by which t-RA causes HBE cells to exit the cell cycle, and blockade of these signaling events may contribute to the development of retinoid resistance in NSCLC cells.

Topics: Anticarcinogenic Agents; Bronchi; Carcinoma, Non-Small-Cell Lung; Cell Cycle Proteins; Cyclin D1; Cyclin E; Cyclin-Dependent Kinase Inhibitor p27; Cyclin-Dependent Kinases; Epithelial Cells; Genes, Reporter; Humans; Lung Neoplasms; Microtubule-Associated Proteins; Phosphorylation; Protein Processing, Post-Translational; Retinoblastoma Protein; Signal Transduction; Tretinoin; Tumor Cells, Cultured; Tumor Suppressor Proteins

This study examined whether or not cyclin D1 expression is associated with the smoking habits of patients with non-small cell lung carcinomas (NSCLC). Immunohistochemistry was used to analyze 181 NSCLC samples for the expression of cyclin D1. Expression of cyclin D1 protein was found in 130 out of 181 cases (72%). A significant relationship between cyclin D1 expression and stage or histological classification was not observed. The carcinomas of smokers expressed cyclin D1 in 77% of the cases while carcinomas of non-smokers expressed this protein only 57% of the time (P < 0.01, Fisher's exact test). The correlation between smoking and cyclin D1 expression was maintained when the analysis was limited to squamous cell lung carcinomas. However, no correlation was found between cyclin D1 expression and the smoking habits of patients with adenocarcinomas. This can be explained by the fact that the development of adenocarcinomas--in contrast to squamous cell lung carcinomas--is not closely related to tobacco smoke.

Topics: Adenocarcinoma; Adult; Aged; Carcinoma, Non-Small-Cell Lung; Carcinoma, Squamous Cell; Cyclin D1; Female; Humans; Immunohistochemistry; Lung Neoplasms; Male; Middle Aged; Smoking

Cyclin D1 is one of the G1 cyclins that control cell cycle progression by allowing G1 to S transition. Overexpression of cyclin D1 has been postulated to play an important role in the development of human cancers. We have investigated the correlation between cyclin D1 overexpression and known clinicopathological factors and also its prognostic implication on resected non-small-cell lung cancer (NSCLC) patients. Formalin-fixed and paraffin-embedded tumour tissues resected from 69 NSCLC patients between stages I and IIIa were immunohistochemically examined to detect altered cyclin D1 expression. Twenty-four cases (34.8%) revealed positive immunoreactivity for cyclin D1. Cyclin D1 overexpression is significantly higher in patients with lymph node metastasis (50.0% vs 14.4%, P = 0.002) and with advanced pathological stages (I, 10%; II, 53.8%; IIIa, 41.7%, P = 0.048; stage I vs II, IIIa, P = 0.006). Twenty-four patients with cyclin D1-positive immunoreactivity revealed a significantly shorter overall survival than the patients with negativity (24.0 +/- 3.9 months vs 50.1 +/- 6.4 months, P = 0.0299). Among 33 patients between stages I and II, nine patients with cyclin D1-positive immunoreactivity had a much shorter overall survival (29.7 +/- 6.1 months vs 74.6 +/- 8.6 months, P = 0.0066). These results suggest that cyclin D1 overexpression is involved in tumorigenesis of NSCLCs from early stage and could be a predictive molecular marker for poor prognosis in resectable NSCLC patients, which may help us to choose proper therapeutic modalities after resection of the tumor.

Topics: Adult; Aged; Biomarkers, Tumor; Carcinoma, Non-Small-Cell Lung; Cyclin D1; Female; Humans; Immunohistochemistry; Lung Neoplasms; Male; Middle Aged; Multivariate Analysis; Prognosis; Proliferating Cell Nuclear Antigen; Survival Analysis

In order to test the hypothesis that increased apoptotic activity is connected with neuroendocrine differentiation and low differentiation degree in large cell carcinoma (LCLC) and is regulated by bcl-2 family proteins, we analysed the extent of apoptosis and tumor necrosis and their relation to the expression of bcl-2, bax, bak and mcl-1 in 35 LCLCs, of which 20 were classified as large cell neuroendocrine lung carcinomas (LCNEC) and 15 as large cell non-neuroendocrine lung carcinomas (LCNNEC). The extent of apoptosis was determined by detecting and counting the relative and absolute numbers of apoptotic cells and bodies using in situ 3 -end labelling of the apoptotic DNA. The extent and intensity of expression of the bcl-2, bax, bak and mcl-1 proteins were studied by immunohistochemistry. Also the relative volume density of necrosis was evaluated and correlated with the other parameters. Finally, all the parameters were evaluated as prognostic markers and correlated with data on the survival of the patients. Relatively high apoptotic indices were seen in both tumor types (average for both 2.53%, range 0.09 27.01%). Significantly higher bcl-2 and bak indices were detected more often in LCNECs than in LCNNECs. Immunohistochemically detected bax, bcl-2 and bak expression was independent of apoptotic index in both tumor types, while there was a statistically significant positive association between mcl-1 expression and apoptotic index in LCNNEC but not in LCNEC. There was a statistically significant association between high apoptotic index and shortened survival in LCLC. However, no association was found between tumor stage and apoptosis. The patients with LCNEC and low bcl-2 protein expression had a significantly shorter survival time than those with high bcl-2 indices. There was also a clear association between shortened survival and necrotic LCNNEC. LCLCs show relatively high apoptotic activity, which is associated with shortened survival. The expression of bcl-2, bak and mcl- 1 is associated with neuroendocrine differentiation in LCLC. Finally, our results support some previous reports suggesting that bcl-2 expression in combination with some other markers involved in apoptosis and/or proliferation may be of prognostic value in cases of lung carcinoma with neuroendocrine differentiation.

Topics: Adult; Aged; Apoptosis; bcl-2 Homologous Antagonist-Killer Protein; bcl-2-Associated X Protein; Blotting, Western; Carcinoma, Neuroendocrine; Carcinoma, Non-Small-Cell Lung; Cell Differentiation; Cyclin D1; Female; Follow-Up Studies; Humans; Immunohistochemistry; Lung Neoplasms; Male; Membrane Proteins; Middle Aged; Myeloid Cell Leukemia Sequence 1 Protein; Necrosis; Neoplasm Proteins; Prognosis; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Survival Rate

The retinoblastoma protein (pRB), p16, and cyclin D1 are major components of the RB pathway, which controls the G1 checkpoint of the cell cycle. Proper regulation of this pathway is crucial for normal cell proliferation. Abnormal forms of these proteins have been found in various types of malignant tumours. In the present report, immunohistochemical techniques were applied to study the expression of pRB, p16, and cyclin D1 in 161 samples of primary small cell lung cancer (SCLC) and 20 samples of non-small cell lung cancer (NSCLC). While pRB and cyclin D1 staining was negative in 161 specimens of SCLC, expression of p16 was observed in 153 samples. In contrast to SCLC, 16 out of 20 NSCLC cases exhibited pRB expression and 15 showed cyclin D1 expression, but only very weak p16 staining was found in five samples. These observations could provide additional criteria for the distinction between SCLC and NSCLC. Furthermore, these findings, based on primary tissues, implicate different mechanisms in the tumourigenesis of SCLC and NSCLC.

Topics: Adenocarcinoma; Adult; Aged; Aged, 80 and over; Carcinoma, Non-Small-Cell Lung; Carcinoma, Small Cell; Carcinoma, Squamous Cell; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p16; Diagnosis, Differential; Female; Humans; Immunoenzyme Techniques; Lung Neoplasms; Male; Middle Aged; Neoplasm Proteins; Retinoblastoma Protein

Cyclin D1 gene amplification is an important event in many cancers, but it is rarely found in non-small-cell lung cancer (NSCLC). This study was conducted in an attempt to clarify any other mechanisms related to cyclin D1 involvement in the malignant transformation of NSCLC, and we clearly showed for the first time that cyclin D1-related kinases are activated in NSCLC, especially in adenocarcinoma but not in squamous cell carcinoma. The results of this study strongly suggest that enhanced cyclin D1-related kinase activity could contribute to a progression of adenocarcinoma in NSCLC.

Topics: Adenocarcinoma; Adult; Aged; Blotting, Western; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase 6; Cyclin-Dependent Kinases; Enzyme Activation; Female; Humans; Immunoblotting; Immunohistochemistry; Lung Neoplasms; Male; Middle Aged; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Retinoblastoma Protein

Among the major regulators of the G1 restriction point are cyclin D1 and the retinoblastoma gene product (RB). In non-small cell lung cancer (NSCLC), the cyclin D1 gene is amplified/over-expressed in almost 50% of cases, and RB is inactivated in 6-32% of cases. It is of interest to evaluate concurrently the alterations of both genes on the same series of NSCLCs, to investigate whether cyclin D1 and RB alterations are alternative pathways leading to inactivation of the G1 restriction point or if they can occur in the same tumor, possibly exerting an additive effect on cancer progression. We investigated a series of 57 NSCLCs, analyzing cyclin D1 and RB at the gene and protein levels by Southern blot, Northern blot and immunohistochemistry. The cyclin D1 gene was amplified in 18 cases, cyclin D1 immunoreactivity was seen in 25 tumors. Amplification and expression were significantly associated. RB immunohistochemical expression was absent in 9 of 42 informative cases. RB mRNA expression was low to absent in 9 of 45 informative cases, cyclin D1 amplification was associated with normal RB mRNA, and cyclin D1 over-expression was associated with normal RB immunoreactivity, supporting the hypothesis that alterations of cyclin D1 and RB are alternative mechanisms by which tumor cells may escape the G1 restriction point. A concurrent alteration of RB and cyclin D1 was seen in a small subset of NSCLCs. Abnormalities of cyclin D1 and/or RB at the gene and/or expression level were present in more than 90% of cases, stressing that cyclin D1 and/or RB alterations represent an important step in lung tumorigenesis.

Topics: Carcinoma, Non-Small-Cell Lung; Cyclin D1; Genes, Retinoblastoma; Humans; Immunohistochemistry; Lung Neoplasms; RNA, Messenger

To clarify the events leading to the disruption of cell growth control that occurs during the development of pulmonary adenocarcinoma (AC), we used immunohistochemistry to evaluate the expression of G1 cycle regulators, cyclin D1, Rb protein (pRb), and p16 MTS1 protein and the tumour proliferation marker, Ki 67, both in AC of the lung and in its precursor lesion, atypical adenomatous hyperplasia (AAH). The frequency of lesions with cyclin D1 overexpression was relatively high in AAH (47-89%), but was decreased in early AC (28%) and overt AC (35%). The loss of pRb expression was rare in both AAH (0-18%) and early AC (0%), and was infrequent even in overt AC (13%). The loss of p16 expression was also relatively infrequent in both the premalignant and the malignant lesions (11-25%). Our results suggest that overexpression of cyclin D1 is an early event and plays an important part in tumorigenesis in the case of lung AC. However, cyclin D1 overexpression is not required for the development and maintenance of a malignant phenotype. It is likely that some cyclin D1-independent pathways other than Rb and p16 abnormalities have an important role in the malignant transformation from AAH to early AC.

Topics: Adenocarcinoma; Adenoma; Adult; Aged; Aged, 80 and over; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p16; Female; Humans; Hyperplasia; Immunohistochemistry; Ki-67 Antigen; Lung; Lung Neoplasms; Male; Middle Aged; Retinoblastoma Protein

The pathway consisting of retinoblastoma protein (pRB), cyclin D1 and p16 (RB pathway) which is involved in the phosphorylation of pRB plays an important role in G1/S progression. The disruption of this RB pathway has been reported in several types of human neoplasm. An immunohistochemical study of 101 non-small-cell lung cancers (NSCLCs) showed loss of p16 is in 47 tumors (46.5%) and loss of pRB in 42 tumors (41.6%). In 79 of 101 NSCLCs (78.2%), the expression of p16 and pRB was complementary (p < 0.0001). Methylation of the cdkn2 gene was detected in 50% of p16-negative tumors and in 11% of p16-positive tumors. Aberrant expression of cyclin D1 was found in 45 tumors (44.5%). The cyclin-D1-positive tumors had significantly higher Ki-67 indices than the cyclin-D1-negative tumors irrespective of the tumor p16 or pRB expression. Thus, 91 (90%) of 101 NSCLCs showed disturbed expression of at least 1 of the 3 components of the RB pathway. Our results suggest that the disruption of the RB pathway plays an important role in tumorigenesis in NSCLCs and that increased cyclin-D1 expression leads to strong proliferative activity which may over-ride the suppressive effect of p16 and pRB.

Topics: Adenocarcinoma; Carcinoma, Large Cell; Carcinoma, Non-Small-Cell Lung; Carcinoma, Squamous Cell; Cell Cycle; Cell Division; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p16; Female; Humans; Immunohistochemistry; Ki-67 Antigen; Lung Neoplasms; Male; Neoplasm Staging; Polymerase Chain Reaction; Polymorphism, Single-Stranded Conformational; Retinoblastoma Protein

Previously, we demonstrated that inostamycin, an inhibitor of phosphatidylinositol turnover, caused cell cycle arrest at the G1 phase, inhibiting the expression of cyclins D1 and E in normal cells. In the present study, we examined the effects of inostamycin on cell cycle progression and apoptosis in human small cell lung carcinoma Ms-1 cells. Treatment of exponentially proliferating Ms-1 cells with low concentrations of inostamycin caused cells to accumulate in the G1 phase. We found that inostamycin decreased cyclin D1, and increased cyclin-dependent kinase inhibitors such as p21WAF1 and p27KIP1 in Ms-1 cells. On the other hand, higher concentrations of inostamycin induced morphological apoptosis and DNA fragmentation in Ms-1 cells without affecting the expression of p53, Bcl-2 and Bax. Inostamycin-induced apoptosis was suppressed by an inhibitor of caspase-3, and a 17 kDa fragment of activated caspase-3 was detected following inostamycin treatment. Therefore, caspase-3(-like) would appear to be involved in inostamycin-induced apoptosis. On the other hand, an inhibitor of caspase-3(-like) proteases did not affect the inhibitory effect of inostamycin on cyclin D1 expression, suggesting that caspase-3(-like) proteases were not responsible for inostamycin-induced G1 arrest.

Topics: Anti-Bacterial Agents; Apoptosis; Carcinoma, Small Cell; Caspase 3; Caspases; Cell Division; Cyclin D1; Cysteine Endopeptidases; DNA Fragmentation; Furans; Humans; Interphase; Lung Neoplasms; Time Factors; Tumor Cells, Cultured

To observe the expression of p16, pRb, cdk4 and cyclinD1 in non-small cell lung cancers, 104 cases of resected lung cancers were collected, which included squamous cell carcinomas, adenocarcinomas and large cell carcinomas. Immunohistochemistry assay was carried out. The results showed that 67% of squamous cell carcinomas and 46% of adenocarcinomas expressed p16, 64% of squamous cell carcinomas and 85% of adenocarcinomas expressed pRb and 66% of cancers expressed p16 or pRb. About 70% of the tumors expressed cyclinD1. More than 90% of the tumors expressed cdk4 and there was an increased trend with decreasing differentiation of both squamous cell carcinomas and adenocarcinomas. Sixty-seven percent of the highly differentiated and 100% of the poorly differentiated squamous cell carcinomas expressed cdk4. The aberrant p16 and pRb gene product expression played a significant role in the development and histological subtype of lung cancers by conditioning the biological behavior of NSCLC. cdk4 was an important factor in histological differentiation.

Topics: Adenocarcinoma; Carcinoma, Large Cell; Carcinoma, Non-Small-Cell Lung; Carcinoma, Squamous Cell; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase Inhibitor p16; Cyclin-Dependent Kinases; Humans; Lung Neoplasms; Neoplasm Proteins; Proto-Oncogene Proteins; Retinoblastoma Protein

In the present study, we found that inostamycin increased the ability of paclitaxel to induce apoptosis in Ms-1 cells. A considerably higher concentration of paclitaxel was required for the induction of apoptosis in Ms-1 cells than in other cell lines tested. Treatment of Ms-1 cells with inostamycin, an inhibitor of phoshatidylinositol (PI) synthesis, reduced the dosage of paclitaxel required to induce cell death by apoptosis. This effect of inostamycin is specific to Ms-1 cells, and inostamycin did not increase the cytotoxicity of other antitumor drugs such as adriamycin, vinblastine, methotrexate, cisplatin, etoposide, or camptothecin in Ms-1 cells. Addition of inostamycin to paclitaxel-treated cells caused a significant increase in the sub G1 peak, representing apoptosis, which was accompanied by a decrease in the G2/M peak seen in paclitaxel-treated Ms-1 cells, without affecting paclitaxel-inhibited tubulin depolymerization. Moreover, paclitaxel did not enhance inostamycin-inhibited PI synthesis. The expression levels of Bcl-2, Bax, and Bcl-XL were not changed following the co-treatment with inostamycin plus paclitaxel, whereas the activated form of caspase-3 was markedly increased. Thus, inostamycin is a chemosensitizer of paclitaxel in small cell lung carcinoma Ms-1 cells.

Topics: Antineoplastic Combined Chemotherapy Protocols; Apoptosis; bcl-2-Associated X Protein; bcl-X Protein; Carcinoma, Small Cell; Caspase 3; Caspases; Cyclin D1; Drug Synergism; Furans; Humans; Lung Neoplasms; Paclitaxel; Phosphatidylinositols; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Tumor Cells, Cultured

The CDKN2 locus expresses two different mRNA transcripts, designated alpha and beta. The protein product of the alpha transcript is the cell cycle inhibitor and tumour suppressor p16INK4a. The beta transcript is translated in an alternate reading frame (ARF) and in humans encodes a 15 kDa protein (p19ARF). Immunohistochemical and Western analysis of p16INK4a has shown that the protein is downregulated in a significant number of tumours, but less is known on the expression of the p19ARF. We have examined the expression of p16INK4a and p19ARF in resectable non-small cell lung cancer (NSCLC) by immunostaining (n=49) and multiplex RT-PCR (n=28). In order to investigate the mechanism responsible for p16INK4a downregulation, exon 1alpha methylation was analysed in a PCR-based assay. Of 49 tumours examined by immunostaining, 24 and 20 tumours expressed p16INK4a and p19ARF at nil to low levels, respectively. p19ARF was localized primarily to the nuclei of tumour cells, but was also seen to varying degrees in nuclei of lymphocytes, chondrocytes, fibroblasts, and epithelial cells. No tumour with normal p16INK4a had decreased p19ARF expression. Among 16 tumours with nil to low p16INK4a expression, 11 tumours exhibited full methylation of at least one site within exon 1alpha and these tumours showed normal p19ARF expression. In contrast, no methylation of exon 1alpha was observed in five tumours which also lacked p19ARF. In normal lung, p16INK4a and p19ARF were not expressed at detectable levels, the multiplex RT-PCR results were balanced, and sites within exon 1alpha were strongly methylated. In tumours, imbalanced multiplex RT-PCR data (p16INK4a

Topics: Aged; Animals; Carcinoma, Non-Small-Cell Lung; COS Cells; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p16; DNA Methylation; DNA, Neoplasm; Exons; Female; G1 Phase; Gene Expression Regulation, Neoplastic; Genes, Overlapping; Genes, p16; Genes, p53; Genes, Tumor Suppressor; HeLa Cells; Humans; K562 Cells; Lung Neoplasms; Male; Middle Aged; Neoplasm Proteins; Proteins; Retinoblastoma Protein; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Neoplasm; Tumor Cells, Cultured; Tumor Suppressor Protein p14ARF; Tumor Suppressor Protein p53

Recent allelotyping of chemical-induced lung tumors in hybrid mice has detected loss of heterozygosity on chromosome 4 in a region involving the interferon-alpha (IFN-alpha gene cluster that is syntenic to human chromosome 9p21-22, the location of the p16INK4a (p16) and p15INK4b (p15) tumor suppressor genes. The purpose of the current investigation was to characterize the expression of p16 and p15 in lung tumors and tumor-derived cell lines induced in A/J mice by exposure to the tobacco-specific nitrosamine, 4-methylnitrosamino-1-(3-pyridyl)-1-butanone (NNK). Expression of p16 and p15 was detected in all primary lung tumors; however, levels of expression of p16 differed by up to 15-fold between tumors. This is the first study to note a marked difference in the expression of the p16 gene in primary lung tumors. The apparent low levels of expression seen in approximately half of the tumors was not attributed to deletion, mutation or methylation of the p16 gene. Conversely, the high levels of p16 expression were not the result of effects on the retinoblastoma gene (Rb) or cyclin D1 proteins but most likely in response to a dysfunction elsewhere within this pathway. In contrast to the detection of p16 expression in primary tumors, this gene was deleted in all four cell lines. Three of four cell lines also showed loss of the p15 gene. Mapping of these homozygous deletions on chromosome 4 revealed that the p16 gene resides near the D4MIT77 marker, which is located approximately 12 cM proximal to the IFN-alpha gene cluster, thereby implicating the p16 gene as one of the targets within the allelic deletions detected previously in primary lung tumors from hybrid mice.

Topics: Animals; Carrier Proteins; Cell Cycle Proteins; Chromosome Mapping; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p15; Cyclin-Dependent Kinase Inhibitor p16; Cyclins; Gene Deletion; Gene Expression Regulation, Neoplastic; Genes, Tumor Suppressor; Lung Neoplasms; Methylation; Mice; Mice, Inbred A; Mutation; Oncogene Proteins; Retinoblastoma Protein; RNA, Messenger; Transcription Factors; Tumor Suppressor Proteins

Tumours develop through the accumulation of genetic alterations associated with a progressive increase of the malignant phenotype. In lung cancer, chronic exposure of bronchial epithelium to carcinogens in cigarette smoke may lead to multiple dysplastic and hyperplastic lesions scattered throughout the tracheobronchial tree. Little is known about the genetic alterations in such lesions. This study was carried out to examine cyclin D1 (CCND1) and retinoblastoma (RB1) gene expression in the bronchial epithelium of patients with lung cancer. Lung tumours and their corresponding tumour-free resection margins from 33 patients who underwent resection of non-small-cell lung cancer (NSCLC) were examined by immunostaining with monoclonal antibodies against cyclin D1 (DCS-6; Novocastra) and pRb (NCL Rb-1; Novocastra). Examination of the resection margins revealed four carcinomas in situ, 19 hyperplasias and ten sections showing apparently normal bronchial epithelium. A control group of patients, without lung tumours and who had never smoked, revealed no or weak cyclin D1 and positive pRb staining within bronchial epithelia. Increased cyclin D1 and diminished pRb expression were found in 76% (n = 25) and 27% (n = 9) of the resection margins respectively, and in 12% (n = 4) both cyclin D1 and pRb expression were altered. In the corresponding tumours, 48% (n = 16) were normal, while altered expression was found for cyclin D1 in 33% (n = 11), pRb in 27% (n = 9) and both in 9% (n = 3) of cases. It appears that altered expression of cyclin D1 and pRb is an early event in NSCLC development in almost half of cases analysed. Further investigations are needed to determine the significance of immunostaining of bronchial specimens in individuals at risk of lung cancer, with the possibility that the observations are of importance in the early diagnosis of NSCLC.

Topics: Adult; Aged; Antibodies, Monoclonal; Biomarkers, Tumor; Carcinoma in Situ; Carcinoma, Non-Small-Cell Lung; Cyclin D1; Cyclins; Data Interpretation, Statistical; Epithelium; Female; Genes, Retinoblastoma; Humans; Hyperplasia; Immunohistochemistry; Lung; Lung Neoplasms; Male; Middle Aged; Oncogene Proteins; Prognosis; Staining and Labeling

Lung cancer is a worldwide problem and in many countires it is the most lethal malignancy. Because relapse is frequent after resection of non small cell lung cancer, an urgent need exists to define prognostic factors which could help in choosing the best therapeutic approach. We performed immunohistochemistry on 60 formalin-fixed paraffin-embedded non small cell lung cancer specimens in order to evaluate the frequency of cyclin D1 overexpression, and to relate it to the degree of malignancy of these tumors and to the overall survival time of the patients. All specimens were positive for cyclin D1 immunostaining. We found cyclin D1 overexpression in 30 (50%) of our specimens, with no significant difference among the different histological types. Cyclin D1 overexpression correlates in a statistical manner with short-term patient survival. Mantel-Cox analysis of these data generated a significant P value = 0.003. The mean survival time and the five-year survival rate also differed statistically. We did not find any statistically significant correlation between cyclin D1 overexpression and histological grading, tumor stage or TNM status. We concluded that cyclin D1 overexpression in 30 patients is a frequent event in non small cell lung cancer pathogenesis and may have prognostic relevance.

Topics: Biomarkers, Tumor; Carcinoma, Non-Small-Cell Lung; Cyclin D1; Cyclins; Humans; Immunohistochemistry; Lung Neoplasms; Middle Aged; Neoplasm Staging; Oncogene Proteins; Prognosis; Survival Rate

To investigate the role of cyclin D1 in the regulation of lung cancer cell growth, we created five stably transfected cell lines carrying a cyclin D1 antisense construct. The transfected cells exhibited a marked decrease in the rate of cell growth, in contrast to the original lines (A549 and NCI-H441). The expression of several cell cycle-regulating proteins, including cyclin A, the cyclin-dependent kinases (cdk) 2 and cdk4, in addition to cyclin D1 itself, was markedly decreased. The expression of one cdk inhibitor, p21WAF1/CIP1, increased in the A549-derived cell lines. A specific target of cyclin D1 activity, the growth-suppressing product of the retinoblastoma gene, pRb, exhibited decreased expression and a decreased level of phosphorylation in the transfected cells. Decreased expression of pRb due to a significant increase in its turnover rate suggested that the stability of the protein may depend on phosphorylation by cyclin D1-dependent cdk activity. In addition to the impact on pRb stability, decreased expression of cyclin D1 induced susceptibility to cell death after withdrawal of exogenous growth factors in the antisense transfected cell lines, a response that was not observed in the original cancer cell lines. We conclude that abrogation of cyclin D1 overexpression in lung cancer cells disrupts several key pathways that are required for uncontrolled cell growth and induces those that lead to cell death after growth factor deprivation. Therefore, we speculate that use of antisense cyclin D1 expression in appropriate gene vectors could be a useful method for retarding lung cancer cell growth in accessible tumors such as those of the lung epithelium.

Topics: CDC2-CDC28 Kinases; Cell Death; Cell Division; Culture Media, Serum-Free; Cyclin A; Cyclin D1; Cyclin D2; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinases; Cyclins; Gene Expression Regulation, Neoplastic; Humans; Kinetics; Lung Neoplasms; Phosphorylation; Polymerase Chain Reaction; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Recombinant Proteins; Retinoblastoma Protein; RNA, Antisense; Transfection; Tumor Cells, Cultured

Cell-cycle-dependent phosphorylation of the tumor-suppressor protein product of the retinoblastoma gene (RB) is mediated by a family of cyclin-dependent kinases and cyclins. We examined the expressions of RB protein and cyclin A protein in 13 small-cell lung cancer (SCLC) lines and 14 non-small-cell lung cancer (NSCLC) lines by immunoblotting. RB protein was not present or was of a mutant type in 77% of the SCLC lines (10/13) but was present in all the NSCLC lines. Cyclin A was expressed in 38% of the SCLC lines (5/13) and in 86% of the NSCLC lines (12/14). A positive correlation (P = 0.0034) between expression of cyclin A and wild-type RB protein was found by Fisher's exact probability test. Densitometric analysis of the expression of RB protein in RB(+) lung cancer lines showed that the phosphorylated form was predominant in 2/3 of the SCLC and 8/14 of the NSCLC lines. The positive correlation between the expressions of RB protein and cyclin A suggests that RB protein in most RB(+) lung cancer cell lines is a target of cyclin-A-dependent kinase and that the tumor-suppressor function may be inactivated by phosphorylation.

Topics: Adenovirus E1A Proteins; Blotting, Western; Carcinoma, Non-Small-Cell Lung; Carcinoma, Small Cell; Cyclin A; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase Inhibitor p16; Cyclin-Dependent Kinases; Humans; Leukemia; Lung Neoplasms; Phosphorylation; Proto-Oncogene Proteins; Retinoblastoma Protein; Tumor Cells, Cultured

This study was conducted to evaluate the prognostic significance of cyclin D1 and retinoblastoma (Rb) expression in combination with abnormal p53 accumulation in primary, resected non-small cell lung cancers (NSCLCs). We evaluated immunohistochemically the expression of cyclin D1 and Rb in 208 NSCLC patients whose resections were consecutively performed between January 1984 and December 1988 and determined their prognostic significance by comparison with follow-up data. Expression of cyclin D1 and Rb was detected immunohistochemically in 39 and 80% of the 208 NSCLCs, respectively. The Kaplan-Meier survival curve demonstrated that absence of cyclin D1 expression was significantly associated with shortened survival (P = 0.01 by the log-rank test), particularly in adenocarcinomas (n = 100; P = 0.004). Expression status of the Rb protein was not significantly associated with clinical outcome in any of the cohorts. The predictive power of these prognosticators was also assessed in combination with findings for abnormal p53 accumulation by multivariate analysis using the Cox proportional hazards modeling. Patients with cyclin D1-negative tumors had a significantly greater risk of earlier death than those with cyclin D1-positive tumors (risk ratio, 1.61; P = 0.03), particularly in adenocarcinomas (risk ratio, 2.49; P = 0.002). Cases showing abnormal p53 accumulation tended to have a shortened survival only when the tumors were adenocarcinomas (risk ratio, 1.75; P = 0.06). Absence of cyclin D1 expression may be a useful prognosticator for shortened survival in primary, resected NSCLCs with particular significance for adenocarcinomas. In contrast, Rb expression status is not a useful prognostic factor for any type of NSCLC.

Topics: Adenocarcinoma; Aged; Carcinoma, Non-Small-Cell Lung; Carcinoma, Squamous Cell; Chromosome Aberrations; Chromosome Disorders; Cyclin D1; Female; Genes, p53; Humans; Lung Neoplasms; Male; Middle Aged; Neoplasm Staging; Prognosis; Retinoblastoma Protein; Smoking; Survival Analysis

Amplification of the CCDN1 gene encoding cyclin D1 was examined by Southern blotting and multiplex polymerase chain reaction (PCR) and occurred in 8 of 53 patients (15%) with primary resected non-small-cell lung cancer (NSCLC). These tumours and 17 additional tumours with a normal gene copy number showed overexpression of cyclin D1 (25/53, 47%), as assessed by immunostaining using a monoclonal antibody. In 22/25 cases, cyclin D1 was localised in the cytoplasm, but some (7/25) had simultaneous nuclear staining. This result is in marked contrast to that reported in breast, hepatocellular and colorectal carcinoma studies where immunostaining was invariably nuclear. Examination of a restriction fragment length polymorphic (RFLP) site within the 3'untranslated region of the cDNA following reverse transcriptase (RT)-PCR (29/53 informative cases) showed a strong association between cytoplasmic staining and imbalance in allele-specific message levels. Cyclin D1 overexpression was associated with a poorly differentiated histology (P = 0.04), less lymphocytic infiltration of the tumour (P = 0.02) and a reduction in local relapse rate (P = 0.01). The relative risk of local relapse was 9.1 in tumours without cyclin D1 overexpression (P = 0.01, Cox regression analysis). We conclude that genetic alteration of cyclin D1 is a key abnormality in lung carcinogenesis and may have diagnostic and prognostic importance in the treatment of resectable NSCLC.

Topics: Aged; Alleles; Base Sequence; Carcinoma, Small Cell; Cyclin D1; Cyclins; DNA Primers; Female; Gene Amplification; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Male; Middle Aged; Molecular Sequence Data; Oncogene Proteins; Prognosis; Risk Factors; RNA, Messenger

Cyclin D1, a G1 cyclin, has been implicated in the oncogenesis of various types of malignancies via deregulation of cell cycles. Amplification of cyclin D1 as a part of 11q13 amplicon has been reported in lung cancer as well as a subset of carcinomas arising from various organs including breast, head and neck, and esophagus. In addition to its role as an oncogene, several recent studies have suggested that amplification is indicative of poor prognosis. In this study we examined the cyclin D1 protein expression in 102 consecutive cases of lung cancers using the microwave enhanced immunohistochemical staining method and correlated the data with the histologic subtype and grade, Ki-67 (MIB-1) labeling index, and survival. Nuclear positive staining was observed in 18 cases (18 %) of lung cancers. Although squamous cell carcinoma demonstrated a higher rate of expression (12 /58, 21%), three of 33 adenocarcinomas (9%) revealed overexpression and both adenocarcinoma and squamous cell carcinoma components within the adenosquamous carcinoma showed nuclear staining. There was no correlation between cyclin D1 overexpression and histologic grade, Ki-67 (MIB-1) labeling index, and survival. These observations indicate that cyclin D1 protein overexpression might be implicated in the oncogenesis of the various histologic types of non-small cell lung carcinomas but it has no usefulness as a prognostic marker.

Topics: Cyclin D1; Cyclins; Humans; Immunohistochemistry; Lung Neoplasms; Neoplasm Staging; Oncogene Proteins

Expression of the neural cell adhesion molecule, NCAM, in frozen sections has been associated with decreased postoperative survival in non-small cell lung carcinoma. Of the various isoforms of NCAM described, the highly sialylated isoform plays a role in the migration of embryonal cells from the neural crest and is expressed by highly malignant tumours such as small cell lung carcinomas. We investigated the clinical significance of expression of this NCAM isoform as a prognostic factor in a series of 96 non-small cell lung carcinomas resected with curative intent. We also evaluated the effect of microwave pre-treatment of formalin-fixed, paraffin-embedded sections on the NCAM immunostaining and related the outcome to the postoperative clinical course of disease. In addition, in an attempt to extend our search for possible molecular markers of unfavourable prognosis in lung cancer, we evaluated increased immunostaining for p53 and cyclin D1 in the same series. We did not find a significant relation between expression of NCAM or its highly sialylated isoform and the length of postoperative survival. The numbers of positive cases (9 and 14, respectively) were relatively low. Increased p53 and cyclin D1 immunostaining (50 and 55 of the 96 tumours) failed to show a significant relation with postoperative survival. In our material, tumour stage was the only significant prognostic factor.

Topics: Antibodies, Monoclonal; Biomarkers, Tumor; Carcinoma, Non-Small-Cell Lung; Cyclin D1; Cyclins; Female; Formaldehyde; Humans; Immunohistochemistry; Lung Neoplasms; Male; Middle Aged; Neural Cell Adhesion Molecules; Oncogene Proteins; Paraffin Embedding; Predictive Value of Tests; Prognosis; Tissue Fixation; Tumor Suppressor Protein p53

Cyclin D1 is part of the molecular system regulating the cell cycle G1 to S transition point. Its overexpression, a common finding in carcinomas of the breast, oesophagus, and head and neck, has also been demonstrated in a high percentage of non-small cell lung carcinomas (NSCLCs). The role of cyclin D1 in NSCLC has been studied by correlating its immunoreactivity with the Ki67 labelling index in paraffin-embedded, autoclaved surgical samples of 56 NSCLC cases. In addition, flow cytometric determination of ploidy and cell cycle status was carried out on 172 fresh tumour samples from the same cases. Twenty-four (42.8 per cent) NSCLCs showed positive cyclin D1 immunostaining, a finding which showed no relationship to ploidy pattern, cell cycle phase, histological subtype, or lymph node metastasis, but was significantly associated with the Ki67 labelling index (P = 0.03) and with poor cytoplasmic differentiation (P = 0.01). Cyclin D1-positive nuclei were abundant in poorly differentiated zones and absent in the best differentiated areas, particularly in heavily keratinized fields. These data indicate that in NSCLC, cyclin D1 overexpression is not only associated with a high cell proliferation rate, but also seems to play a role in the process of tumour differentiation.

Topics: Adult; Aged; Aged, 80 and over; Carcinoma, Non-Small-Cell Lung; Cell Differentiation; Cell Division; Cyclin D1; Cyclins; Cytoplasm; Female; Flow Cytometry; Humans; Immunoenzyme Techniques; Ki-67 Antigen; Lung Neoplasms; Male; Middle Aged; Neoplasm Proteins; Oncogene Proteins

Using Northern blotting and PCR analysis of cDNA derived from a range of cell lines and tissues, alternate splicing of the cyclin D1 gene (CCND1) mRNA has been demonstrated. The variant transcript shows no splicing at the downstream exon 4 boundary, encoding a protein with an altered carboxy-terminal domain. Investigation of mRNA extracted from mononuclear cells, lung tumour and normal tissue suggests that both transcripts are invariably expressed. However, splicing to produce the two forms of mRNA is modulated, in the heterozygote, by a frequent A/G polymorphism located within the splice donor region of exon 4. Preliminary analysis of patients with resectable non-small cell lung cancer suggests that genotype is associated with shortened event free survival and greater risk of local relapse.

Topics: Aged; Alternative Splicing; Amino Acid Sequence; Base Sequence; Blotting, Northern; Carcinoma, Non-Small-Cell Lung; Cyclin D1; Cyclins; Female; Humans; Lung Neoplasms; Male; Middle Aged; Molecular Sequence Data; Oncogene Proteins; Polymerase Chain Reaction; Polymorphism, Genetic; RNA, Messenger

Employing Northern blot analysis and the polymerase chain reaction, we investigated PRAD1 gene overexpression in the tumour tissues of 58 patients with B-cell lymphoma. These findings were then examined in relation to the patients' clinical and immunohistological characteristics. The over-expression of this gene was detected in 6/8 patients with mantle cell lymphoma (MCL) and in only 1/50 other lymphomas, indicating its close association with MCL. The patients with MCL had common clinical findings of advanced disease with generalized lymphadenopathy on admission, and they had a CD5+CD10-IgD+ phenotype. The patients with chronic lymphocytic leukaemia (CLL) also showed findings indicating a distinctive disease entity: a CD5+CD10-IgD+ phenotype and lack of PRAD1 over-expression. In contrast, most patients with diffuse low-grade lymphoma other than MCL and CLL had localized extranodal disease, expressed a CD5-CD10-IgD- phenotype, and lacked PRAD1 over-expression. These findings suggest that extranodal low-grade lymphomas differ from nodal MCL and are not part of the spectrum of CLL.

Topics: Base Sequence; Blotting, Northern; Cyclin D1; Cyclins; Gene Expression; Humans; Leukemia, Lymphocytic, Chronic, B-Cell; Lung Neoplasms; Lymphoma, B-Cell; Lymphoma, Non-Hodgkin; Molecular Sequence Data; Oncogene Proteins; Polymerase Chain Reaction

The cyclin-dependent kinases and their associated regulatory cyclins control cell cycle progression and cell growth. Antibodies against these proteins were used to determine their levels in several lung tumor-derived cell lines and a "normal" immortalized bronchoepithelial cell line in order to investigate their potential roles in the etiology of lung cancer. All the cell lines expressed roughly equal levels of cdk-1; cdk-2; PSTAIRE-sequence containing kinases; proliferating cell nuclear antigen; and cyclins A, B1, and E. Cyclin D1, however, was present at 4- to 100-fold higher levels in 11 of 12 non-small cell lung cancer cell lines than in the bronchoepithelial line and all but one of the small cell lung cancer lines. Furthermore, immunoblots of the retinoblastoma gene product, pRB, revealed a perfect correlation between pRB levels and tumor type with normal levels of phosphorylation-competent pRB in all of the non-small cell lung cancer lines and undetectable levels of pRB in all of the small cell lung cancer lines. These data suggest the possibility that small cell and non-small cell lung cancer may evade normal growth controls by different mechanisms: loss of the proliferation inhibitor pRB in small cell lung cancer and overexpression of the growth promoting cyclin D1 in non-small cell lung cancer.

Topics: Carcinoma, Non-Small-Cell Lung; Carcinoma, Small Cell; Cell Cycle; Cyclin D1; Cyclins; Humans; Lung Neoplasms; Oncogene Proteins; Protein Kinases; Retinoblastoma Protein; Tumor Cells, Cultured

Cyclin D1, which is suggested to have a role in G1 control during the cell cycle, is genetically linked to BCL-1 and is widely overexpressed in parathyroid, breast, and squamous cancer cells. We postulated that cyclin D1 regulation may also be important in lung cancer. Therefore, we characterized the cell cycle-dependent expression of cyclin D1 at both mRNA and protein levels in synchronized human A549 lung carcinoma cells. Monospecific anti-cyclin D1 C-terminal peptide antibodies recognized both p36cyclinD1 and an as-yet uncharacterized 45 kD protein (p45). A549 cells were synchronized with well-studied drugs. Cyclin D1 mRNA expression remained relatively constant, with less than a twofold fluctuation during the cell cycle and with a minor peak at M phase. However, the p36cyclinD1 protein fluctuated during the A549 cell cycle and was expressed at very low levels in late G1 and at the G1/S boundary, but then increased in S phase and peaked at M phase. In contrast, p45 protein was expressed at relatively high levels in late G1 and reached maximal levels at the G1/S boundary, was expressed at decreased levels in S phase, and then had disappeared by M phase. Moreover, p45 was highly expressed only in transformed alveolar epithelial cells, but not in normal rat alveolar epithelial cells or fetal rat lung fibroblasts in primary cultures. In mink Mv1Lu cells, the expression of p45 was totally blocked by transforming growth factor-beta 1 treatment or contact inhibition. p45 protein was phosphorylated on serine, threonine, and tyrosine residues in A549 cells in culture. The phosphorylation of the p45 protein was cell cycle-regulated and reached its maximal levels at G2/M phase. The p45 protein had a different peptide map from p36cyclinD1 after cleavage with N-chlorosuccinimide. Immunoprecipitation studies showed that p45 was also anti-ubiquitin immunoreactive during the cell cycle. We conclude that p36cyclinD1 and the p45 protein are differentially regulated in a cell cycle-dependent manner in A549 cells. Although p45 is antigenically related to p36cyclinD1, it is probably not a closely cyclin-related protein. We speculate that p45 may be associated with malignant transformation and may play a distinct role from p36cyclinD1 in regulation of the cell cycle in A549 cells.

Topics: Amino Acid Sequence; Amino Acids; Animals; Carcinoma; Cell Transformation, Neoplastic; Cells, Cultured; Cyclin D1; Cyclins; Gene Expression Regulation, Neoplastic; Humans; Interphase; Lung; Lung Neoplasms; Molecular Sequence Data; Molecular Weight; Oligopeptides; Oncogene Proteins; Phosphorylation; Protein Biosynthesis; Proteins; Rats; RNA, Messenger; RNA, Neoplasm; Tumor Cells, Cultured