cyclin-d1 and Nasopharyngeal-Neoplasms

Multiple primary malignant tumors (MPMTs), usually associated with worse malignant behavior and prognosis comparing to a single primary tumor, and have recently been found to have an increasing incidence globally. However, the pathogenesis of MPMTs remains to be clarified. Here, we report a unique case of the coexistence of malignant melanoma (MM), papillary thyroid carcinoma (PTC), and clear-cell renal cell carcinoma (ccRCC) along with our perceptions on its pathogenesis.. The case reported is of a 59-year-old male patient with unilateral nasal obstruction as well as a renal occupying lesion. Positron emission tomography-computed tomography (PET-CT) revealed a palpable mass of 32 × 30 mm on the posterior and left walls of the nasopharynx. In addition, an isodense nodule was observed in the right superior renal pole, approximately 25 mm in diameter, as well as a slightly hypodense shadow in the right leaf of the thyroid, approximately 13 mm in diameter. Nasal endoscopy and magnetic resonance imaging (MRI) confirmed the existence of a nasopharyngeal neoplasm. Afterward, biopsies of the nasopharyngeal neoplasm, thyroid gland and kidney were performed, and the patient was diagnosed with MM, PTC, and ccRCC according to the pathological and immunohistochemical results. Moreover, mutation of BRAF. This is the first reported case of a patient with the co-existence of MM, PTC and ccRCC undergoing chemotherapy with a favorable prognosis. Herein, we suggest that such a combination may be non-random, as for mutation of BRAF

Topics: Carcinoma; Carcinoma, Renal Cell; Cyclin D1; Humans; Kidney Neoplasms; Male; Melanoma, Cutaneous Malignant; Middle Aged; Mutation; Nasopharyngeal Neoplasms; Neoplasms, Multiple Primary; Positron Emission Tomography Computed Tomography; Proto-Oncogene Proteins B-raf; Thyroid Cancer, Papillary; Thyroid Neoplasms

Cyclin D1 (CCND1) plays a key role in cell cycle regulation. It is a well-established human oncogene which is frequently amplified or overexpressed in cancers. The association between CCND1 G870A polymorphism and cancer risk has been widely assessed. However, a definitive conclusion between CCND1 G870A polymorphism and risk of nasopharyngeal carcinoma (NPC) remains elusive.. We firstly performed a hospital-based case-control study involving 165 NPC cases and 191 cancer-free controls in central-south China, and then conducted a meta-analysis with six case-control studies to evaluate the association between NPC risk and CCND1 G870A polymorphism.. The case-control study found a significant association between CCND1 G870A polymorphism and NPC risk in various comparison models (AA vs. GG: OR = 2.300, 95% CI 1.089-4.857, p = 0.029; AG vs. GG: OR = 2.832, 95% CI 1.367-5.867, p = 0.005; AA/AG vs. GG: OR = 2.597, 95% CI 1.288-5.237, p = 0.008; AA vs.. OR = 0.984, 95% CI 0.638-1.518, p = 0.944). Further meta-analysis showed that there was no significant association between CCND1 G870A polymorphism and NPC risk in overall analysis. In the stratified analysis by race, however, significant associations were only found in Caucasians (for the allele model A vs. G: OR = 0.75, 95% CI 0.59-0.97, p = 0.03; for the co-dominant model AA vs. GG: OR = 0.52, 95% CI 0.32-0.86, p = 0.01; for the dominant model AA/AG vs. GG: OR = 0.49, 95% CI 0.32-0.74, p<0.01; for the recessive model AA vs.. OR = 0.90, 95% CI 0.61-1.34, p = 0.60).. A significant association between CCND1 G870A polymorphism and NPC risk was found in the central-southern Chinese population. The meta-analysis indicated that CCND1 G870A polymorphism may contribute to the development of NPC in Caucasians.

Topics: Adult; Aged; Asian People; Carcinoma; Case-Control Studies; China; Cyclin D1; Female; Genetic Association Studies; Genetic Predisposition to Disease; Humans; Male; Middle Aged; Nasopharyngeal Carcinoma; Nasopharyngeal Neoplasms; Polymorphism, Single Nucleotide; White People

Recently, there have been a number of studies on the association between cyclin D1 G870A polymorphism and nasopharyngeal carcinoma risk. However, the results of previous reports remain controversial and ambiguous. Thus, we performed a meta-analysis to explore more precisely the association between cyclin D1 G870A polymorphism and the risk of nasopharyngeal carcinoma. No significant association was found between cyclin D1 G870A polymorphism and nasopharyngeal carcinoma risk in total population analysis. In the subgroup meta-analysis by ethnicity, a negative association was shown in Caucasian subgroup, and no significant association in any genetic models among Asians was observed. In summary, positive results have been shown on the search for polymorphic variants influencing the risk of NPC. This meta-analysis provides evidence of the association between CCND1 G870A polymorphism and NPC risk, supporting the hypothesis that CCND1 870A allele probably acts as an important NPC protective factor in Caucasians but not in Asians. Since the results of our meta-analysis are preliminary and may be biased by the relatively small number of subjects, they still need to be validated by well-designed studies using larger samples in the future.

Topics: Asian People; Carcinoma; Cyclin D1; Genetic Predisposition to Disease; Humans; Nasopharyngeal Neoplasms; Polymorphism, Single Nucleotide; White People

Compound Kushen injection (CKI) is a representative medication of Chinese herbal injection and is often used in the adjuvant treatment of nasopharyngeal carcinoma (NPC), but its antitumor mechanism is poorly understood.. To preliminarily elucidate the effects and possible mechanisms of CKI on NPC.. In this work, we explored the possible molecular mechanisms of CKI against NPC by using network pharmacology and molecular docking. In addition, proteomics was used to explore the localization and quantitative information of protein in NPC C666-1 cells after the intervention of CKI, and enrichment analysis was used to obtain the potential targets and pathways. Finally, the effect and the core targets of CKI in the intervention of NPC were explored in vitro experiments.. Network pharmacology analysis identified three active components of CKI and 13 key targets. Molecular docking analysis showed that TNF, PTEN, CCND1, MAPK3, IL6, HIF1A, MYC had high affinity with corresponding components. Then the key pathway, cell cycle and the core targets MYC, CCND1, and P15 related to the key pathway were obtained. The results of in vitro experiments showed that CKI could inhibit the proliferation, migration, and invasion of NPC 5-8F cells and C666-1 cells, induce apoptosis of C666-1 cells, and arrest cell cycle G0/G1 phase. In addition, RT-qPCR and western blot showed that the expression of P15 was up-regulated and E2F4, E2F5, c-Myc, CCND1, and P107 was down-regulated in 5-8F cells and C666-1 cells intervened by CKI.. The key pathway, cell cycle and the corresponding core targets MYC, CCND1, and P15 were obtained from network pharmacology, molecular docking, and proteomics. CKI could inhibit the proliferation, migration, and invasion of NPC cells, induce apoptosis of C666-1 cells. Especially CKI may arrest cell cycle G0/G1 phase through regulating targets MYC/P15/CCND1 of cell cycle pathway.

Topics: Antineoplastic Agents; Cyclin D1; Drugs, Chinese Herbal; Humans; Molecular Docking Simulation; Nasopharyngeal Carcinoma; Nasopharyngeal Neoplasms; Signal Transduction

Nucleosome assembly protein 1-like 1 (NAP1L1) is significantly involved in the development of various cancers. However, its role in the molecular mechanism of nasopharyngeal carcinoma (NPC) remains undetermined. In this study, we detected the upregulated expression of NAP1L1 mRNA and protein levels by quantitative polymerase chain reaction and Western blot analysis in NPC cell lines. Results of the immunohistochemistry analysis of NPC tissue biopsies showed that upregulated NAP1L1 protein expression promoted NPC progression and negatively correlated with poor prognosis in NPC patients. Suppression of NAP1L1 expression by small interfering RNA (siRNA) or small hairpin RNA (shRNA) methods significantly decreased cell proliferation in vivo and in vitro. Mechanism analysis revealed that the regulation of cell growth was enriched by Gene Set Enrichment Analysis based on RNA sequencing data. Cell cycle-induced genes CCND1 and E2F1 were downregulated in NAP1L1 knockdown NPC cells. Reduced NAP1L1 suppressed the recruitment of hepatoma-derived growth factor (HDGF) and decreased its expression. Knockdown of HDGF reduced the expression of c-JUN, a key oncogenic transcription factor that can induce the expression of cyclin D1 (CCND1), reducing cell cycle progression and suppressing cell growth in NPC. Transfecting HDGF or c-JUN could reverse the growth-suppressive effects in NAP1L1-downregulated NPC cells. The data obtained in this study suggest that NAP1L1 acts as a potential oncogene by activating HDGF/c-JUN/CCND1 signaling in NPC.

Topics: Animals; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Databases, Genetic; Female; G1 Phase Cell Cycle Checkpoints; Gene Expression Regulation, Neoplastic; Humans; Intercellular Signaling Peptides and Proteins; Male; Mice, Inbred BALB C; Mice, Nude; Middle Aged; Nasopharyngeal Carcinoma; Nasopharyngeal Neoplasms; Nucleosome Assembly Protein 1; Proto-Oncogene Proteins c-jun; RNA Interference; Signal Transduction; Tumor Burden; Xenograft Model Antitumor Assays

Cyclin D1 is an essential part of oncogenic transformation. We previously proved that cyclin D1 was upregulated in nasopharyngeal carcinoma (NPC) and promoted the NPC cell proliferation. But the association between cyclin D1 and the clinical outcome of NPC has not yet been determined. The study explores the possible relevance between the cyclin D1 expression and clinical parameters and its predictive value of prognosis in NPC patients.. We analyzed the clinical data from 379 NPC patients and 112 non-NPC patients in our previous study, which made further statistics. Receiver operating curve (ROC) was applied to select the optimal cutoff points. By analyzing the clinical data from 101 NPC patients using Chi-squared test, we estimated the relationship between the cyclin D1 expression level and clinicopathological parameters. We also used Kaplan-Meier method and log-rank test assess and compared the disease-free survival (DFS) rate and overall survival (OS) rate. The Cox proportional hazards model was adopted to perform the univariate and multivariate analyses.. Receiver operating curve analysis reported that cyclin D1 was used to differentiate between NPC patients and non-NPC patients (P < .001, sensitivity: 53.6%, specificity: 85.7%, AUC = 0.752). Cyclin D1 was positively correlated with lymph node metastasis (P = .015). A survival analysis of the 101 NPC patients indicated that the positive expression of cyclin D1 was predictive of a good prognosis (DFS: P = .010, OS: P = .019). Multivariate analysis showed that cyclin D1 could be used independently to predict NPC patients' prognosis (DFS: P = .038).. The overexpression of cyclin D1 is a good prognostic marker for NPC.

Topics: Adult; Aged; Biomarkers, Tumor; Cyclin D1; Female; Humans; Male; Middle Aged; Nasopharyngeal Carcinoma; Nasopharyngeal Neoplasms; Prognosis; Survival Analysis; Up-Regulation; Young Adult

nasopharyngeal cancer (NPC) is the most common type of head and neck cancer in Indonesia (28.4%). Reports showed that 18.9% of cases came with advanced stage. Chemotherapy play important role in advanced stage. However, patients with the same stage of the disease may have different treatment response, likely due to the different tumor biological characteristics. Cyclin D1 is a protein involved in the cell cycle, which will stimulate proliferation. This study aimed to examine the proportion of cyclin D1 in NPC and its association with treatment response.. a retrospective cohort study was conducted on advanced NPC patients that underwent chemotherapy at Cipto Mangunkusumo Hospital from 2015 until 2018. Cyclin D1 immunohistochemistry staining was done by antigen retrieval methods using the cyclin D1 NovocastraTM monoclonal antibody. The cyclin D1 expression was evaluated with h-score. Treatment response was reviewed based on the RECIST 1.1 criteria.. fifteen subjects (48.4%) had a positive expression of cyclin D1. Higher proportion of cyclin D1 positive was found in responsive group compare with non-responsive group (66.7% vs. 33.3%, p = 0.032). Statistically significant difference in mean h-score was observed between the subjects who responded and those who did not respond (116.24 SD57.80 vs. 77.97 SD45.27, p = 0.048).. this study suggests that a higher expression of cyclin D1 is associated with a good treatment response in NPC patients.

Topics: Adult; Antineoplastic Agents; Biomarkers, Tumor; Cisplatin; Cyclin D1; Female; Fluorouracil; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Indonesia; Male; Middle Aged; Nasopharyngeal Carcinoma; Nasopharyngeal Neoplasms; Retrospective Studies; Treatment Outcome

Long non-coding RNAs (lncRNAs) exhibit important roles in a variety of biological properties of tumors. LncRNA HCG18 (HCG18) is a newly identified lncRNA whose roles in tumor progression remains largely unclear. The objective of our current research was to explore the roles and the underlying mechanisms of HCG18 in nasopharyngeal carcinoma (NPC).. Reverse Transcription-Polymerase Chain Reaction (RT-PCR) was used to determine the levels of HCG18 in NPC tissue and cell lines. Clinical significances and prognostic values of HCG18 were analyzed using the statistical methods. Cell Counting Kit-8 (CCK-8) assays, clonogenic survival assays, flow cytometry, wound-healing assays, and transwell assays were used to examine the tumorigenesis functions of HCG18 in vitro. Insights of the mechanism of ceRNAs were gained from bioinformatic methods and Luciferase analysis. Western blot was performed to determine the expression of tumor-related pathways.. We found that HCG18 expression was upregulated in both NPC specimens and cell lines. Higher levels of HCG18 were associated with positive lymph node metastasis and poor prognosis of NPC patients. Importantly, the multivariate analysis confirmed that HCG18 was an independent risk factor for outcome. Functionally, the downregulation of HCG18 exhibited tumor-suppressive effects via the inhibition of cell proliferation and metastasis. Mechanistically, HCG18 may directly bind to miR-140 and effectively act as a ceRNA for miR-140 to increase the expression of cyclin D1 (CCND1). In addition, HCG18 may contribute to NPC progression via modulating Wnt/β-catenin signaling and Hedgehog pathway.. Our findings suggested that HCG18 served as an oncogenic lncRNA in NPC progression, which may provide a novel biomarker of an unfavorable outcome and a potential therapeutic target for NPC.

Topics: Cell Line, Tumor; Cyclin D1; Disease-Free Survival; Female; Gene Expression Regulation, Neoplastic; Hedgehog Proteins; Humans; Kaplan-Meier Estimate; Lymphatic Metastasis; Male; MicroRNAs; Middle Aged; Nasopharyngeal Carcinoma; Nasopharyngeal Neoplasms; Nasopharynx; Pharyngectomy; Prognosis; Risk Factors; RNA, Long Noncoding; Up-Regulation; Wnt Signaling Pathway

The transcription factor Krüppel-like factor 6 (KLF6) regulates various cellular functions, such as metabolism, cell proliferation, and differentiation. KLF6 plays a key role in the development and progression of multiple human cancers.. Fifty primary biopsies and 10 normal nasopharyngeal mucosae were used to analyze by RT-QPCR the expression and the copy number of wtKLF6 and the spliced variants (KLF6-SV1, KLF6-SV2, and KLF6-SV3) in Tunisian patients with nasopharyngeal carcinoma. The expression analysis of E-cadherin and cyclin D1 was conducted by RT-QPCR and Western blot, respectively.. The wtKLF6 was significantly downexpressed in tumors compared to normal tissues (. The wtKLF6 was downexpressed in contrast with the oncogenic variants. Overexpression of KLF6-SV1 is associated with young patients, and loss of E-cadherin suggests that this variant correlated with the aggressiveness of NPC.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Alternative Splicing; Antigens, CD; Cadherins; Carcinoma; Cyclin D1; DNA Copy Number Variations; Female; Humans; Kruppel-Like Factor 6; Male; Middle Aged; Nasopharyngeal Carcinoma; Nasopharyngeal Neoplasms; Young Adult

Patient-derived xenograft (PDX) tumor model has become a new approach in identifying druggable tumor mutations, screening and evaluating personalized cancer drugs based on the mutated targets.. We established five nasopharyngeal carcinoma (NPC) PDXs in mouse model. Subsequently, whole-exome sequencing (WES) and genomic mutation analyses were performed to search for genetic alterations for new drug targets. Potential drugs were applied in two NPC PDX mice model to assess their anti-cancer activities. RNA sequencing and transcriptomic analysis were performed in one NPC PDX mice to correlate with the efficacy of the anti-cancer drugs.. A relative high incident rate of copy number variations (CNVs) of cell cycle-associated genes. Among the five NPC-PDXs, three had cyclin D1 (CCND1) amplification while four had cyclin-dependent kinase inhibitor CDKN2A deletion. Furthermore, CCND1 overexpression was observed in > 90% FFPE clinical metastatic NPC tumors (87/91) and was associated with poor outcomes. CNV analysis disclosed that plasma CCND1/CDKN2A ratio is correlated with EBV DNA load in NPC patients' plasma and could serve as a screening test to select potential CDK4/6 inhibitor treatment candidates. Based on our NPC PDX model and RNA sequencing, Palbociclib, a cyclin-dependent kinase inhibitor, proved to have anti-tumor effects by inducing G1 arrest. One NPC patient with liver metastatic was treated with Palbociclib, had stable disease response and a drop in Epstein Barr virus (EBV) EBV titer.. Our integrated information of sequencing-based genomic studies and tumor transcriptomes with drug treatment in NPC-PDX models provided guidelines for personalized precision treatments and revealed a cyclin-dependent kinase inhibitor Palbociclib as a novel candidate drug for NPC.

Topics: Adolescent; Adult; Animals; Carcinoma; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p16; Cyclin-Dependent Kinase Inhibitor p18; DNA Copy Number Variations; Exome Sequencing; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Herpesvirus 4, Human; Humans; Male; Mice; Middle Aged; Nasopharyngeal Carcinoma; Nasopharyngeal Neoplasms; Piperazines; Protein Kinase Inhibitors; Pyridines; Xenograft Model Antitumor Assays

Nasopharyngeal carcinoma (NPC) is rare worldwide but remains highly prevalent in endemic regions, notably in southern China. Radiotherapy remains the treatment of choice for NPC, but radioresistance has been identified as a major cause of therapeutic failure. The Wnt/β-catenin signaling has been found to be involved in NPC radioresistance; however, the effect of β-catenin overexpression on radioresistance remains unknown in NPC until now. This study aimed to examine the impact of β-catenin overexpression on the radiosensitivity of human NPC CNE-2 cells.. Immunohistochemistry was performed to detect the β-catenin expression in normal nasopharyngeal specimens and NPC specimens. The human NPC CNE-2 cell line overexpressing β-catenin was modeled by transfection with the pcDNA3.1/Hygro(+)/β-catenin recombinant vector (transfection group), while cells transfected with the pcDNA3.1/Hygro(+) vector served as negative controls and non-transfected cells served as blank controls. The expression of key molecules of the Wnt/β-catenin signaling pathway was determined using Western blotting and qPCR assays, and the changes of radiation sensitivity were measured with a colony-formation assay. Cell viability was measured by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5 -diphenyltetrazolium bromide) assay. In addition, the cell cycle and apoptosis was detected using flow cytometry and the TCF/LEF transcriptional activity was measured with a Dual Luciferase Reporter Assay System.. Immunohistochemical staining showed high β-catenin expression in radioresistant NPC specimens, and low expression in radiosensitive NPC specimens and normal nasopharyngeal specimens. Western blotting and qPCR assays detected higher β-catenin expression in the transfection group than in the negative and blank controls (P < 0.01). Down-regulation of GSK-3β expression (P < 0.05) and up-regulation of Cyclin D1 expression (P < 0.01) was detected in β-catenin overexpressing NPC cells exposed to X-ray radiation relative to negative and blank controls. Colony-formation assay revealed higher D0, Dq and SF in the transfection group than in the negative and blank control groups post-radiation, and the SER in the transfection group was 0.75-fold and 0.68-fold greater than that in the blank and negative control groups, respectively. MTT assay revealed that the viability of CNE-2 cells was significantly higher in the transfection group (96% ± 8.72%) than in the negative control group (74.67 ± 7.05%) and the blank control group (75.33% ± 7.02%) 24 h post-exposure to 6 Gy X-ray radiation (P < 0.05). X-ray radiation led to a lower proportion of CNE-2 cells at the G2/M phase and a lower apoptotic rate in the transfection group than in the negative and blank control groups (P < 0.05). In addition, the TCF/LEF transcriptional activity was higher in the transfection group than in the negative and blank control groups (P < 0.01), and 6 Gy X-ray radiation elevated the TCF/LEF transcriptional activity relative to 0 Gy radiation in the transfection group (P < 0.01).. β-catenin overexpression may decrease the radiation sensitivity in NPC CNE-2 cells through activating the downstream transcriptional factors of β-catenin, and reducing G2/M arrest and cell apoptosis.

Topics: beta Catenin; Carcinoma; Cell Line, Tumor; Cell Survival; Cyclin D1; Down-Regulation; G2 Phase Cell Cycle Checkpoints; Glycogen Synthase Kinase 3 beta; Humans; M Phase Cell Cycle Checkpoints; Nasopharyngeal Carcinoma; Nasopharyngeal Neoplasms; Radiation Tolerance; Up-Regulation; Wnt Signaling Pathway; X-Rays

Nasopharyngeal carcinoma (NPC) is one of the most common malignant tumor in Southeast Asia, its regulatory mechanism is still to be understood. miR-519 inhibits the progression of several tumors, including cervical cancer, ovarian cancer and gastric cancer. But its role in NPC hasn't been studied. In present study, we found miR-519 was downregulated in NPC cells, its overexpression inhibited NPC cell proliferation and arrested cell cycle at G0/G1 phase, while its knockdown promoted NPC cell proliferation and cell cycle progression. An oncogene URG4/URGCP (upregulated gene-4/upregulator of cell proliferation) was the target of miR-519, URG4 was upregulated in NPC cells, miR-519 inhibited URG4 expression by directly binding to the 3'UTR of URG4. miR-519 inhibited Cyclin D1 expression and the phosphorylation level of Rb, and increased p21 and p27 expression, confirming miR-519 blocked G1/S transition. Moreover, miR-519 level was negative correlated with URG4 level in NPC tissues. In summary, we found miR-519 NPC cell proliferation by inhibiting URG4.

Topics: Carcinoma; Cell Line, Tumor; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase Inhibitor p27; G1 Phase; Gene Expression Regulation, Neoplastic; Genes, Tumor Suppressor; Humans; MicroRNAs; Nasopharyngeal Carcinoma; Nasopharyngeal Neoplasms; Neoplasm Proteins; Retinoblastoma Protein; RNA, Neoplasm; S Phase; Up-Regulation

miR-374a has been reported to function as an oncogene during tumor pathogenesis. In this study, miR-374a is observed to reduce nasopharyngeal carcinoma (NPC) cell proliferation, migration, invasion, metastasis and cisplatin (DDP) resistance in vitro and in vivo. Mechanistic analyses indicate that miR-374a directly targets CCND1 to inactivate pPI3K/pAKT/c-JUN forming a negative feedback loop, as well as suppressing downstream signals related to cell cycle progression and epithelial-mesenchymal transition (EMT). Interestingly, we also observed that miR-374a direct targeting of CCND1 is modulated by tumor suppressor PDCD4 via suppressing pPI3K/pAKT/c-JUN signaling. In clinical specimens, miR-374a was positively and negatively correlated with expression of PDCD4 and CCND1, respectively. Our studies are the first to demonstrate that the miR-374a-CCND1-pPI3K/AKT-c-JUN feedback loop induced by PDCD4 supresses NPC cell growth, metastasis and chemotherapy resistance.

Topics: Animals; Apoptosis Regulatory Proteins; Carcinoma; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cisplatin; Cyclin D1; Drug Resistance, Neoplasm; Epithelial-Mesenchymal Transition; Feedback, Physiological; Gene Expression Regulation, Neoplastic; Humans; Mice; MicroRNAs; Nasopharyngeal Carcinoma; Nasopharyngeal Neoplasms; Neoplasm Metastasis; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; RNA-Binding Proteins; Signal Transduction

Nasopharyngeal carcinoma (NPC) is an epithelial malignancy usually associated with overexpression of both epidermal growth factor receptor (EGFR) and β-catenin. FM807 is a novel curcumin analogue with antitumor activity against both poorly and well-differentiated NPC cell lines as well as good selectivity for tumor cells. FM807 actions were shown to include inhibition of cell growth, induction of necrotic/late apoptotic cell death, and G1 arrest in NPC cells. Crucially, it exhibited potent antitumor effects both in vitro and in vivo. Binding of FM807 to the N-terminus of Hsp90 disrupted Hsp90/client complexes, resulting in degradation of the Hsp90 client protein EGFR and inhibition of the downstream Raf/MEK/ERK and PI3K/AKT pathway. FM807 also depleted levels of the intranuclear transcription factors β-catenin, Cyclin D1 and c-Myc levels by inhibiting Hsp90 chaperoned nuclear transport. In conjunction with its low toxicity in NPC xenograft mice, these results provide a sound preclinical basis for further development of FM807 as a novel therapeutic agent in the treatment of NPC.

Topics: Animals; Antineoplastic Agents; Apoptosis; beta Catenin; Blotting, Western; Cell Line, Tumor; Cell Proliferation; Curcumin; Cyclin D1; ErbB Receptors; G1 Phase Cell Cycle Checkpoints; HSP90 Heat-Shock Proteins; Humans; Male; MAP Kinase Signaling System; Mice, Inbred BALB C; Mice, Nude; Nasopharyngeal Neoplasms; Protein Binding; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; Xenograft Model Antitumor Assays

Cyclin-dependent kinase 4 (CDK4) is a member of cyclin-dependent kinase family which regulates G1 to S cell cycle transition. CDK4 activity is increased in many tumor types. Here, we report a negative automodulatory feedback loop between CDK4 and miR-16 that regulates cell cycle progression in nasopharyngeal carcinoma (NPC). By miRNA array and real-time PCR, we identified upregulation of tumor suppressor miR-16a, which inhibited cell cycle progression and sensitized NPC cells to chemotherapy. CDK4 knockdown reduced the expression of c-Myc, the latter of which directly suppresses the miR-16 expression by directly binding to the miR-16 promoter. Moreover, we found that miR-16 upregulation could reduce CDK4 expression by repressing CCND1 and thus forms a feedback loop via the CDK4/c-Myc/miR-16/CCND1 pathway. Finally, miR-16 was negatively correlated with CDK4 expression in NPC biopsies. In summary, our results define a double-negative feedback loop involving CDK4 and miR-16 mediated by c-Myc that modulates NPC cell growth and chemotherapy sensitivity.

Topics: Animals; Antineoplastic Agents; Carcinoma; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase 4; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Genes, Tumor Suppressor; Humans; Male; Mice; Mice, Inbred BALB C; Mice, Nude; MicroRNAs; Nasopharyngeal Carcinoma; Nasopharyngeal Neoplasms; Proto-Oncogene Proteins c-myc; Up-Regulation

Nasopharyngeal carcinoma (NPC) is a peculiar Epstein Barr virus (EBV)-associated malignancy that is prevalent in South-East Asia. Peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (PIN1) isomerizes specific phosphorylated amino acid residues, which makes it an important regulator in cell survival and apoptosis. In this study, we investigated the contribution made by PIN1 in NPC tumorigenesis and PIN1's potential role as a therapeutic target.. The expression of PIN1 was examined in a panel of NPC cell lines, xenografts and primary tumors. The functional roles of PIN1 in NPC cells were elucidated by the knockdown and overexpression of PIN1 in in vitro and in vivo nude mice models by siRNA and lenti-viral transfection, respectively. The antitumor effects of the PIN1 inhibitor Juglone in NPC cells were also evaluated.. We revealed the consistent overexpression of PIN1 in almost all EBV-associated NPC cell lines, xenografts and primary tumors. PIN1 suppression was capable of inhibiting cyclin D1 expression and activating caspase-3 in NPC cells. It positively regulated NPC cell proliferation, colony formation and anchorage-independent growth. The inhibition of PIN1 suppressed tumor growth in vitro and in vivo.. This study demonstrates the oncogenic role of PIN1 in NPC tumorigenesis, and shows that its overexpression can enhance tumor cell growth via the upregulation of cyclinD1. Our findings inform the development of novel treatments targeting PIN1 for NPC patients.

Topics: Animals; Carcinoma; Caspase 3; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Female; HeLa Cells; Herpesvirus 4, Human; Humans; Immunohistochemistry; In Vitro Techniques; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Middle Aged; Naphthoquinones; Nasopharyngeal Carcinoma; Nasopharyngeal Neoplasms; NIMA-Interacting Peptidylprolyl Isomerase; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction

Nasopharyngeal carcinoma (NPC) is an Epstein-Barr virus (EBV)-associated epithelial malignancy that exhibits distinct geographical and ethnic prevalence. Although the contemporary therapeutic approach of radio-/chemotherapy provides excellent results for patients with early-stage disease, it is far from satisfactory for those with disease remission and distant metastasis. Promising therapeutic strategies for advanced and relapsed NPC are still lacking. We recently identified and characterized a cancer stem-like cell (CSC) subpopulation in NPC that appeared to play an important role in tumor progression. Microarray analysis revealed downregulation of several stemness-inhibiting miRNAs in these CSC cells. Among these miRNAs, miR-96 and miR-183 showed the highest fold change and were selected to elucidate their role in repressing NPC CSC properties.. MiR-96 and miR-183 expression in NPC CSCs was detected by qRT-PCR. Transient and stable transfection was performed in EBV-positive NPC C666-1 cells to examine the effects of ectopic expression of miR-96 and miR-183 on repressing cell growth and CSC properties. Anchorage-dependent (colony formation) and anchorage-independent (tumor sphere formation) growths of these miR-96 and miR-183 expressing cells were determined. Expression of multiple CSC markers and related molecules were accessed by flow cytometry and Western blotting. The tumorigenicity of the stable miR-96- and miR-183-transfected NPC cells was examined in an in vivo nude mice model.. Downregulation of miR-96 and miR-183 was confirmed in NPC spheroids. Using transient or stable transfection, we showed that ectopic expression of miR-96 and miR-183 suppressed cell growth and tumor sphere formation in NPC. Reduced NICD3 and NICD4 in miR-96- and miR-183-expressing NPC cells suggests the involvement of the NOTCH signaling pathway in their tumor suppressive function. Finally, we showed that the tumorigenicity of cells stably expressing miR-183 was significantly inhibited in the in vivo nude mice model.. miR-183 is a tumor-suppressive miRNA in EBV-associated NPC. Its abilities to suppress CSC properties in vitro and effectively reduce tumor growth in vivo shed light on its role as a potential therapeutic target.

Topics: Animals; Blotting, Western; Cell Line, Tumor; Cyclin D1; Epstein-Barr Virus Infections; Female; Gene Expression Regulation, Neoplastic; Herpesvirus 4, Human; Host-Pathogen Interactions; Humans; Mice, Inbred BALB C; Mice, Nude; MicroRNAs; Nasopharyngeal Neoplasms; Neoplastic Stem Cells; Oligonucleotide Array Sequence Analysis; Proto-Oncogene Proteins; Receptor, Notch3; Receptor, Notch4; Receptors, Notch; Reverse Transcriptase Polymerase Chain Reaction; Spheroids, Cellular; Transplantation, Heterologous

It was shown in this study that knockdown of ClC-3 expression by ClC-3 siRNA prevented the activation of hypotonicity-induced chloride currents, and arrested cells at the G0/G1 phase in nasopharyngeal carcinoma CNE-2Z cells. Reconstitution of ClC-3 expression with ClC-3 expression plasmids could rescue the cells from the cell cycle arrest caused by ClC-3 siRNA treatments. Transfection of cells with ClC-3 siRNA decreased the expression of cyclin D1, cyclin dependent kinase 4 and 6, and increased the expression of cyclin dependent kinase inhibitors (CDKIs), p21 and p27. Pretreatments of cells with p21 and p27 siRNAs depleted the inhibitory effects of ClC-3 siRNA on the expression of CDK4 and CDK6, but not on that of cyclin D1, indicating the requirement of p21 and p27 for the inhibitory effects of ClC-3 siRNA on CDK4 and CDK6 expression. ClC-3 siRNA inhibited cells to progress from the G1 phase to the S phase, but pretreatments of cells with p21 and p27 siRNAs abolished the inhibitory effects of ClC-3 siRNA on the cell cycle progress. Our data suggest that ClC-3 may regulate cell cycle transition between G0/G1 and S phases by up-regulation of the expression of CDK4 and CDK6 through suppression of p21 and p27 expression.

Topics: Carcinoma; Cell Cycle; Cell Line, Tumor; Chloride Channels; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase 6; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase Inhibitor p27; Gene Expression Regulation, Neoplastic; Humans; Nasopharyngeal Carcinoma; Nasopharyngeal Neoplasms; RNA, Small Interfering; S Phase; Transcriptional Activation

Long non-coding RNA (lncRNA) Ewing sarcoma associated transcript 1 (EWSAT1) has been identified as an oncogene, and its dysregulation is closed corrected with tumor progression in Ewing sarcoma. Recently, high-through put analysis reveals that EWSAT1 is also highly expressed in human nasopharyngeal carcinoma (NPC). However, whether the aberrant expression of EWSAT1 in NPC is corrected with malignancy or prognosis has not been expounded. Herein, we identified that EWSAT1 was up-regulated in NPC tissues and cell lines, and higher expression of EWSAT1 resulted in a markedly poorer survival time. EWSAT1 over-expression facilitated, while EWSAT1 silencing impaired cell growth in NPC. In addition, mechanistic analysis demonstrated that EWSAT1 up-regulated the expression of miR-326/330-5p clusters targeted gene cyclin D1 through acting as a competitive 'sponge' of miR-326/330-5p clusters. Collectively, our data revealed that EWSAT1 promotes NPC cell growth in vitro through up-regulating cyclin D1 partially via 'spongeing' miR-326/330-5p clusters.

Topics: Carcinoma; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Gene Expression Regulation, Neoplastic; Humans; MicroRNAs; Nasopharyngeal Neoplasms; RNA-Binding Protein EWS

To observe the effect of high-mobility group box-1 protein (HMGB1) on the proliferation of human nasopharyngeal carcinoma cell line C666-1 and explore the possible underlying mechanisms.. Cultured C666-1 cells were treated with a siRNA targeting HMGB1 gene. The changes in the cell proliferation were detected by CCK8 analysis, the cell cycle distribution was assayed with flow cytometry, and the expressions of cyclin D1, CDK6 and related pathway proteins were detected with Western blotting. The effect of a HMGB1 plasmid carrying the reporter gene GFP on the proliferation of C666-1 cells was tested with CCK8 and EDU analysis.. Compared with the control cells, the cells transfected with the siRNA targeting HMGB1 showed obviously suppressed cell proliferation (P<0.001), cell cycle arrest in G1 phase (P<0.001), and down-regulated expressions of cyclin D1, CDK6, STAT3 and P-STAT3. Overexpression of HMGB1 in cells transfected with the HMGB1 plasmid showed a significantly increased ratio of S phase cells (P<0.05) and obviously enhanced cell proliferation (P<0.001).. HMGB1 can promote the proliferation of human nasopharyngeal carcinoma cell line C666-1 by up- regulating cyclin D1 and CDK6 via the STAT3 signaling pathway.

Topics: Carcinoma; Cell Cycle; Cell Cycle Checkpoints; Cell Division; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase 6; Down-Regulation; HMGB1 Protein; Humans; Nasopharyngeal Carcinoma; Nasopharyngeal Neoplasms; RNA, Small Interfering; Signal Transduction; STAT3 Transcription Factor; Transfection; Up-Regulation

Tumor-associated macrophage (TAM)-related chronic inflammation and interleukin-6 (IL-6) contribute to the progression of nasopharyngeal carcinoma (NPC). In this study, we characterized TAMs and IL-6 expression in 212 biopsied NPC and 119 non-tumor nasopharyngeal epithelium (NPE) tissues by tissue array. In comparison with that in the NPE tissues, more TAM infiltrates and a higher density of IL-6 expression were detected in NPC tissues, which were associated with the poor survival of NPC patients. In contrast, little or no LPLUNC1, a regulator of inflammation, expression was detected in NPC tissues, and the levels of LPLUNC1 expression in the NPC were associated negatively with the numbers of TAMs and the levels of IL-6 expression, but positively with the survival of NPC patients. Induction of LPLUNC1 overexpression in NPC cells mitigated lipopolysaccharide (LPS)-induced IL-6, IL-8, tumor necrosis factor-α and IL-1β expression or treatment of THP-1 macrophages with LPLUNC1 inhibited spontaneous and LPS-induced IL-6 expression in vitro. IL-6-promoted NPC cell proliferation in a dose- and time-dependent manner, accompanied by increasing cyclin D1 and Bcl-2 expression and the Stat3 activation, but inhibiting Bax and p21 expression. Induction of LPLUNC1 overexpression inhibited NPC cell proliferation, induced NPC cell arrest, promoted NPC cell apoptosis even after IL-6 stimulation and inhibited the growth of implanted NPC tumors in vivo, which were associated with decreasing cyclin D1 and Bcl-2 expression and the Janus kinase 2 (JAK2)/Stat3 activation, but enhancing Bax and p21 expression. These results suggest that LPLUNC1 can inhibit inflammation and NPC growth by downregulating the Stat3 pathway.

Topics: Animals; Autoantigens; Blotting, Western; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Dose-Response Relationship, Drug; Fatty Acid-Binding Proteins; Female; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Interleukin-6; Lipopolysaccharides; Macrophages; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Middle Aged; Nasopharyngeal Neoplasms; Proteins; Proto-Oncogene Proteins c-bcl-2; Reverse Transcriptase Polymerase Chain Reaction; STAT3 Transcription Factor; Time Factors; Transplantation, Heterologous

Long non-coding RNAs (lncRNAs) are aberrantly expressed and have important functions in pathological processes. The present study investigated the lncRNA profiles and the effects of curcumin (Cur) on the radiosensitivity of nasopharyngeal carcinoma (NPC) cells. The lncRNA and mRNA profiles of each cell group were described by microarray analysis. Numerous differentially expressed genes were observed by microarrays in three cell groups. Cur significantly reversed the IR-induced lncRNA and mRNA expression signatures, shown by clustering analysis. Moreover, 116 of these IR-induced and Cur-reversed differentially expressed lncRNAs were obtained. Six lncRNAs (AF086415, AK095147, RP1-179N16.3, MUDENG, AK056098 and AK294004) were confirmed by qPCR. Furthermore, functional studies showed that lncRNA AK294004 exhibited a negative effect on cyclin D1 (CCND1), indicating that CCND1 might be a direct target of AK294004. IR-induced differentially expressed lncRNAs were reversed during Cur-enhanced radiosensitization in NPC cells, suggesting that lncRNAs have important functions in IR-induced radioresistance. Thus, Cur could serve as a good radiosensitizer.

Topics: Carcinoma; Cell Line, Tumor; Curcumin; Cyclin D1; Gene Expression Regulation, Neoplastic; Humans; Nasopharyngeal Carcinoma; Nasopharyngeal Neoplasms; Radiation Tolerance; RNA, Long Noncoding; RNA, Messenger

The association between cyclin D1 and survivin protein expressions with radiotherapy sensitivity in patients with nasopharyngeal carcinoma was investigated. Biopsy specimens of 72 patients with nasopharyngeal carcinoma were collected before the initiation of radiotherapy (49 cases were in the radiation-sensitive group and 23 cases were in the radiation-insensitive group). Conventional hematoxylin and eosin staining was used for tissue typing. The immunohistochemical SP method was used to detect cyclin D1 and survivin protein expression levels. The IBM SPSS Statistics 20 statistical software was applied for conducting the chi-squared test and the Spearman correlation analysis. In the 72 cases, the high expression rates of cyclin D1 were 28.6% (14/49) and 69.6% (16/23) in the radiotherapy-sensitive group and in the radiotherapy-insensitive group, respectively, and the differences between groups were statistically significant (P<0.05). The high expression rates of survivin were 34.7% (17/49) and 73.9% (17/23) in the radiotherapy-sensitive group and in the radiotherapy-insensitive group, respectively, which differed significantly (P<0.05). The protein expressions of cyclin D1 and survivin were positively correlated (Spearman's r=0.353, P<0.05). Cyclin D1 and survivin expression levels were negatively correlated with the radiosensitivity of nasopharyngeal carcinoma. Cyclin D1 and survivin may be used as molecular markers to predict the sensitivity of radiotherapy.

Topics: Biomarkers, Tumor; Carcinoma; Cyclin D1; Female; Gene Expression Regulation, Neoplastic; Genetic Association Studies; Humans; Inhibitor of Apoptosis Proteins; Male; Middle Aged; Nasopharyngeal Carcinoma; Nasopharyngeal Neoplasms; Prognosis; Radiation Tolerance; Survivin

To determine the expression and function of B cell translocation gene 1 (BTG1) in nasopharyngeal carcinoma. Nasopharyngeal samples were taken from cancer lesions (n = 75) and adjacent normal tissue (n = 33) in nasopharyngeal cancer patients immediately after endoscopic biopsy. BTG1 expression was determined by immunohistochemistry and Western blotting. The effect of BTG1 overexpression was examined in vitro utilizing a human nasopharyngeal cancer cell line CNE2 stably transfected with a recombinant lentivirus (LeBTG1 cells) and compared to empty vector-transfected controls (LeEmpty). BTG1 overexpression was verified by real-time reverse transcriptase polymerase chain reaction and Western blot. The expression of proteins involved in cell cycle regulation (cyclin D1), apoptosis (Bcl-2) and cell migration (MMP-9) in LeBTG1 cells were analyzed by Western blot. The effect of BTG1 overexpression on cell viability and proliferation was assessed by an MTT assay in LeBTG1 and LeEmpty cells. Flow cytometric analyses were used to evaluate the effect of BTG1 expression on cell cycle distribution and apoptosis. The migration and invasion potential of LeBTG1 cells was examined by plating cells in Matrigel-coated chambers. BTG1 protein expression was significantly lower in nasopharyngeal cancer tissue biopsies than normal tissue as measured by immunohistochemistry (36.0 vs. 81.8 % of tissues; P < 0.05) and Western blotting (0.221 ± 0.019 vs. 0.652 ± 0.055; P < 0.05). Decreased expression of BTG1 was significantly correlated with nasopharyngeal cancer tumor stage, lymph node metastasis, clinical stage and pathologic differentiation (P < 0.05), as well as with reduced overall five-year survival rates compared to patients with higher expression levels (31.2 vs. 70.2 %; P < 0.05). In vitro analyses revealed that LeBTG1 cells had a reduced survival fraction compared to control LeEmpty cells, with higher rates of apoptosis (9.3 ± 0.7 vs. 2.3 ± 0.3 %; P < 0.05). The proportion of LeBTG1 cells in G0/G1 stage and S phase was also significantly different from LeEmpty cells (82.6 ± 3.8 and 10.1 ± 1.0 %, vs. 62.2 ± 2.4 and 28.9 ± 2.0 %, respectively; Ps < 0.05), and the migration and invasion of LeBTG1 cells was significantly impaired with respect to LeEmpty cells (96.0 ± 13.0 and 91.0 ± 11.0 vs. 158.0 ± 17.0 and 142.0 ± 15.0, respectively; Ps < 0.05). These effects were accompanied by decreased protein expression of cyclin D1, Bcl-2 and MMP-9 in LeBTG1 cells (0.231 ± 0.021, 0.413

Topics: Adult; Aged; Apoptosis; Biomarkers, Tumor; Carcinoma; Cell Cycle; Cell Movement; Collagen; Cyclin D1; Down-Regulation; Drug Combinations; Gene Expression Regulation, Neoplastic; Humans; Laminin; Lymphatic Metastasis; Matrix Metalloproteinase 9; Middle Aged; Nasopharyngeal Carcinoma; Nasopharyngeal Neoplasms; Neoplasm Proteins; Proteoglycans; Proto-Oncogene Proteins c-bcl-2

To assess the effect of small interfering RNA (siRNA)-mediated suppression of CDK6 expression on the proliferation and cell cycles of nasopharyngeal carcinoma (NPC) cells in vitro.. QRT-PCR was used to examine the differential expression of CDK6 in 30 NPC tissues and 18 normal nasopharyngeal tissues. A siRNA targeting CDK6 was transfected in NPC CNE2 cells, and MTT assay and flow cytometry were used to analyze the changes in cell proliferation and cell cycle distribution. Western blotting was used to examine the expressions of the cell cycle-related factors.. Compared with normal nasopharyngeal tissues, NPC tissues showed an increased expression of CDK6 mRNA. Knocking down CDK6 expression obviously inhibited tumor cell growth and cell cycle transition from G1 to S phase and caused reduced expressions of CDK4, CCND1, and E2F1 and enhanced expression of the tumor suppressor p21.. NPC tissues overexpress CDK6. Knocking down CDK6 expression inhibits the growth and cell cycle transition of NPC cells in vitro by inhibiting the expressions of CDK4, CCND1, and E2F1 and upregulating tumor suppressor p21 expression.

Topics: Carcinoma; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase 6; Cyclin-Dependent Kinase Inhibitor p21; E2F1 Transcription Factor; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Humans; Nasopharyngeal Carcinoma; Nasopharyngeal Neoplasms; RNA, Messenger; RNA, Small Interfering; Transfection; Up-Regulation

The primary treatment for nasopharyngeal carcinoma (NPC) is radiotherapy, with or without concurrent chemotherapy. However, resistance to radiotherapy is not uncommon. The aim of the present study was to establish a radioresistant NPC cell line to study the molecular mechanisms of radioresistance by measuring the expression of cell cycle control proteins src homology 2 domain-containing phosphatase (SHP)-1/2, p16, CDK4 and cyclin D1. Human nasopharyngeal carcinoma CNE‑2 cells were cultured, divided into two groups (CNE-2S1 and CNE-2S2) and irradiated with a dose of 6 Gy x5 or 2 Gy x15, respectively. The cells were subcultured between doses of irradiation. The surviving sublines (CNE-2S1 and CNE-2S2 clones) were then passaged for three months and their radiosensitivity was determined. The cell cycle distribution and protein expression of SHP-1/2, p16, CDK4 and cyclin D1 in parental and progenitor cell lines were measured. Small interfering (si)RNA-mediated knockdown of SHP-1 and SHP‑2 in the NPC cells was used to further examine their roles in radiosensitivity and cell cycle distribution. CNE-2S1, a radio‑resistant cell line, had a significantly higher percentage of cells in S phase and a lower percentage of cells in G1 phase, enhanced expression levels of SHP-1, CDK4 and cyclin D1, and reduced expression of p16, respectively, as compared with the parent cells. Stable suppression of SHP-1 mRNA in CNE‑2 cells resulted in increased radiosensitivity compared with the parental cells, a decrease in the number of cells in S phase and an increase in the expression of p16. The results suggested that the SHP‑1/p16/cyclin D1/CDK4 pathway may have a role in regulating radiosensitivity and cell cycle distribution in nasopharyngeal cells.

Topics: Carcinoma; Cell Line, Tumor; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase Inhibitor p16; G1 Phase Cell Cycle Checkpoints; Gamma Rays; Humans; Nasopharyngeal Carcinoma; Nasopharyngeal Neoplasms; Protein Tyrosine Phosphatase, Non-Receptor Type 11; Protein Tyrosine Phosphatase, Non-Receptor Type 6; Radiation Tolerance; RNA Interference; RNA, Small Interfering; Up-Regulation

To study the expression of Cyclin D1 in nasopharyngeal carcinoma cells processed by epigallocatechin gallate(EGCG) and it's significance, and revealed the anti-tumor mechanism of EGCG against nasopharyngeal carcinoma.. CNE-2 cells were treated by EGCG at different concentrations, the morphological changes of CNE-2 cells were observed by inverted microscope; the inhibition ratio of cell proliferation was detected by MTT colorimetric method, flow cytometry was used to analyze the changes of cell cycle. The expression of Cyclin D1 mRNA was detected by RT-PCR.. After treated by EGCG, the CNE2 cells decreased in amount and density, some of which became roll and small; Floating and dead cells can be seen in the inverted microscopy; cell proliferation was significantly inhibited in a time and dose dependent (P < 0.05). CNE-2 cells were arrested at G1/G0 phase. The expression of Cyclin D1 mRNA was down-regulated by EGCG with concentration and action time dependent (P < 0.05).. EGCG resisted nasopharyngeal carcinoma by inhibiting the cell proliferation, The down regulation of Cyclin D1 mRNA expression in a time and dose dependent may be the possible mechanisms.

Topics: Carcinoma; Catechin; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Humans; Nasopharyngeal Carcinoma; Nasopharyngeal Neoplasms

Recent studies have revealed that long non-coding RNAs participate in all steps of cancer initiation and progression by regulating protein-coding genes at the epigenetic, transcriptional, and post-transcriptional levels. Long non-coding RNAs are in turn regulated by other genes, forming a complex regulatory network. The regulation networks between the p53 tumor suppressor and these RNAs in nasopharyngeal carcinoma remains unclear. The aims of this study were to investigate the regulatory roles of the TP53 gene in regulating long non-coding RNA expression profiles and to study the function of a TP53-regulated long non-coding RNA (LOC401317) in the nasopharyngeal carcinoma cell line HNE2. Long non-coding RNA expression profiling indicated that 133 long non-coding RNAs were upregulated in the human NPC cell line HNE2 cells following TP53 overexpression, while 1057 were downregulated. Among these aberrantly expressed long non-coding RNAs, LOC401317 was the most significantly upregulated one. Further studies indicated that LOC401317 is directly regulated by p53 and that ectopic expression of LOC401317 inhibits HNE2 cell proliferation in vitro and in vivo by inducing cell cycle arrest and apoptosis. LOC401317 inhibited cell cycle progression by increasing p21 expression and decreasing cyclin D1 and cyclin E1 expression and promoted apoptosis through the induction of poly(ADP-ribose) polymerase and caspase-3 cleavage. Collectively, these results suggest that LOC401317 is directly regulated by p53 and exerts antitumor effects in HNE2 nasopharyngeal carcinoma cells.

Topics: Animals; Apoptosis; Carcinoma; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Gene Expression Regulation, Neoplastic; Humans; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Nasopharyngeal Carcinoma; Nasopharyngeal Neoplasms; RNA, Long Noncoding; Tumor Suppressor Protein p53; Up-Regulation

Long-palate, lung and nasal epithelium clone 1 (LPLUNC1) gene expression is relatively tissue specific. It is highly expressed in nontumor nasopharyngeal epithelial tissues, but its expression is reduced in nasopharyngeal carcinoma (NPC), indicating that LPLUNC1 may be associated with the tumorigenesis of NPC. To study the effects of LPLUNC1 on NPC tumorigenesis, a full-length LPLUNC1 expression plasmid was stably transfected into the NPC cell line, 5-8F. Our data indicated that LPLUNC1 inhibited NPC cell proliferation in vitro and tumor formation in vivo. LPLUNC1 also delayed cell cycle progression from G1 to S phase and inhibited the expression of cyclin D1, cyclin-dependent kinase 4 (CDK4) and phosphorylated Rb. To further investigate the molecular mechanisms underlying the suppressive effects of LPLUNC1 on NPC tumorigenesis, cDNA microarray was performed. These studies revealed that LPLUNC1 inhibited the expression of certain mitogen-activated protein (MAP) kinases (MAPK) kinases and cell cycle-related molecules. Western blotting confirmed that the expression of MEK1, phosphorylated ERK1/2, phosphorylated JNK1/2, c-Myc and c-Jun were inhibited by LPLUNC1. Furthermore, the transcriptional activity of AP-1 was down-regulated by LPLUNC1, suggesting that the MAPK signaling pathway is regulated by LPLUNC1. Taken together, the present study indicates that LPLUNC1 delays NPC cell growth by inhibiting the MAPK and cyclin D1/E2F pathways and suggests that LPLUNC1 may represent a promising candidate tumor suppressor gene associated with NPC.

Topics: Animals; Autoantigens; Carcinogenesis; Carcinoma; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Down-Regulation; E2F Transcription Factors; Fatty Acid-Binding Proteins; Gene Expression; Gene Expression Regulation, Neoplastic; Humans; Lipopolysaccharides; Male; MAP Kinase Signaling System; Mice; Mice, Inbred BALB C; Mice, Nude; Nasopharyngeal Carcinoma; Nasopharyngeal Neoplasms; Neoplasm Transplantation; Proteins; Tissue Array Analysis

This study aimed to determine the molecular mechanisms underlying the effect of the LMP1-targeted DNAzyme 1 (DZ1) on cell cycle progression in nasopharyngeal carcinoma (NPC) cells. We showed that the active DZ1 inhibited the expression of latent membrane protein 1 (LMP1) and induced a G1 phase arrest. In addition, this cell cycle deregulation was shown to be accompanied by upregulation of the DNA damage marker γ-H2AX, downregulation of the DNA damage response factor p-p53-Ser15 and cell proliferation inhibition. To investigate what affected the cell cycle progression, we examined the expression of two checkpoint-related cyclins and cyclin-dependent kinases (CDKs). We found a decrease of cyclin D1 and cyclin E protein levels at 24 h from the DZ1 treatment. Moreover, we observed inhibition of CDK4 activity and decreased cyclin D1 expression in the complexes immunoprecipitated with CDK4 antibody. We also found a reduction in cdc2 phosphorylation at Thr161 which partially stands for the cdc2 kinase activity in DZ1-treated CNE1-LMP1 cells, although the downregulation of LMP1 expression had no effect on the cyclin B1 and cdc2 expression. Further, we analyzed changes in cdc2 kinase activity induced by DZ1 and found that the downregulation of the LMP1 expression resulted in a 5-fold reduction in cdc2 kinase activity in CNE1-LMP1. The data suggest that the downregulation of the LMP1 expression by DZ1 was able to induce DNA damage, which then further inhibited the cell proliferation and resulted in malfunction of cell cycle checkpoints that led to G1 phase arrest and the decrease in number of cells in G2/M phase.

Topics: Apoptosis; Blotting, Western; Carcinoma; Carcinoma, Squamous Cell; CDC2 Protein Kinase; Cell Cycle Checkpoints; Cell Proliferation; Cyclin B; Cyclin D1; Cyclin E; Cyclin-Dependent Kinases; DNA Damage; DNA, Catalytic; Fluorescent Antibody Technique; Histones; Humans; Immunoenzyme Techniques; Immunoprecipitation; Nasopharyngeal Carcinoma; Nasopharyngeal Neoplasms; Oncogene Proteins; Phosphorylation; Tumor Cells, Cultured; Viral Matrix Proteins

The principal Epstein-Barr virus (EBV) oncoprotein, latent membrane protein 1 (LMP1) is strongly associated with nasopharyngeal carcinoma (NPC), a prevalent cancer in China. The epidermal growth factor receptor (EGFR) is important in carcinogenesis, as it is a ubiquitously expressed receptor tyrosine kinase. Signal transducer and activator of transcription 3 (STAT3) is a master transcriptional regulator in proliferation and apoptosis. Our previous study demonstrated that the nuclear EGFR could bind to the cyclin D1 promoter directly in the presence of LMP1, and the correlation between EGFR and STAT3 in NPC remains to be further explored. Here, we have shown that the interaction of EGFR and STAT3 increased in the nucleus in the presence of LMP1. LMP1 promoted both EGFR and STAT3 binding to the promoter region of cyclin D1, in turn, enhancing the promoter activity of cyclin D1. Furthermore, we demonstrated that both transcriptional activity and mRNA levels of cyclin D1 were decreased by small molecule interference of EGFR and STAT3 activity. These findings may provide a novel linkage between the EGFR and STAT3 signaling pathways and the activation of cyclin D1 by LMP1 in the carcinogenesis of NPC.

Topics: Cell Growth Processes; Cell Line, Tumor; Cyclin D1; ErbB Receptors; Herpesvirus 4, Human; Humans; Nasopharyngeal Neoplasms; Promoter Regions, Genetic; Signal Transduction; STAT3 Transcription Factor; Transfection; Viral Matrix Proteins

It has been demonstrated previously by us that cyclin D1 and ClC-3 play important roles in regulation of the cell cycle in nasopharyngeal carcinoma cells. The action of cyclin D1 on the functional activities and expression of chloride channels were investigated in nasopharyngeal carcinoma CNE-2Z cells in this study. The results indicated that enhanced cyclin D1 expression increased the activation of volume-activated chloride currents and promoted the expression of ClC-3 chloride channel proteins. The fluorescence resonance energy transfer (FRET) experiments demonstrated that the distance between cyclin D1 and ClC-3 was less than 10nm, and there existed interaction between the two proteins. ClC-3 was partially colocalized with cyclin D1 and CDK4/6. Dialyzing CDK4 antibodies into cells via recording pipettes activated a chloride current, but dialysis of CDK6 antibodies inhibited basal and volume-activated Cl(-) currents. The CDK4/6 inhibitor fascaplysin chloride hydrate (highly selective for CDK4/cyclin D1 with IC(50) = 0.35 μM and less selective for CDK6/D1 with IC(50) = 3.4 μM) activated a chloride current in low concentration, but did not show significantly facilitative effects on the current in high concentration. In conclusion, our data suggest that the ClC-3 chloride channel is an important target of cyclin D1. Cyclin D1 may regulate the functional activities of the chloride channel via CDK4 and CDK6, and/or the expression of the chloride channel. Cyclin D1-CDK4 complexes may phosphorylate chloride channels resulting in inhibition or inactivation of the channels, and cyclin D1-CDK6 complexes may facilitate the activation of chloride channels.

Topics: Carcinoma; Cell Line, Tumor; Chloride Channels; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase 6; Fluorescence Resonance Energy Transfer; Gene Expression Regulation, Neoplastic; Humans; Nasopharyngeal Neoplasms; Phosphorylation

Galectin-3 was shown to be involved in various biological events, including cell growth, adhesion, differentiation, angiogenesis, apoptosis, tumorigenesis, and metastasis. The prognostic significance of galectin-3 expression has already been evaluated in several cancers. However, its prognostic role has not been investigated in nasopharyngeal carcinoma. The loss of cell cycle control is one of the critical steps in the development of nasopharyngeal carcinoma. Cyclin D1 is one of the key proteins involved in cell cycle control and is essential for G1/S phase transition. Overexpression of cyclin D1 has been observed in several human cancers. In the present study, the expression of galectin-3 and cyclin D1 was evaluated with immunohistochemical analysis in 45 patients diagnosed as undifferentiated nasopharyngeal carcinoma and expression of these proteins was correlated with clinicopathological parameters and prognosis. Multivariate analysis showed that older age (>50 vs. ≤50) (P = 0.028), distant metastasis at presentation (M(1) vs. M(0)) (P = 0.001), and increased galectin-3 expression (>5% vs. ≤5%) (P = 0.025) were independently correlated with poor overall survival. We found no statistically significant correlation between cyclin D1 immunoexpression and disease outcome. The Spearman's correlation coefficient revealed a significant correlation between galectin-3 and cyclin D1 expression (r = 0.425; P = 0.004). Our findings suggested that the immunohistochemical analysis of galectin-3 might be useful in predicting prognosis in nasopharyngeal carcinoma.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Carcinoma; Cyclin D1; Female; Follow-Up Studies; Galectin 3; Humans; Immunoenzyme Techniques; Male; Middle Aged; Nasopharyngeal Neoplasms; Neoplasm Metastasis; Neoplasm Recurrence, Local; Neoplasm Staging; Prognosis; Survival Rate; Young Adult

The cell cycle regulator cyclin D1 (CCND1) is a critical regulator of the G1/S phase transition and plays an important part in several tumor types. This study aimed at investigating the association of CCND1 with and examining the interaction among CCND1 genotype and individual smoking habit in nasopharyngeal carcinoma susceptibility.. A total of 352 native Taiwanese consisting of 176 cases and 176 controls were enrolled in this hospital-based study, and CCND1 A870G (rs9344) and C1722G (rs678653) genotyping were analyzed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and partially verified by direct sequencing.. The results showed that there were significant differences between nasopharyngeal carcinoma and control groups in the distribution of the genotypic (p=0.0222) and allelic (p=0.0322) frequencies in the CCND1 A870G genotype. Individuals who carried at least one G allele (GG or AG) had a 0.71-fold lower risk of developing nasopharyngeal carcinoma compared to those who had the AA genotype (95% confidence interval=0.53-0.96). In addition, there is an obvious joint effect of CCND1 A870G genotype with smoking habit on nasopharyngeal carcinoma susceptibility.. These findings support the conclusion that the cell cycle regulation may play a role in nasopharyngeal carcinoma development and that CCND1 A870G polymorphism maybe a useful biomarker for nasopharyngeal carcinoma progression.

Topics: Adult; Base Sequence; Case-Control Studies; Cyclin D1; DNA Primers; Female; Genetic Predisposition to Disease; Genotype; Humans; Male; Middle Aged; Nasopharyngeal Neoplasms; Polymerase Chain Reaction; Polymorphism, Restriction Fragment Length; Taiwan

Nasopharyngeal carcinoma (NPC) is a highly malignant and frequently metastasized tumor, and the prognosis is very poor when distant metastases occur. Recently, immunotherapy is becoming a promising therapeutic approach. Interferon-α (IFN-α) represents the cytokines exhibiting the longest record of use in clinical oncology. In this study, we examined the antitumor effects of IFN-α1b on NPC. The results showed that recombinant human IFN-α1b (hIFN-α1b) suppressed cell growth, induced a G1-phase cell cycle arrest in vitro, increased the expression of p16 and pRb, and decreased the expression of CCND1 and CDK6. In vivo analyses showed that either recombinant adeno-associated virus (rAAV)-IFN-α1b or hIFN-α1b treatment inhibited tumor growth and metastasis, reduced intratumoral microvessel density, increased cell apoptosis and necrosis, and induced prolonged survival. Notably, rAAV-IFN-α1b or hIFN-α1b treatment led to significantly higher serum levels of IL-12 and GM-CSF in mice compared to respective controls. Our findings suggest that IFN-α1b acts as a multifunctional antitumor agent in NPC, which may have important therapeutic implications.

Topics: Animals; Antineoplastic Agents; Apoptosis; Carcinoma; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase 6; Cyclin-Dependent Kinase Inhibitor p16; G1 Phase; Gene Expression; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Interferon-alpha; Interleukin-12; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Nasopharyngeal Carcinoma; Nasopharyngeal Neoplasms; Necrosis; Neoplasm Metastasis; Recombinant Proteins; Retinoblastoma Protein

To investigate the mechanisms underlying the anticancer effect of celecoxib on nasopharyngeal carcinoma (NPC).. NPC cell lines, HNE1 and CNE1-LMP1, were treated with various concentrations of celecoxib for 48 h. The antiproliferative effect of celecoxib was assessed using MTT assay. Both cell cycle profiles and apoptosis were analyzed using flow cytometry. Western blot was used to measure the levels of signal transducer and activator of transcription 3 (STAT3), phosphorylated STAT3(Y705) (pSTAT3(Y705)), COX-2, Survivin, Mcl-1, Bcl-2 and Cyclin D1.. Celecoxib (10-75 μmol/L) inhibited the proliferation of the NPC cell lines in a dose-dependent manner. Celecoxib (25 and 50 μmol/L) induced apoptosis and cell-cycle arrest at the G(0)/G(1) checkpoint in the NPC cell lines, which was associated with significantly reduced STAT3 phosphorylation. The genes downstream of STAT3 (ie, Survivin, Mcl-1, Bcl-2 and Cyclin D1) were significantly down-regulated after exposure to celecoxib (25 and 50 μmol/L).. The anticancer effects of celecoxib on NPC cell lines results from inducing apoptosis and cell cycle arrest, which may be partly mediated through the STAT3 pathway.

Topics: Antineoplastic Agents; Apoptosis; Blotting, Western; Carcinoma; Celecoxib; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclooxygenase 2; Dose-Response Relationship, Drug; Flow Cytometry; Humans; Interleukin-6; Myeloid Cell Leukemia Sequence 1 Protein; Nasopharyngeal Carcinoma; Nasopharyngeal Neoplasms; Phosphorylation; Protein Processing, Post-Translational; Proto-Oncogene Proteins c-bcl-2; Pyrazoles; Signal Transduction; STAT3 Transcription Factor; Sulfonamides; Time Factors

Tumor suppressor genes function to regulate and block tumor cell proliferation. To explore the mechanisms underlying the tumor suppression of BLU/ZMYND10 gene on a frequently lost human chromosomal region, an adenoviral vector with BLU cDNA insert was constructed.. BLU was re-expressed in nasopharyngeal carcinoma cells by transfection or viral infection. Clonogenic growth was assayed; cell cycle was analyzed by flow cytometry-based DNA content detection; c-Jun N-terminal kinase (JNK) and cyclin D1 promoter activities were measured by reporter gene assay, and phosphorylation was measured by immunoblotting. The data for each pair of groups were compared with Student t tests.. BLU inhibits clonogenic growth of nasopharyngeal carcinoma cells, arrests cell cycle at G1 phase, downregulates JNK and cyclin D1 promoter activities, and inhibits phosphorylation of c-Jun.. BLU inhibits growth of nasopharyngeal carcinoma cells by regulation of the JNK-cyclin D1 axis to exert tumor suppression.

Topics: Adenoviridae; Carcinoma; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cytoskeletal Proteins; Down-Regulation; G1 Phase; Gene Expression Regulation, Neoplastic; Genetic Vectors; Humans; JNK Mitogen-Activated Protein Kinases; Nasopharyngeal Neoplasms; Phosphorylation; Promoter Regions, Genetic; Tumor Suppressor Proteins

The microRNA miR-138 is dysregulated in several human cancers, but the underlying mechanism remains largely unknown. Here, we report that miR-138 is commonly underexpressed in nasopharyngeal carcinoma (NPC) specimens and NPC cell lines. The ectopic expression of miR-138 dramatically suppressed cell proliferation and colony formation in vitro and inhibited tumorigenesis in vivo. Moreover, we identified the cyclin D1 (CCND1) gene as a novel direct target of miR-138. In consistent with the knocked-down expression of CCND1, overexpression of miR-138 inhibited cell growth and cell cycle progression in NPC cells. Furthermore, CCND1 was widely upregulated in NPC tumors, and its mRNA levels were inversely correlated with miR-138 expression. Taken together, our findings suggest that miR-138 might be a tumor suppressor in NPC, which is exerted partially by inhibiting CCND1 expression. The identification of functional miR-138 in NPC and its direct link to CCND1 might provide good candidates for developing diagnostic markers and therapeutic applications for NPC.

Topics: 3' Untranslated Regions; Animals; Carcinoma; Cell Line, Tumor; Cell Proliferation; Cyclin D1; G1 Phase Cell Cycle Checkpoints; Humans; Mice; Mice, Nude; MicroRNAs; Nasopharyngeal Carcinoma; Nasopharyngeal Neoplasms; RNA Interference; RNA, Small Interfering; Transplantation, Heterologous; Up-Regulation

STGC3 is a potential tumor suppressor in nasopharyngeal carcinoma. We previously found that CNE2 cells that re-expressed STGC3 formed smaller tumors in female mice than in male mice. Here, we investigated the sexual dimorphism of STGC3 as a tumor-suppressor in female and male nude mice injected subcutaneously with pcDNA3.1(+)-STGC3/CNE2 cells. ER-α was positively expressed in vitro in the CNE2 cells. The pcDNA3.1(+)-STGC3/CNE2 cell growth rate decreased after treatment with β-estradiol in vitro. There were significant differences in tumor size or mass between pcDNA3.1(+)-STGC3/CNE2 and control cases (P < 0.05), but there were significant differences in tumor size between female and male nude mice in the STGC3 transfection groups, and the pcDNA3.1(+)-STGC3/CNE2 tumor growth rate in the female nude mice was the lowest in all cases (P < 0.05). There were no significant differences between female and male nude mice in control groups. Furthermore, a greater number of cells were blocked in the G(0)/G(1) phase in pcDNA3.1(+)-STGC3/ CNE2 tumor xenografts in the female mice. Protemic analysis found 9 differentially expressed proteins in the pcDNA3.1-STGC3/CNE2 xenograft tissues in females and males. A heat shock 70 protein 8 isoform 2 variant was identified as a down-regulated protein associated with cell cycle control and its downstream factor cyclin D1 was also decreased in STGC3-repressed xenografts in female mice. The data above suggest that STGC3 and its associated proteins play an important role in nasopharyngeal carcinoma gender differences.

Topics: Animals; Carcinoma; Cell Line, Tumor; Cyclin D1; Estradiol; Estrogen Receptor alpha; Female; Genes, Tumor Suppressor; HSC70 Heat-Shock Proteins; Humans; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Nasopharyngeal Carcinoma; Nasopharyngeal Neoplasms; Neoplasm Transplantation; Proteins; Resting Phase, Cell Cycle; Sex Characteristics; Sex Factors; Transcriptome; Tumor Burden

Undifferentiated nasopharyngeal carcinomas (NPCs) are commonly present with latent EBV infection. However, events regulating EBV infection at early stages of the disease and the role of EBV in disease pathogenesis are largely undefined. Genetic alterations leading to activation of cyclin D1 signaling in premalignant nasopharyngeal epithelial (NPE) cells have been postulated to predispose cells to EBV infection. We previously reported that loss of p16, a negative regulator of cyclin D1 signaling, is a frequent feature of NPC tumors. Here, we report that early premalignant lesions of nasopharyngeal epithelium overexpress cyclin D1. Furthermore, overexpression of cyclin D1 is closely associated with EBV infection. Therefore we investigated the potential role of cyclin D1 overexpression in dysplastic NPE cells in vitro. In human telomerase reverse transcriptase-immortalized NPE cells, overexpression of cyclin D1 or a p16-resistant form of CDK4 (CDK4(R24C)) suppressed differentiation. This suppression may have implications for the close association of EBV infection with undifferentiated NPC. In these in vitro models, we found that cellular growth arrest and senescence occurred in EBV-infected cell populations immediately after infection. Nevertheless, overexpression of cyclin D1 or a p16-resistant form of CDK4 or knockdown of p16 in the human telomerase reverse transcriptase-immortalized NPE cell lines could counteract the EBV-induced growth arrest and senescence. We conclude that dysregulated expression of cyclin D1 in NPE cells may contribute to NPC pathogenesis by enabling persistent infection of EBV.

Topics: Base Sequence; Cell Cycle; Cell Differentiation; Cell Line, Tumor; Cell Transformation, Neoplastic; Cell Transformation, Viral; Cells, Cultured; Cellular Senescence; Cyclin D1; DNA, Viral; Epithelial Cells; Epstein-Barr Virus Infections; Gene Expression; Genes, bcl-1; Genes, Viral; Herpesvirus 4, Human; Humans; Nasopharyngeal Neoplasms; Nasopharynx; Precancerous Conditions; Signal Transduction; Telomerase

It has been reported that metformin, a biguanide derivative widely used in type II diabetic patients, has antitumor activities in some cancers by activation of AMP-activated protein kinase (AMPK). But its role in nasopharyngeal carcinoma (NPC) is not known. Here, we reported for the first time that 1-50 mM of metformin in a dose- and time-dependent manner suppressed cell proliferation and colony formation in NPC cell line, C666-1. Further studies revealed that the protein level of cyclin D1 decreased and the percentage of the cells in G0/G1 phase increased by 5 mM metformin treatment. Metformin also induced the phosphorylation of AMPK (T172) in a time-dependent manner. Mammalian target of rapamycin complex 1 (mTORC1), which is negatively regulated by AMPK and plays a central role in cell growth and proliferation, was inhibited by metformin, as manifested by dephosphorylation of its downstream targets 40S ribosomal S6 kinase 1 (S6K1) (T389), the eukaryotic translation initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1) (T37/46) and S6 (S235/236) in C666-1 cells. In a summary, metformin prevents proliferation of C666-1 cells by down-regulating cyclin D1 level and inducing G1 cell cycle arrest. AMPK-mediated inhibition of mTORC1 signaling may be involved in this process.

Topics: Adaptor Proteins, Signal Transducing; AMP-Activated Protein Kinases; Antineoplastic Agents; Carcinoma; Cell Cycle Proteins; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Dose-Response Relationship, Drug; G1 Phase Cell Cycle Checkpoints; Humans; Mechanistic Target of Rapamycin Complex 1; Metformin; Multiprotein Complexes; Nasopharyngeal Carcinoma; Nasopharyngeal Neoplasms; Phosphoproteins; Phosphorylation; Proteins; Proto-Oncogene Proteins c-akt; Ribosomal Protein S6 Kinases, 70-kDa; Signal Transduction; Time Factors; TOR Serine-Threonine Kinases

To explore biomolecular characteristics of a group of patients with nasopharyngeal carcinoma from European (Spanish) hospitals, addressing the pathogenesis of the tumor and the response to treatment.. Cyclin D1 and p16 expression were evaluated immunohistochemically in 33 tissue samples of nasopharyngeal carcinoma. CCDN1 gene amplification and p16 gene deletion were studied by fluorescence in situ hybridization. Patient clinical data were examined, and tissues were evaluated histologically using hematoxylin-eosin staining.. Cyclin D1 overexpression was found in 19 cases, and p16 expression was undetected in 30 cases. An association was observed between impaired p16 expression and cyclin D1 overexpression (p = 0.034). Eleven patients displayed p16 gene deletion and CCDN1 gene amplification.. Cyclin D1 overexpression and CCDN1 amplification, loss of p16 expression and p16 deletion may be among the genetic alterations involved in the pathogenesis of nasopharyngeal carcinoma.

Topics: Adult; Aged; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p16; Female; Humans; Immunohistochemistry; In Situ Hybridization, Fluorescence; Male; Middle Aged; Nasopharyngeal Neoplasms; Retrospective Studies; Spain

To construct the eukaryotic expression vectors of human cyclin D1 gene and express them in poorly differentiated nasopharyngeal carcinoma cells (CNE-2Z cells).. The full-length cyclin D1 was cloned from CNE-2Z cells by RT-PCR. The cDNA fragments were inserted into pIRES2-EGFP plasmids and pEGFP-C2 plasmids and confirmed by restriction enzyme digestion, PCR and sequencing. The recombinant vectors were transfected into CNE-2Z cells via Lipofectamine 2000, and the expression of cyclin D1 in the cells was examined by immunofluorescence and Western blotting.. Agarose gel electrophoresis showed a 918 bp band of the RT-PCR products, which matched the expected size. Restriction enzyme digestion, PCR and sequencing demonstrated successful construction of the recombinant vectors. CNE-2Z cells transfected with the recombinant vectors expressed cyclin D1 protein or cyclin D1-GFP protein as were verified by immunofluorescence and Western blotting.. We have cloned cyclin D1 gene and constructed its eukaryotic expression vectors that can be expressed in nasopharyngeal carcinoma cells, which may facilitate the study of the role of cyclin D1 in the development of nasopharyngeal carcinoma.

Topics: Cell Line, Tumor; Cloning, Molecular; Cyclin D1; Genetic Vectors; Green Fluorescent Proteins; Humans; Nasopharyngeal Neoplasms; Plasmids; Recombinant Proteins; Transfection

Luteolin, a plant flavonoid is known to possess multiple biological activities such as anti-inflammation, anti-allergy, anti-oxidant as well as anti-cancer. At present, the anti-proliferative potential of luteolin has not been fully understood. In this study, we focused on the effect of luteolin on cell cycle regulation in human nasopharyngeal carcinoma (NPC) cells. First, we found that luteolin inhibited cell cycle progression at G1 phase and prevented entry into S phase in a dose- and time-dependent manner. Next, it was found that luteolin treatment led to down-regulation of cyclin D1 via enhanced protein phosphorylation and proteasomal degradation, leading to reduced CDK4/6 activity and suppression of retinoblastoma protein (Rb) phosphorylation, and subsequently inhibition of the transcription factor E2F-1. In search of the molecular mechanisms underlying luteolin-mediated cyclin D1 down-regulation, it was found that luteolin was capable of suppressing Akt phosphorylation and activation, resulting in de-phosphorylation and activation of glycogen synthase kinase-3β (GSK-3β). Activated GSK-3β then targeted cyclin D1, causing phosphorylation of cyclin D1 at Thr(286) and subsequent proteasomal degradation. The above findings were reinforced by the fact that luteolin was able to abrogate the effect of insulin on the Akt/GSK-3β/Cyclin D1 pathway, resulting in suppression of insulin-induced cell proliferation. Since Akt is often over-activated in many human cancers including NPC, it is thus believed that data from this study support the potential application of luteolin as a chemotherapeutic or chemopreventive agent in human cancer.

Topics: Apoptosis; Cell Cycle; Cell Line, Tumor; Cyclin D1; Dose-Response Relationship, Drug; Down-Regulation; E2F1 Transcription Factor; Flow Cytometry; G1 Phase; Gene Expression Regulation, Neoplastic; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Humans; Immunoblotting; Luteolin; Nasopharyngeal Neoplasms; Phosphorylation; Proto-Oncogene Proteins c-akt; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Time Factors

Activated mTOR was implicated to play a role in the carcinogenesis of nasopharyngeal carcinoma (NPC). However, the mechanism of activated mTOR/Complex1(mTORC1) signaling pathway in NPC development has not been well established. In this study, we correlated the expression of mTORC1 signal molecules and Cyclin D1 in NPC. We also investigated the effect of blocking mTORC1 signal with rapamycin and mTOR siRNA on Cyclin D1 expression in CNE-2 cells, as well as cell apoptosis and viability. We found a positive association of mTORC1 signal molecules and Cyclin D1 in NPC. Also, we found blockage mTORC1 inhibited Cyclin D1 expression in CNE-2 cells and enhanced cell apoptosis. Our results suggested that mTORC1 signal pathway might be a potential target for NPC therapy.

Topics: Adaptor Proteins, Signal Transducing; Adult; Apoptosis; Carcinoma; Case-Control Studies; Cell Cycle Proteins; Cell Line, Tumor; Cell Survival; Cyclin D1; Dose-Response Relationship, Drug; Female; Humans; Male; Middle Aged; Multiprotein Complexes; Nasopharyngeal Neoplasms; Phosphoproteins; Phosphorylation; Protein Kinases; RNA Interference; RNA, Small Interfering; Signal Transduction; Sirolimus; Time Factors; TOR Serine-Threonine Kinases; Transfection; Up-Regulation

Cell cycle dysregulation resulting in expression of antiapoptotic genes and uncontrolled proliferation is a feature of undifferentiated nasopharyngeal carcinoma. The pharmacodynamic effects of seliciclib, a cyclin-dependent kinase (CDK) inhibitor, were studied in patients with nasopharyngeal carcinoma.. Patients with treatment-naïve locally advanced nasopharyngeal carcinoma received seliciclib at 800 mg or 400 mg twice daily on days 1 to 3 and 8 to 12. Paired tumor samples obtained at baseline and on day 13 were assessed by light microscopy, immunohistochemistry, and transcriptional profiling using real-time PCR low-density array consisting of a panel of 380 genes related to cell cycle inhibition, apoptosis, signal transduction, and cell proliferation.. At 800 mg bd, one patient experienced grade 3 liver toxicity and another had grade 2 vomiting; no significant toxicities were experienced in 13 patients treated at 400 mg bd. Seven of fourteen evaluable patients had clinical evidence of tumor reduction. Some of these responses were associated with increased tumor apoptosis, necrosis, and decreases in plasma EBV DNA posttreatment. Reduced protein expression of Mcl-1, cyclin D1, phosphorylated retinoblastoma protein pRB (T821), and significant transcriptional down-regulation of genes related to cellular proliferation and survival were shown in some patients posttreatment, indicative of cell cycle modulation by seliciclib, more specifically inhibition of cdk2/cyclin E, cdk7/cyclin H, and cdk9/cyclin T.. Brief treatment with this regimen of seliciclib in patients with nasopharyngeal carcinoma is tolerable at 400 mg bd and associated with tumor pharmacodynamic changes consistent with cdk inhibition, and warrants further efficacy studies in this tumor.

Topics: Administration, Oral; Adult; Aged; Antineoplastic Agents; Apoptosis; Cell Cycle; Cyclin D1; DNA, Viral; Female; Gene Expression Profiling; Humans; Immunohistochemistry; Male; Middle Aged; Myeloid Cell Leukemia Sequence 1 Protein; Nasopharyngeal Neoplasms; Proto-Oncogene Proteins c-bcl-2; Purines; Roscovitine

Previous studies have revealed that Epstein-Barr virus (EBV) was closely associated with nasopharyngeal carcinoma (NPC). This study aimed to characterize the global pathways affected in the EBV-associated NPC. Combined with microdissection, gene expression profiles in 22 NPCs and 10 non-tumor nasopharyngeal epithelial (NPE) tissue samples were analyzed. All NPC specimens served in the microarray analysis were positive for EBV, as judged by identification of the expression of EBV nuclear antigen 1 (EBNA1). Through gene set enrichment analysis (GSEA), we found that cell cycle pathway was the most disregulated pathway in NPC (P=0.000, false discovery rate q-value=0.007), which included some aberrant expressed components. We first found that overexpression of CDK4, cyclin D1, and Rb proteins, and loss of expression of proteins p16, p27, and p19 were statistically significant in NPC tissues compared with non-cancerous NPE (P<0.05) by real-time RT-PCR and tissue microarray. EBV-encoded small RNA-1 (EBER-1) hybridization signals in the NPC showed significant associations with the overexpression of Rb (P=0.000), cyclin D1 (P=0.000), CDK4 (P=0.000), and the loss of expression of p16 proteins (P=0.039). In the final logistic regression analysis model, EBER-1 and abnormal expression of p16, Rb, cyclin D1, and E2F6 were independent contributions to nasopharyngeal carcinogenesis. Through survival analysis, only cyclin D1 could predict the prognosis of NPC patients. These results suggested that cell cycle pathway was the most disregulated pathway in the EBV-associated NPC, and EBER-1 was closely associated with p16, CDK4, cyclin D1, and Rb.cyclin D1 could be the prognosis biomarker for NPC.

Topics: Cell Cycle; Cell Cycle Proteins; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase Inhibitor p16; Cyclin-Dependent Kinase Inhibitor p19; Cyclin-Dependent Kinase Inhibitor p27; E2F6 Transcription Factor; Epstein-Barr Virus Infections; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Herpesvirus 4, Human; Host-Pathogen Interactions; Humans; Immunohistochemistry; In Situ Hybridization; Kaplan-Meier Estimate; Logistic Models; Nasopharyngeal Neoplasms; Oligonucleotide Array Sequence Analysis; Retinoblastoma Protein; Reverse Transcriptase Polymerase Chain Reaction; RNA, Viral; Tissue Array Analysis

The aim of this study was to investigate the susceptibility and prognostic implications of the cyclin D1 gene (CCND1) G870A polymorphism to nasopharyngeal carcinomas (NPC) in Han population in Yunnan China.. Two hundred and forty one cases with NPC and 271 matched cancer-free controls were genotyped for the CCND1 G870A polymorphism by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and sequencing. The adjusted odds ratios (OR) and 95% confidence intervals (CI) were calculated by using unconditional logistic regression model. Overall survival was assessed using univariate and multivariate analyses.. Contrast with homozygous CCND1 G870G, A allele significantly increasing risk of NPC was associated with homozygous A870A (OR = 4.79, 95% CI 2.77 - 8.28, P < 0.001) and heterozygous A870G (OR = 1.72, 95% CI 1.10 - 2.68, P = 0.017). The subjects at least having one CCND1 870A allele had OR of 2.40 (95% CI 1.59 - 3.63, P < 0.001). Furthermore, smoking may increase the risk of developing NPC interacting with CCND1 G870A polymorphism. Kaplan-Meier analysis and Cox regression analysis demonstrated that the five-year survival rate of subjects with AA, AG and GG genotype was 56.2%, 78.5% and 81.4% (AA vs GG, P = 0.003; AA vs AG, P = 0.012; AG vs GG, P = 0.132), but not independent prognostic factor in NPC (P = 0.501).. The CCND1 870A allele is associated with the NPC in Han population in Yunnan China, meanwhile, showed a significant prognosis for those patients.

Topics: Adolescent; Adult; Aged; Carcinoma, Squamous Cell; Cyclin D1; Female; Gene Frequency; Genetic Predisposition to Disease; Genotype; Humans; Male; Middle Aged; Nasopharyngeal Neoplasms; Polymorphism, Single Nucleotide; Prognosis; Young Adult

Lactotransferrin (LTF) has been shown to regulate tumorogenesis. However, little is known about the role of LTF in regulating the development of human nasopharyngeal carcinoma (NPC). The aim of our study was to investigate whether LTF could regulate the development of NPC by characterizing the pattern of LTF expression in human NPC tissues using cDNA and tissue microarrays. Loss of LTF expression was observed in a significantly higher frequency of NPC tissues compared to that in nontumor nasopharyngeal epithelial tissues. While 61.25% of NPC tissues at the T1/T2 stage were positive for LTF expression, only 40.82% of NPC at the T3/T4 stage were stained by anti-LTF. Similarly, 41.58% of NPC with local lymph node metastasis displayed LTF expression, a value significantly lower than the 46.36% in primary tumors (p < 0.05). These findings suggest that LTF may negatively regulate the development and metastasis of NPC in vivo. Furthermore, overexpression of or treatment with LTF inhibited the proliferation of NPC cells and promoted cell cycle arrest at the G(0)/G(1) phase in vitro. While LTF treatment downregulated expression of cyclin D1 and phosphorylation of retinoblastoma protein (Rb), expression of p21 and p27 in 5-8F NPC cells was enhanced. Moreover, LTF treatment modulated the mitogen-activated protein kinase (MAPK) pathway, but did not affect p53 and STAT3 expression in 5-8F NPC cells. Thus LTF is likely to be a candidate tumor suppressor and downregulates the development of NPC by inhibiting NPC proliferation through induction of cell cycle arrest and modulation of the MAPK signaling pathway. Therefore, our findings provide new insights in understanding the mechanism(s) underlying the action of LTF in regulating the development of human NPC.

Topics: Cell Cycle; Cell Line, Tumor; Cell Proliferation; Chromosomes, Human, Pair 3; Cyclin D1; Humans; Lactoferrin; MAP Kinase Signaling System; Nasopharyngeal Neoplasms; Oligonucleotide Array Sequence Analysis; Phosphorylation; Retinoblastoma Protein; Tissue Array Analysis; Tumor Suppressor Proteins

BRD7 is a novel gene which involved NPC in our lab. Our previous studies showed that BRD7 was expressed at high level in normal nasopharyngeal epithelial tissues, but at low level in nasopharyngeal carcinoma biopsies and cell lines. In these papers, we found that ectopic expression of BRD7 can decrease cell proliferation and capability to form colonies in soft agar. FCM (Flow cytometry) assay indicated that the cell cycle progression from G1 to S phase was inhibited and the expression of cyclinD1 was significantly decreased after being transfected with BRD7 in HNE1 cells (NPC cells). To further investigate the molecular mechanism of BRD7 suppression of NPC cells growth, the cDNA microarray was performed to detect difference in gene expression profile induced by BRD7. The results indicated that 21 genes expression were changed after being transfected with BRD7 and the differentially expressed gene including alpha-catenin, cyclinD1, E2F3 was confirmed by western-blot. Next, we found that even though no obvious changes of the total expression of beta-catenin were observed, the accumulation of beta-catenin in nucleus was blocked. In addition, it was found that the expression of beta-catenin was up-regulated in the complex composed of beta-catenin and alpha-catenin in HNE1 cells induction of BRD7. So, we concluded that over-expression of BRD7 increased the expression of alpha-catenin which "hold" beta-catenin in the complex and inhibited its accumulating in nucleus. At last, we demonstrated the c-jun, p-MEK, and p-ERK1/2 expression were down-regulated, and the Ap-1 promoter activity was inactive after being transfected with BRD7. We also found that over-expression of BRD7 can inactivate the c-jun and p-ERK1/2 after being treated with EGF in HNE1 cells. These results indicated that BRD7 played a negative role in ERK1/2 pathway. Taken together, our present results provide new insights for BRD7 function to inhibit NPC cells growth through negative regulating beta-catenin and ERK1/2 pathways.

Topics: beta Catenin; Cell Cycle; Cell Nucleus; Cell Proliferation; Chromosomal Proteins, Non-Histone; Cyclin D1; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Immunoprecipitation; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Molecular Sequence Data; Nasopharyngeal Neoplasms; Nuclear Proteins; Oligonucleotide Array Sequence Analysis; Signal Transduction; Transcription, Genetic; Tumor Cells, Cultured

Although activating protein-1 (AP-1) transcription factors play an important role in mediating metastasis for nasopharyngeal carcinoma (NPC), the biological and physiological functions of AP-1, in relation to the oncogenic phenotype of NPC, are not fully understood. Our previous study showed that the latent membrane protein 1 (LMP1) mediated a primary dimer form of c-jun and jun B. In this study, we used a NPC cell line that express a specific inhibitor of AP-1, a dominant-negative c-jun mutant (TAM67), to investigate the role of AP-1 in regulating the NPC oncogenic phenotype. First, we observed that TAM67 inhibited cell growth in vitro and in vivo. Next, with Western blotting, we discovered that TAM67 impaired the cyclin D1/cdk4 complex but had little effect on the cyclin E/cdk2 complex, concomitantly with inhibiting Rb phosphorylation. RT-PCR and luciferase assay results demonstrated that the levels of cyclin D1 mRNA and the promoter activity in TAM67 transfectants were reduced as compared with control cells. Thereby, we show that blockade of AP-1 transcriptional activity has a negative impact on cyclin D1 transcription. We obtained the first evidence that TAM67 prevented NPC growth both in vitro and in vivo. AP-1 appears to be a novel target for treating or preventing LMP1-positive NPC effectively.

Topics: Animals; Cell Cycle; Cell Cycle Proteins; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinases; Humans; Mice; Mice, Nude; Nasopharyngeal Neoplasms; Neoplasm Transplantation; Peptide Fragments; Proto-Oncogene Proteins c-jun; Transcription Factor AP-1; Tumor Cells, Cultured; Viral Matrix Proteins

Epstein-Barr virus (EBV)-encoded latent membrane protein 1 (LMP1) is considered to be the major oncogenic protein of EBV-encoded proteins and has always been the core of the oncogenic mechanism of EBV. Advanced studies on nuclear translocation of the epidermal growth factor receptor (EGFR) family have greatly improved our knowledge of the biological function of cell surface receptors. In this study, we used the Tet-on LMP1 HNE2 cell line as a cell model, which is a dual-stable LMP1-integrated nasopharyngeal carcinoma (NPC) cell line and the expression of LMP1 which could be regulated by the Tet system. We found that LMP1 could regulate the nuclear accumulation of EGFR in a dose-dependent manner quantitatively and qualitatively. We also demonstrated that the nuclear localization sequence of EGFR played some roles in the location of the protein within the nucleus under LMP1 regulation and EGFR in the nucleus could bind to the promoters of cyclinD1 and cyclinE, respectively. We further demonstrated that EGFR is involved in the acceleration of the G1/S phase transition by LMP1 through binding to cyclinD1 and cyclinE directly. These findings provided a novel view that the acceleration of LMP1 on the G1/S transition via the nuclear accumulation of EGFR was critical in the process of nasopharyngeal carcinoma.

Topics: Base Sequence; Cell Line, Tumor; Cell Nucleus; Cyclin D1; Cyclin E; DNA, Neoplasm; Epstein-Barr Virus Infections; ErbB Receptors; G1 Phase; Herpesvirus 4, Human; Humans; Nasopharyngeal Neoplasms; Promoter Regions, Genetic; Protein Binding; S Phase; Viral Matrix Proteins

Recently we confirmed that latent membrane protein 1 (LMP1) encoded by Epstein-Barr virus (EBV) accelerates a newly forming active c-Jun/Jun B heterodimer, a transcription factor, but little is known about the target gene regulated by it. In this paper, results indicated that a c-Jun/Jun B heterodimer induced by LMP1 upregulated cyclin D1 promoters activity and expression, on the contrary, downregulated p16, and maladjustment of cyclin D1 and p16 expression accelerated progression of cell cycle. Firstly, we found a c-Jun/Jun B heterodimer regulated synchronously and directly cyclin D1 and p16 in the Tet-on-LMP1-HNE2 cell line, in which LMP1 expression is regulated by Tet-on system. This paper investigated in depth function of the newly forming active c-Jun/Jun B heterodimer, and built new connection between environmental pathogenic factor, signal transduction and cell cycle.

Topics: Carcinoma; Cell Cycle; Cell Line; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p16; Dimerization; Doxycycline; Humans; Nasopharyngeal Neoplasms; Promoter Regions, Genetic; Protein Binding; Proto-Oncogene Proteins c-jun; Signal Transduction; Transfection; Viral Matrix Proteins

To study the effect of NP9 on the growth of transplanted nasopharyngeal carcinoma (NPC) in nude mice and explore the mechanisms involved.. Recombinant pRc/CMV2-NP9 plasmid was constructed and transfected into the NPC cell lines by lipofectamine 2000. Cell clones stably expressing NP9 were obtained by detecting the mRNA expression of NP9 in G418-resistant clones with RT-PCR. The tumorigenicity and size of transplanted tumors were assessed after inoculation of NPC cells and their transgene clones into Balb/C mice. The expression of PCNA and cyclin D1 in transplanted tumors was detected by immunohistochemistry.. The expression of NP9 was detected in some of NP9 gene-transfected G418-resistant clones of CNE1 and SUNE1. In vivo experiments showed that the tumorigenicity of CNE19 clone was decreased significantly compared to that of CNE1 and its vector control, and the transplanted tumors grew more slowly from SUNE1/NP9 than from SUNE1 and SUNE1/vector. Compared with the vector control, the expression of cyclin D1 and PCNA in CNE1/NP9 transplants was decreased.. NP9 inhibits tumorigenicity and growth of NPC transplanted tumor by down-regulating the expression of cyclin D1 and PCNA.

Topics: Animals; Cyclin D1; Endogenous Retroviruses; Female; Gene Products, env; Genes, Tumor Suppressor; Humans; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Nasopharyngeal Neoplasms; Neoplasm Transplantation; Proliferating Cell Nuclear Antigen; Tumor Cells, Cultured

Celecoxib is a cyclooxygenase-2 (COX-2) selective non-steroidal anti-inflammatory drug (NSAID) which has been shown to be capable of inhibiting the growth of various cancer cell lines. However, studies of its effect on the growth of nasopharyngeal carcinoma (NPC) cells are scarce. In this study, we investigated the effect of celecoxib on cell growth using three NPC cell lines: HK-1, Hone-1 and CNE-2. Our results showed that while all 3 cell lines expressed the COX-2 mRNA as determined by reverse transcription-polymerise chain reaction (RT-PCR), western blot analysis showed that only HK-1 expressed the COX-2 protein. Using MTT assay, celecoxib was found to inhibit growth in all 3 cell lines in a dose dependent manner. The IC(50) of celecoxib were 41.04 +/- 1.22, 49.68 +/- 1.12 and 51.74 +/- 3.89 microM for CNE-2, Hone-1 and HK-1 cells, respectively. This growth inhibitory effect was found to be independent of the cell line's COX-2 protein expression level of the cells lines. In HK-1 cells, the expression of the cell cycle regulatory protein, cyclin D1, was down regulated by incubation with 80 microM celecoxib for 24 hrs.

Topics: Blotting, Western; Celecoxib; Cell Division; Cell Line, Tumor; Cyclin D1; Cyclooxygenase 2; Cyclooxygenase 2 Inhibitors; Dose-Response Relationship, Drug; Humans; Nasopharyngeal Neoplasms; Pyrazoles; Reverse Transcriptase Polymerase Chain Reaction; Sulfonamides; Tetrazolium Salts; Thiazoles

We cloned NP9 gene (GenBank, BF718797) in a previous study which was down-regulated in nasopharyngeal carcinoma (NPC). To clarify the function of NP9 gene, we cloned the coding sequence (CDS) of NP9 gene and investigated the influence of NP9 on cyclin D1 expression.. A full-length cDNA sequence was obtained by Blast NP9 EST, then the complete CDS was cloned into an eukaryotic expressing vector pRc/CMV2. The plasmid was transfected into CNE1 cells (NPC cells) with lipofectamine and positive cell clones which stably expressed NP9 CDS were established by G418 screening. The luciferase report plasmid with cyclin D1 promoter or NF-kappaB-Luc report plasmid was transfected into positive clones and the luciferase activity was detected. At last, the NP9 CDS was subcloned into pEGFP-C1 and cellular localization of NP9 protein was observed through transfecting pEGFP-C1-NP9 into CNE1 cells.. The fusion GFP was located in cell nuclei. The positive cell clones screened by G418 expressed NP9 CDS at different levels. Compared with control, the luciferase activities in NP9 positive clones were decreased 46% and 63% after 48 hours of transfecting the luciferase report plasmid with cyclin D1 promoter or NF-kappaB-Luc plasmid.. NP9 protein is a nuclear protein. NP9 gene can down-regulate the transcription activity of cyclin D1 and NF-kappaB.

Topics: Base Sequence; Cell Line, Tumor; Cloning, Molecular; Cyclin D1; Down-Regulation; Humans; Molecular Sequence Data; Nasopharyngeal Neoplasms; NF-kappa B; Transcription, Genetic

The cyclin D1/p16/Rb pathway plays a critical role in tumorigenesis and each component of this pathway may be affected in various malignancies. The purpose of this study was to investigate the expression and prognostic significance of these proteins in nasopharyngeal carcinoma (NPC).. Sixty-five patients undergoing radiotherapy for NPC were analyzed. The expression of cyclin D1, p16 and pRb was evaluated with immunohistochemical analysis of archived pretreatment tumor materials and expression of these proteins was correlated with clinicopathological parameters.. Positive expression of cyclin D1 was observed in 43 of 65 NPCs (66%). p16 and pRb inactivation was identified in 42 of 65 (65%) and four of 65 (6%) tumors, respectively. All but seven tumors (58 of 65, 89%) contained at least one alternation in the cyclin D1/p16/Rb pathway. Loss of cyclin D1 as well as p16 was closely related to local recurrence after radiotherapy for NPC (P = 0.015 and 0.047). No association between pRb expression and clinicopathological outcome was apparent.. The study's results suggest that the cyclin D1/p16/Rb pathway plays an important role in NPC tumorigenesis. We also find that cyclin D1 and p16 protein levels in NPC may be of use clinically as a predictor of local tumor control.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Carcinoma, Squamous Cell; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p16; Female; Humans; Immunoenzyme Techniques; Lymph Nodes; Male; Middle Aged; Nasopharyngeal Neoplasms; Neoplasm Recurrence, Local; Neoplasm Staging; Prognosis; Retinoblastoma Protein

NPC-associated gene NAG7 was a novel candidate tumor suppressor gene associated with nasopharyngeal carcinoma cloned in our laboratory. This study was designed to investigate the potential effect of NAG7 on the cell cycle and apoptosis of nasopharyngeal carcinoma cell line HNE1 and its molecular mechanism.. NAG7 gene was introduced into HNE1 cells using lipofectin transfection technique. The expression level of NAG7 gene was analyzed by Northern blot. Cell cycle, cyclins, and cell apoptosis were detected by flow cytometry, and the expressions of cyclin D1 and cyclin E were detected by Western blot.. NAG7 gene was re-expressed in NAG7 transfected HNE1 cells. Compared with HNE1 cells and vector transfected HNE1 cells, NAG7 transfected HNE1 cells arrested in G0/G1 phase increased (P < 0.05) and cells in S phase decreased (P < 0.05), the apoptosis cells increased (P < 0.05), and the levels of cyclins of A, B1, D1, and E decreased. Furthermore, the expression of cyclin D1 and E decreased in NAG7 transfected HNE1 cells.. NAG7 gene re-expression could inhibit overproliferation of NPC cell by delaying the progression of G1 into S in cell cycle and inducing cell apoptosis.

Topics: Apoptosis; Cell Cycle; Cell Cycle Proteins; Cyclin A; Cyclin D1; Cyclin E; G1 Phase; Gene Expression; Genes, Tumor Suppressor; Humans; Membrane Proteins; Nasopharyngeal Neoplasms; RNA, Long Noncoding; RNA, Untranslated; Transfection; Tumor Cells, Cultured; Tumor Suppressor Proteins

Cyclin D1 expression and the rate of apoptosis have been reported to serve as important prognostic indicators in human cancers. The purpose of the present study was to determine the prognostic significance of both initial cyclin D1 expression and the apoptotic index in nasopharyngeal carcinoma.. Cohort study.. Cyclin D1 protein levels and apoptosis in tumors and their corresponding adjacent, histologically normal tissues were determined at the time of initial diagnosis using immunohistochemical staining, Western blot analysis, and in situ end labeling, respectively, in 64 patients with T1-T4/N0-N2, poorly differentiated squamous cell carcinoma of the nasopharynx. All cases were treated by routine radiation therapy with a total median dose of 70 Gy and followed up for 10 years.. High levels of cyclin D1 were found in 35 of 64 tumor specimens (54.7%); no cyclin D1--positive cells were determinable in normal epithelium of the nasopharynx. Rates of early local recurrence (within 5 y) were significantly higher (P <.01) for patients with high levels of cyclin D1 before radiation therapy (24 of 35 patients [68.6%]) as compared with patients with low or no expression (3 of 29 [10.3%]). Furthermore, patients bearing high levels of cyclin D1 had a poorer prognosis concerning 10-year survival than the others (P <.001), whereas overexpression of cyclin D1 did not correlate with the initial TMN classification (P >.05). According to the rate of spontaneous apoptosis in tumors below or above the median, patients were divided into two groups. There was no statistically significant difference for the overall survival between the two groups (P >.05).. The present study demonstrates that cyclin D1 can be used as an indicator of recurrence and subsequent prognosis in nasopharyngeal carcinoma after radiation therapy. At the same time, the apoptotic state before radiation therapy is of no value in predicting the prognosis of patients with nasopharyngeal carcinoma.

Topics: Adult; Aged; Analysis of Variance; Biomarkers, Tumor; Biopsy, Needle; Blotting, Western; Carcinoma; China; Cohort Studies; Culture Techniques; Cyclin D1; Female; Humans; Immunohistochemistry; Male; Middle Aged; Nasopharyngeal Neoplasms; Neoplasm Recurrence, Local; Neoplasm Staging; Probability; Prognosis; Sensitivity and Specificity

Cyclin D1 is a key cell cycle regulator and a candidate proto-oncogene, whose deregulation has been implicated in pathogenesis of several types of cancers, including NPC. A common A/G polymorphism (A870G) in exon 4 of the cyclin D1 gene, CCND1, is associated with the presence of 2 distinct mRNA transcripts for this G1/S regulatory protein, and CCND1 genotype has been related to some phenotypes of several tumors. To investigate the influence of cyclin D1 genotypes on the genetic susceptibility in humans from Southern China to sporadic nasopharyngeal carcinoma, cyclin D1 genotyping was performed by denaturing high performance liquid chromatography (DHPLC) and DNA sequencing analysis of the PCR products from 84 NPC cases and 91 normal controls. Gene frequency distribution was tested by Hardy-Weinberg equilibrium and comparison of cyclin D1 gene frequencies between the patient and control groups was performed by chi 2 test. Results showed that in NPC patients, the AA genotype of CCND1 was significantly lower (20.24%) than in normal controls (38.46%), and the GG and AG genotypes (GG + AG) were significantly higher in NPC group than in the control group (chi 2 = 6.946, P corrected = 0.016, OR = 2.463, 95% CI = 1.249-4.859). These suggest that the A/G polymorphism of CCND1 was associated with the susceptibility to NPC, and the GG and AG genotypes in NPC patients were significantly higher than those in normal controls.

Topics: Adult; Aged; Asian People; Chromatography, High Pressure Liquid; Cyclin D1; Female; Gene Frequency; Genetic Markers; Genetic Predisposition to Disease; Genotype; Humans; Male; Middle Aged; Nasopharyngeal Neoplasms; Polymorphism, Genetic; Proto-Oncogene Mas

Nasopharyngeal carcinoma (NPC) is a malignant tumor with a high incidence in east Asian countries. Inactivation of cyclin-dependent kinase (CDK) inhibitors (CKIs) and overexpression of G1 cyclin has been thought to be important for tumor development. To determine whether reduction of CKI (p16 and p27) expression was associated with NPC development, we performed immunohistochemical staining of NPC specimens from 20 patients. We found that p16 and p27 proteins were negative in 8 of 20 and 16 of 20 cases, respectively; that either p16 or p27 proteins were negative in 17 of 20; and that both p16 and p27 were negative in 7 of 20. Excepting the cases in which both CKIs were negative, negativity of p27 alone was statistically higher than that of p16 (9/20 versus 1/20, P = .022), suggesting that the reduction of p27 protein is an important event for the multi-step process of NPC development.

Topics: Carcinoma, Squamous Cell; Cell Cycle Proteins; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p16; Cyclin-Dependent Kinase Inhibitor p27; Cyclin-Dependent Kinases; Enzyme Inhibitors; Genes, Tumor Suppressor; Humans; Immunoenzyme Techniques; Nasopharyngeal Neoplasms; Nasopharynx; Tumor Suppressor Proteins

In the present study, we tried to further elucidate the expression and function of cyclin D1 in human nasopharyngeal carcinoma (NPC) cell line.. Western blot was used in the analysis of D-type cyclin expression profile. The distribution of this protein was determined in the double parameter flow cytometry (FCM). The function of cyclin D1 in nasopharyngeal carcinoma cell line was determined in the antisense-oligonucleotied-cyclin D1 and antibody mediated knock-out experiments.. The immunoblot analysis of two NPC cell lines showed that the spectra of D-type in NPC cell lines founded their expressions in D1, D2, and D3. It also suggested that the overexpression of cyclin D1 could lead to deregulation of G1/S control. An additional double parameter FCM showed a characteristic variation in HNE1 which was consistent with the cell-cycle oscillation and the peak level expressed in G0/G1 phase and the lowest level in the S-phase. Two experimental approaches aimed at specific functional knock-out of cyclin D1 were employed; the cyclin D1-ASPODN experiment showed an inhibition of cyclin D1 expression at mRNA level and a down-regulated cyclin D1 partial inhibition of progression into S phase, resulting in cell growth arrest while the electroporation of neutralizing antibodies demonstrated a specific delay of S-phase entry in HNE1 and the requirement for the cyclin D1 in cell cycle progression of HNE1 cell line in regulation of G1/S.. These data reveal that the cell-cycle regulatory function of cyclin D1 is essential for cell cycle progression in the G1 of nasopharyngeal carcinoma cell line and suggest that cyclin D1 play an important role in NPC carcinogenesis.

Topics: Cell Cycle; Cell Division; Cyclin D1; Humans; Nasopharyngeal Neoplasms; RNA, Messenger; S Phase; Tumor Cells, Cultured

To investigate the effect of exogenous p16 on Cyclin D1, CDK4 and pRb, and to explore the mechanism of the growth suppression of p16 in nasopharyngeal carcinoma cell lines.. The curve of cell growth rate in three kinds of HNE1, # 3-2 and # 4-2 cell lines was analyzed and their double time was compared. Then the distribution of the cell cycle was detected by flow cytometry. The expression of p16 and the effect of exogenous p16 expression on CDK4, Cyclin D1 and pRb are studied by means of Western Blot.. As compared with HNE1, # 3-2 and # 4-2 showed a longer double time(23.4 h vs 28.8 h, 31.2 h). # 4-2 showed a significant accumulation of cells in G0/G1 phase(P < 0.01) and decreasing in S phase(P < 0.05) while HNE1 and # 3-2 had no obvious difference(P > 0.05). Cyclin D1 expression was upregulated in # 4-2 but downregulated in # 3-2 by exogenous expressed p16. No obvious difference on CDK4 expression was found. Hypophosphorylated pRb was detected in three cell lines. The expression was stronger in # 4-2, and # 3-2 than that in HNE1 and Hela. Hyperphosphorylated pRb was also detected in HNE1.. Exogenous p16 expression may arrest cell cycle in G0/G1 phase and suppress cell growth. The major mechanism is not to regulate the level of the expression of CDK4. There might be a threshold in p16 regulating Cyclin D1 expression. However, the final result contributes to the inhibition of pRb phosphorylation.

Topics: Cell Cycle; Cell Division; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase Inhibitor p16; Cyclin-Dependent Kinases; Humans; Nasopharyngeal Neoplasms; Phosphorylation; Proto-Oncogene Proteins; Retinoblastoma Protein; Tumor Cells, Cultured