cyclin-d1 and Colorectal-Neoplasms

Accumulating evidence indicates that the expression and/or variants of several genes play an essential role in the progress of colorectal cancer (CRC). The current study is a meta-analysis undertaken to estimate the prognosis and survival associated with. The authors searched PubMed, EMBASE, and Science Direct for relevant reports published between 2000 and 2020 and analyzed them to determine any relationship between the (immunohistochemically/sequencing-detected) gene expression and variants of the selected genes and the survival of CRC patients.. The analysis included 34,074 patients from 64 studies. To evaluate association, hazard ratios (HRs) were estimated for overall survival (OS) or disease-free survival (DFS), with a 95% confidence interval (CIs). Pooled results showed that. The present meta-analysis indicates that overexpression or underexpression and variants of

Topics: beta Catenin; Colorectal Neoplasms; Cyclin D1; Genes, bcl-1; Humans; Prognosis; Smad3 Protein; Tumor Suppressor Protein p53

CyclinD1 (CCND1) is a key cell cycle regulatory protein. A large number of epidemiological studies have assessed the potential correlation between the CCND1 G870A polymorphism and the risk of colorectal cancer (CRC), but their findings have been inconsistent. To obtain a more precise understanding of the association between the G870A polymorphism in the CCND1 gene and the CRC risk, we conducted a more comprehensive meta-analysis.. We searched PubMed, Ovid, Springer, Weipu, China National Knowledge Infrastructure (CNKI), and Wanfang databases, covering all publications (the last search was updated on January 10, 2017). The pooled odds ratios (ORs) with 95% confidence intervals (CIs) were derived from a fixed effect or random effect model. Statistical analyses were performed using Review Manager 5.3 and STATA 10.0 software.. A total of 7276 CRC patients and 9667 controls from 27 publications were included in this meta-analysis. We found that compared with GG homozygote genetic model, AA, AG, AA + AG genetic models of the CCND1 G870A polymorphism were significantly associated with overall CRC risk (AA homozygote genetic model: OR = 1.28, 95% CI = 1.10-1.49; AG heterozygote genetic model: OR = 1.15, 95% CI = 1.06-1.25; AA homozygote + AG heterozygote genetic model: OR = 1.19, 95% CI = 1.07-1.33). Subgroup analyses by ethnicity and cancer location showed that A carriers were consistently associated with a significantly increased risk of CRC in all subsets of participants (Asian and Caucasian; colon cancer and rectal cancer). When stratified by study design, we found a significant association in hospital-based studies (HB), but no significant associations were found in either population-based studies (PB) or family-based studies (FB). According to subgroup analysis by cancer type, the risk of sporadic colorectal cancer (sCRC) and hereditary nonpolyposis colorectal cancer (HNPCC) were not correlated with the CCND1 G870A polymorphism, except AG (AG vs GG: OR = 1.30, 95% CI = 1.11-1.53).. This meta-analysis suggests that the CCND1 G870A polymorphism is associated with an increased risk of CRC, especially that A carriers may be a major risk factor for CRC.

Topics: Adult; Aged; Asian People; Case-Control Studies; Colorectal Neoplasms; Cyclin D1; Female; Genetic Association Studies; Genetic Predisposition to Disease; Humans; Male; Middle Aged; Odds Ratio; Polymorphism, Single Nucleotide; Risk Factors; White People

Cyclin D1 plays a vital role in cancer cell cycle progression and is overexpressed in many human cancers, including colorectal cancer (CRC). However, the prognostic value of cyclin D1 overexpression in colorectal cancer is conflicting and heterogeneous. We conducted a meta-analysis to more precisely evaluate its prognostic significance.. A comprehensive literature search for relevant studies published up to January 2014 was performed using PubMed, EMBASE, and ISI Web of Science. The pooled hazard ratio (HR) with 95% confidence intervals (CI) was used to estimate the effects.. 22 studies with 4150 CRC patients were selected to evaluate the association between cyclin D1 and overall survival (OS), disease-free survival (DFS) and clinicopathological parameters. In a random-effects model, the results showed that cyclin D1 overexpression in CRC was significantly associated with both poor OS (HR = 0.73, 95% CI: 0.63-0.85, P<0.001) and DFS (HR = 0.60, 95% CI: 0.44-0.82, P = 0.001). Additionally, cyclin D1 overexpression was significantly associated with more relative older patients (≥ 60 years) (OR 0.62, 95% CI 0.44-0.89, P = 0.009), T3,4 tumor invasion (OR 0.70, 95% CI 0.57-0.85, P<0.001), N positive (OR 0.75, 95% CI 0.60-0.95, P = 0.016) and distant metastasis (OR 0.60, 95% CI 0.36-0.99, P = 0.047) of CRC.. The meta-analysis results indicated that cyclin D1 is an unfavorable prognostic factor for CRC. Cyclin D1 overexpression might be associated with poor clinical outcome and some clinicopathological factors such as age, T category, N category and distant metastasis in CRC patients.

Topics: Colorectal Neoplasms; Cyclin D1; Disease-Free Survival; Humans; Observational Studies as Topic; Prognosis; Proportional Hazards Models; Publication Bias

To detect the association between G870A polymorphism of cyclin D1 (CCND1) gene and colorectal cancer.. We performed a systematic literature and abstract search using PubMed, EMBase digital database. Keywords included CCND1, cyclin D1, polymorphism, SNP, colon cancer, rectal cancer and colorectal cancer. "And", "OR" and "NOT" were used as conjunction to narrow and widen the search. Data were extracted by two investigators independently, and meta-analysis was carried out by using Review Manager 4.2.8. The following pairwised combinations of genotypes for the CCND1 G870A polymorphism were evaluated: AA vs. GG, AG vs. GG, AA+AG vs. GG. Subsequently, sub-group analyses for cancer type, ethnicity, and the family history were performed. Sensitivity analysis was conducted by excluding the articles deviated from Hardy-Weinberg equilibrium.. Using GG genotype as a reference, A carriers were associated with a significantly increased cancer risk (OR=1.15, 95%CI=1.06-1.25, P=0.001, P(heterogeneity)=0.130), especially with rectal cancer (OR=1.24, 95%CI=1.02-1.51, P=0.030, P(heterogeneity)=0.570) and sporadic colorectal cancer (OR=1.26, 95%CI=1.08-1.46, P=0.003, P(heterogeneity)=0.730). The effect of A carriers on cancer also existed in Caucasians (OR=1.19, 95%CI=1.06-1.32, P=0.002, P(heterogeneity)=0.100).. CCND1 G870A polymorphism is associated with the increased risk of colorectal cancer, especially for sporadic colorectal cancer and in Caucasians.

Topics: Colorectal Neoplasms; Cyclin D1; Humans; Polymorphism, Genetic; Risk Factors; White People

Cyclin D1 (CCND1) plays a vital role in cancer cell cycle progression. Numerous epidemiological studies have evaluated the association between the CCND1 G870A polymorphism and the risk of colorectal cancer. However, these studies have yielded conflicting results. To derive a more precise estimation of this association, we conducted a meta-analysis and systematic review.. A comprehensive search was conducted to identify eligible studies of the CCND1 G870A polymorphism and colorectal cancer risk. Pooled odds ratios (ORs) with 95% confidence intervals (CIs) were derived from a fixed effect or random effect model. We applied a grading system (Venice criteria) that assessed the epidemiological strength of the association. A total of 22 publications that included 6157 cases and 8198 controls were identified. We found that the CCND1 G870A polymorphism was significantly associated with overall colorectal cancer risk (homozygote genetic model: OR = 1.130, 95% CI = 1.023-1.248, P = 0.016; heterozygote genetic model: OR = 1.124, 95% CI = 1.030-1.226, P = 0.009; dominant genetic model: OR = 1.127, 95% CI = 1.037-1.224, P = 0.005). After further stratified analyses, the increased risk was observed only in the subgroups of hospital-based studies, PCR-RFLP genotyping methods, sporadic colorectal cancer, and Caucasian ethnicity.. The available evidence demonstrates that the CCND1 870A allele might be a low-penetrant risk factor for colorectal cancer.

Topics: Case-Control Studies; Colorectal Neoplasms; Cyclin D1; Female; Genes, bcl-1; Genetic Predisposition to Disease; Humans; Male; Polymorphism, Single Nucleotide; Risk Factors; White People

Alternative pre-mRNA splicing allows exons of pre-mRNA to be spliced in different arrangements to produce functionally distinct mRNAs. More than 95% of human genes encode splice isoforms, some of which exert antagonistic functions. Recent studies revealed that alterations of the splicing machinery can cause the development of neoplasms, and understanding the splicing machinery is crucial for developing novel therapeutic strategies for malignancies. Colorectal cancer patients need novel strategies not only to enhance the efficacy of the currently available agents but also to utilize newly identified therapeutic targets. This review summarizes the current knowledge about the splice isoforms of VEGFA, UGT1A, PXR, cyclin D1, BIRC5 (survivin), DPD, K-RAS, SOX9, SLC39A14 and other genes, which may be possible therapeutic targets for colorectal cancer. Among them, the VEGFA splice isoforms are classified into VEGFAxxx and VEGFAxxxb, which have proangiogenic and antiangiogenic properties, respectively; UGT1A is alternatively spliced into UGT1A1 and other isoforms, which are regulated by pregnane X receptor isoforms and undergo further splicing modifications. Recently, the splicing machinery has been extensively investigated and novel discoveries in this research field are being reported at a rapid pace. The information contained in this review also provides suggestions for how therapeutic strategies targeting alternative splicing can be further developed.

Topics: Alternative Splicing; Camptothecin; Colorectal Neoplasms; Cyclin D1; Dihydrouracil Dehydrogenase (NADP); Glucuronosyltransferase; Humans; Inhibitor of Apoptosis Proteins; Irinotecan; Pregnane X Receptor; Protein Isoforms; Receptors, Steroid; Survivin; Vascular Endothelial Growth Factor A

Studies of the association between the cyclin D1 (CCND1) G870A genetic polymorphism and risk of colorectal cancer (CRC) have generated conflicting results. In order to derive a more precise estimation, a meta-analysis was here performed.. An extensive search of relevant studies was carried out as a meta-analysis of twenty studies with 5,975 cases and 8,333 controls.. Overall, a significantly elevated colorectal cancer risk was associated with variant allele 870A when all studies were pooled (AA vs. GG: OR = 1.23, 95%CI = 1.04-1.44; GA vs. GG: OR = 1.13, 95% CI = 1.01-1.26; dominant model: OR = 1.16, 95%CI = 1.03-1.31). In the subgroup analysis by ethnicity, significantly increased risks were detected among Caucasians (AA vs. GG: OR = 1.27, 95%CI = 1.04-1.44; and dominant model: OR = 1.17, 95%CI = 1.02-1.34). With stratification into sporadic CRC and hereditary nonpolyposis colorectal cancer (HNPCC), the former demonstrated increased cancer susceptibility (AA vs. GG: OR = 1.24, 95%CI = 1.04-1.48; dominant model: OR = 1.17, 95%CI = 1.04-1.33). However, no significant associations were found in either Asians or HNPCC patients for any genetic model.. The results suggest that the cyclin D1 870A allele is a low-penetrant risk factor for development of sporadic colorectal cancer, especially among Caucasians.

Topics: Alleles; Case-Control Studies; Colorectal Neoplasms; Colorectal Neoplasms, Hereditary Nonpolyposis; Cyclin D1; Genetic Predisposition to Disease; Genotype; Humans; Polymorphism, Single Nucleotide

Studies investigating the association between genetic polymorphism of cyclin D1 (CCND1) G870A and risk of colorectal cancer (CRC) reported conflicting results. In order to derive a more precise estimation of the relationship, a meta-analysis was performed.. We performed an extensive search of relevant studies and carried out a meta-analysis, including 20 studies with 5,975 cases and 8,333 controls, to obtain a more precise estimate.. Overall, significantly elevated colorectal cancer risk was associated with variant allele 870A when all studies were pooled (AA vs. GG: OR = 1.23, 95% CI = 1.04-1.44; GA vs. GG: OR = 1.13, 95% CI = 1.01-1.26; dominant model: OR = 1.16, 95% CI = 1.03-1.31). In the subgroup analysis by ethnicity, significantly increased risks were detected among Caucasians (AA vs. GG: OR = 1.27, 95% CI = 1.04-1.44; dominant model: OR = 1.17, 95% CI = 1.02-1.34).We also observed sporadic CRC with an increased cancer susceptibility (AA vs. GG: OR = 1.24, 95% CI = 1.04-1.48; dominant model: OR = 1.17, 95% CI = 1.04-1.33), when colorectal cancer was stratified into sporadic CRC and hereditary nonpolyposis colorectal cancer (HNPCC). However, no significant associations were found in both Asians and HNPCC patients for all genetic models.. Result suggests that the cyclin D1 870A allele is a low-penetrant risk factor for developing sporadic colorectal cancer, especially among Caucasians.

Topics: Amino Acid Substitution; Colorectal Neoplasms; Cyclin D1; Genetic Predisposition to Disease; Genetics, Population; Humans; Odds Ratio; Polymorphism, Single Nucleotide; Publication Bias; Risk Factors

Cyclin D1 (CCND1) and E-cadherin (CDH1) have been shown to be important genes of the beta-catenin/LEF pathway that is involved in colorectal carcinogenesis. However, epidemiological studies on relationship between genetic variants of these two genes and colorectal cancer (CRC) have shown inconsistent results. In a population-based case-control study (498 cases and 600 controls), we assessed the association of CCND1 G870A and CDH1 C-160A polymorphisms with CRC risk. Multivariable logistic regression analysis was used to estimate the association between genotypes, environmental exposures and CRC risk, adjusting for potential confounders. Compared to common homozygotes, the OR for heterozygous and homozygote variant genotype was 1.08 (95% CI, 0.80-1.46) in CCND1 and 0.97 (95% CI, 0.75-1.25) in CDH1. Neither tumor stage nor location showed an association with genetic susceptibility. However, a significant interaction between hormone replacement therapy (HRT) and CCND1 genotypes in CRC risk was found among postmenopausal women (p(interaction) = 0.02). The risk reduction associated with HRT was substantial (OR, 0.09; 95% CI, 0.02-0.35) in women who were GG homozygous. A meta-analyses including 11 published studies on CCND1 G870A in addition to our study showed a slightly increased risk of CRC for carriers of the A allele (OR, 1.19; 95% CI, 1.06-1.34); however, there was some indication of publication bias. We conclude that the CCND1 G870A and CDH1 C-160A polymorphisms are not associated with the risk of CRC in the German population. However, the CCND1 G870A polymorphism may modify the protective effect of postmenopausal hormone use on the development of CRC.

Topics: Adult; Aged; Aged, 80 and over; Cadherins; Case-Control Studies; Colorectal Neoplasms; Cyclin D1; Female; Genetic Predisposition to Disease; Genotype; Germany; Humans; Logistic Models; Male; Middle Aged; Multivariate Analysis; Odds Ratio; Polymorphism, Genetic; Publication Bias; Research Design; Risk Factors; White People

Among the targeted therapies used in the treatment of metastatic colorectal cancer (CRC), cetuximab was registered in France in 2004. This chimeric antibody inhibiting the Epidermal Growth receptor (EGFR) has been demonstrated to be efficient in the treatment of irinotecan-resistant metastatic CRC expressing the EGFR. Panitumumab, a fully humanized anti-EGFR antibody should soon be registered after failure of conventional chemotherapies. However, these costly and potentially toxic treatments are efficient in a little proportion of patients. It is so necessary to identify some factors able to better define whose patients will benefit from these treatments. The major potential predictive factors of response to cetuximab and/or panitumumab that have been evaluated in the literature, which are summarized in this review, are molecular factors involved more or less directly in the EGF signaling pathway. Among them, KRAS mutations, EGFR gene copy number and, more recently, epiregulin and amphiregulin expression are those, along with skin toxicity, which appear to be the most relevant and which will have to be evaluated in future clinical trials to be validated before being incorporated in therapeutic strategy of CRC.

Topics: Amphiregulin; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Camptothecin; Cetuximab; Colorectal Neoplasms; Cyclin D1; Drug Eruptions; Drug Resistance, Neoplasm; EGF Family of Proteins; Epidermal Growth Factor; Epiregulin; ErbB Receptors; Gene Dosage; Glycoproteins; Humans; Intercellular Signaling Peptides and Proteins; Irinotecan; Mutation; Neoplasm Proteins; Panitumumab; Polymorphism, Genetic; Proto-Oncogene Proteins; Proto-Oncogene Proteins p21(ras); ras Proteins; Receptors, IgG; Vascular Endothelial Growth Factor A

Barrett's esophagus (BE) represents the most serious histological consequence of gastroesophageal reflux disease (GERD) that develops in 5-10% of patients with GERD. Given that BE is the only known precursor to esophageal adenocarcinoma (EA), a systematic endoscopic biopsy protocol can detect EAs at an early stage. However, endoscopic and histopathological evaluation of BE are not adequate for effective screening of high risk patients. Therefore, molecular abnormalities associated with BE have been considered as surrogate markers and their use as such is proposed. Flow cytometry is the most useful adjunct to histology, and ploidy status of BE is an independent risk factor. Cyclin D1 overexpression is inversely correlated with survival in EA. C-erbB2 (+) patients have poorer prognosis. High plasma adenomatous polyposis coli levels correlate with reduced patient survival. p53 expression allows patient risk for EA stratification. Nuclear factor-kappaB overexpression inversely correlates with good response to adjuvant chemotherapy and radiotherapy in EA. Patients with cyclooxygenase-2 overexpression have reduced survival rates. Increased E-cadherin staining is associated with shorter survival in EA patients who received chemoradiotherapy. Finally, existing data cannot rule out a correlation between EA and colorectal tumors. Seventeen BE molecular alterations yielded noteworthy clinical implications. Apart from endoscopy and histology, these data allow for better risk stratification for patients with BE and for more efficient and timely therapeutic approaches.

Topics: Adenocarcinoma; Adenomatous Polyposis Coli Protein; Barrett Esophagus; Biomarkers; Biomarkers, Tumor; Cadherins; Clinical Trials as Topic; Colorectal Neoplasms; Cyclin D1; Cyclooxygenase 2; Endoscopy, Gastrointestinal; Esophageal Neoplasms; Gastroesophageal Reflux; Gene Expression Regulation, Neoplastic; Humans; Membrane Proteins; NF-kappa B; Prostaglandin-Endoperoxide Synthases; Receptor, ErbB-2; Risk Factors; Survival Rate; Tumor Suppressor Protein p53; Up-Regulation

An important role for beta-catenin pathways in colorectal carcinogenesis was first suggested by the protein's association with adenomatous polyposis coli (APC) protein, and by evidence of dysregulation of beta-catenin protein expression at all stages of the adenoma-carcinoma sequence. Recent studies have, however, shown that yet more components of colorectal carcinogenesis are linked to beta-catenin pathways. Pro-oncogenic factors that also release beta-catenin from the adherens complex and/or encourage translocation to the nucleus include ras, epidermal growth factor (EGF), c-erbB-2, PKC-betaII, MUC1, and PPAR-gamma, whereas anti-oncogenic factors that also inhibit nuclear beta-catenin signaling include transforming growth factor (TGF)-beta, retinoic acid, and vitamin D. Association of nuclear beta-catenin with the T cell factor (TCF)/lymphoid enhancer factor (LEF) family of transcription factors promotes the expression of several compounds that have important roles in the development and progression of colorectal carcinoma, namely: c-myc, cyclin D1, gastrin, cyclooxygenase (COX)-2, matrix metalloproteinase (MMP)-7, urokinase-type plasminogen activator receptor (aPAR), CD44 proteins, and P-glycoprotein. Finally, genetic aberrations of several components of the beta-catenin pathways, eg, Frizzled (Frz), AXIN, and TCF-4, may potentially contribute to colorectal carcinogenesis. In discussing the above interactions, this review demonstrates that beta-catenin represents a key molecule in the development of colorectal carcinoma.

Topics: Adenoma; Adenomatous Polyposis Coli Protein; Animals; ATP Binding Cassette Transporter, Subfamily B, Member 1; beta Catenin; Calcium-Calmodulin-Dependent Protein Kinases; Carcinoma; Colorectal Neoplasms; Cyclin D1; Cyclooxygenase 2; Cytoskeletal Proteins; Gastrins; Glycogen Synthase Kinase 3; Humans; Hyaluronan Receptors; Intestinal Mucosa; Isoenzymes; Matrix Metalloproteinase 7; Membrane Proteins; Prostaglandin-Endoperoxide Synthases; Proto-Oncogene Proteins; Receptors, Cell Surface; Receptors, Urokinase Plasminogen Activator; Signal Transduction; Trans-Activators

Brazilian propolis, a folk medicine, is used worldwide as an alternative medicine to prevent colon cancer. The objective of the study was to test in a small pilot biomarker study in a high-risk group the safety and efficacy of propolis for colon cancer prevention, which has not been evaluated in humans.. Subjects with adenoma polyps recently removed from the colon were randomly assigned to a propolis group of 15 and a placebo group of 16. In a double-blind study, the propolis group received capsules containing 165 μmol artepillin C and 150 μmol other polyphenols per day for 3 months. Prior to and at the end of the experiments, their blood was analyzed using biochemical tests, and specimens from the normal-appearing sigmoid colon mucosa were biopsied endoscopically to examine the levels of 8-hydroxy-2'-deoxyguanosine (8-OHdG) and mRNA expressions of proliferating cell nuclear antigen, cyclin D1, and Bax.. Propolis extract significantly increased the mRNA level of cyclin D1 in the sigmoid colon mucosa, and the other biomarkers remained unchanged. Blood biochemical tests showed significantly higher activity of creatine phosphokinase (CPK), 143 ± 52 units/ml in the propolis group and 104 ± 38 units/ml in the placebo group (p = 0.026), at the end of the study. The increase in CPK activity in the propolis group was due to the increase of the myocardial band form of CPK. On the other hand, laxative treatment prior to endoscopic biopsy significantly increased 8-OHdG levels.. The results from our pilot study did not provide evidence that Brazilian propolis was effective in preventing changes occurring during early stages of colon cancer. In contrast, propolis may have detrimental side effects on muscle tissue, including myocardial cells.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Adenomatous Polyps; Aged; Biomarkers; Brazil; Colon; Colorectal Neoplasms; Creatine Kinase; Cyclin D1; Deoxyguanosine; Double-Blind Method; Female; Humans; Intestinal Polyps; Male; Middle Aged; Phenylpropionates; Pilot Projects; Plant Extracts; Polyphenols; Proliferating Cell Nuclear Antigen; Propolis; RNA, Messenger

Bardoxolone methyl, a novel synthetic triterpenoid and antioxidant inflammation modulator, potently induces Nrf2 and inhibits NF-κB and Janus-activated kinase/STAT signaling. This first-in-human phase I clinical trial aimed to determine the dose-limiting toxicities (DLT), maximum tolerated dose (MTD), and appropriate dose for phase II studies; characterize pharmacokinetic and pharmacodynamic parameters; and assess antitumor activity.. Bardoxolone methyl was administered orally once daily for 21 days of a 28-day cycle. An accelerated titration design was employed until a grade 2-related adverse event occurred. A standard 3 + 3 dose escalation was then employed until the MTD was reached. Single dose and steady-state plasma pharmacokinetics of the drug were characterized. Assessment of Nrf2 activation was examined in peripheral blood mononuclear cells (PBMC) by measuring NAD(P)H:quinone oxidoreductase (NQO1) mRNA levels. Immunohistochemical assessment of markers of inflammation, cell cycle, and apoptosis was carried out on tumor biopsies.. The DLTs were grade 3 reversible liver transaminase elevations. The MTD was established as 900 mg/d. A complete tumor response occurred in a mantle cell lymphoma patient, and a partial response was observed in an anaplastic thyroid carcinoma patient. NQO1 mRNA levels increased in PBMCs, and NF-κB and cyclin D1 levels decreased in tumor biopsies. Estimated glomerular filtration rate (eGFR) was also increased.. Bardoxolone methyl was well tolerated with an MTD of 900 mg/d. The increase in eGFR suggests that bardoxolone methyl might be beneficial in chronic kidney disease. Objective tumor responses and pharmacodynamic effects were observed, supporting continued development of other synthetic triterpenoids in cancer.

Topics: Adult; Aged; Aged, 80 and over; Antineoplastic Agents; Carcinoma, Renal Cell; Colorectal Neoplasms; Cyclin D1; Dose-Response Relationship, Drug; Drug Administration Schedule; Female; Humans; Janus Kinases; Kidney Neoplasms; Lymphoma; Male; Maximum Tolerated Dose; Melanoma; Middle Aged; NAD(P)H Dehydrogenase (Quinone); Neoplasms; NF-E2-Related Factor 2; NF-kappa B; Oleanolic Acid; RNA, Messenger; STAT Transcription Factors; Thyroid Neoplasms; Young Adult

The aim of this study was to investigate the value of the cyclin D1 isoforms D1a and D1b as prognostic factors and their relevance as predictors of response to adjuvant chemotherapy with 5-fluorouracil and levamisole (5-FU/LEV) in colorectal cancer (CRC).. Protein expression of nuclear cyclin D1a and D1b was assessed by immunohistochemistry in 335 CRC patients treated with surgery alone or with adjuvant therapy using 5-FU/LEV. The prognostic and predictive value of these two molecular markers and clinicopathological factors were evaluated statistically in univariate and multivariate survival analyses.. Neither cyclin D1a nor D1b showed any prognostic value in CRC or colon cancer patients. However, high cyclin D1a predicted benefit from adjuvant therapy measured in 5-year relapse-free survival (RFS) and CRC-specific survival (CSS) compared to surgery alone in colon cancer (P=0.012 and P=0.038, respectively) and especially in colon cancer stage III patients (P=0.005 and P=0.019, respectively) in univariate analyses. An interaction between treatment group and cyclin D1a could be shown for RFS (P=0.004) and CSS (P=0.025) in multivariate analysis.. Our study identifies high cyclin D1a protein expression as a positive predictive factor for the benefit of adjuvant 5-FU/LEV treatment in colon cancer, particularly in stage III colon cancer.

Topics: Antineoplastic Combined Chemotherapy Protocols; Biomarkers, Tumor; Colorectal Neoplasms; Combined Modality Therapy; Cyclin D1; Disease-Free Survival; Female; Fluorouracil; Follow-Up Studies; Humans; Immunohistochemistry; Levamisole; Male; Middle Aged; Predictive Value of Tests; Prognosis; Prospective Studies; Recurrence; Treatment Outcome

Low-dose aspirin reduces the incidence of colorectal cancer and recurrence of adenomas. Cyclooxygenase-2 (COX-2), one of its main target enzymes, is reportedly over-expressed in colorectal adenomas.. To assess COX-2 expression, in relation to adenoma recurrence and the protective effect of aspirin, in a large series of colorectal adenomas, recruited from a double-blind randomised controlled trial comparing recurrences after low-dose aspirin or placebo.. Follow-up colonoscopies were performed after 1 and 4 years to assess adenoma recurrence. COX-2 expression was assessed by immunohistochemistry for each adenoma obtained at baseline colonoscopy, separately for epithelium, deep stroma and overall. Architecture, grade of dysplasia, K-ras mutation, p53 and cyclin D1 expression were studied.. COX-2 expression could be assessed in 219 adenomas from 136. 128 adenomas (58%) from 59 patients strongly expressed COX-2. Strong COX-2 expression predominated in adenomas larger than 10 mm (84/129 vs 44/90; p=0.02) and in adenomas showing high-grade dysplasia (22/29 vs 104/188; p=0.04). Deep stromal but not epithelial initial expression of COX-2 predicted adenoma recurrence in the whole population (30/72 patients or 42% strongly expressed deep stromal COX-2 compared with 16/64 or 25% without recurrent adenoma; p=0.04). The protective effect of aspirin was mainly observed in patients in whom COX-2 initial expression was low (RR for recurrence in patients taking aspirin with low COX-2 expression: 0.59; 95% CI 0.39 to 0.90; p=0.02). There was no significant effect of aspirin at the end of the trial.. Over-expression of COX-2 was frequent and predominated in large and high-grade dysplasia adenomas. Deep stromal but not epithelial initial expression of COX-2 predicted recurrence of adenomas. Aspirin did not act preferentially on patients whose initial adenomas strongly expressed COX-2.

Topics: Adenoma; Adolescent; Adult; Aged; Anticarcinogenic Agents; Aspirin; Biomarkers, Tumor; Colorectal Neoplasms; Cyclin D1; Cyclooxygenase 2; Cyclooxygenase Inhibitors; Double-Blind Method; Female; Genes, ras; Humans; Male; Middle Aged; Mutation; Neoplasm Proteins; Neoplasm Recurrence, Local; Tumor Suppressor Protein p53; Young Adult

To evaluate the effects of perioperative total parenteral nutrition on cyclin D1, recurrence and metastasis of colorectal cancer cells.. A total of 120 patients with colorectal carcinoma were randomly divided into two groups, namely group A(total parenteral nutrition, TPN,60 cases) and group B(non total parenteral nutrition, NTPN, 60 cases). In group A, the patients were given with TPN(including glucose, intralipid, amino acid, and vitamins, etc.) for 10 days perioperation (7 days preoperatively and 3 days postoperatively). In group B, the patients did not receive any nutrition support perioperative nutrition support. The samples were obtained by colonoscopy preoperatively or during operation. Apoptosis was detected by terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) technique,expression of proliferating cell nuclear antigen (PCNA) by immunohistochemical staining, and the expression of cyclin D1 by in situ hybridization. The apoptotic index (AI), the proliferating index (PI), and the expression of cyclin D1 were calculated perioperatively and postoperatively.. After perioperative nutrition support, the expression rates of cyclin D1, PI and AI in group A and group B were (35.23+/-5.12)% and (37.53+/-5.31)%, (7.21+/-2.56)% and (8.75+/-3.84)%, (53.45+/-7.74)% and (56.74+/-8.02)% respectively. There were no significant difference of PI, AI and the expression of cyclin D1(all P>0.05) between two groups. The 3-year recurrent rates in two groups were 16.7% and 15.0%( P>0.05).. Perioperative TPN can not promote proliferation and apoptosis of carcinoma cells, and has no significant impact on the expression of cyclin D1, recurrence or metastasis of colorectal cancer.

Topics: Adult; Aged; Colorectal Neoplasms; Cyclin D1; Female; Humans; Intraoperative Period; Male; Middle Aged; Neoplasm Metastasis; Neoplasm Recurrence, Local; Parenteral Nutrition, Total; Prognosis

The molecular basis for most non-HNPCC familial colorectal cancer cases is unknown, but there is increasing evidence that common genetic variants may play a role. We investigated the contribution of polymorphisms in two genes implicated in the pathogenesis of colorectal cancer, cyclin D1 (CCND1) and E-cadherin (CDH1), to familial and sporadic forms of the disease. The CCND1 870A/G polymorphism is thought to affect the expression of CCND1 through mRNA splicing and has been reported to modify the penetrance of HNPCC. Inactivation of E-cadherin is common in colorectal cancer, and truncating germline mutations have been reported to confer susceptibility to colorectal as well as diffuse gastric cancer. The -160A/C CDH1 polymorphism appears to affect expression of CDH1 and may therefore also confer an increased risk. We found a significantly higher frequency of CCND1 870A allele in 206 familial cases compared to 171 controls (P=0.03). Odds ratios in heterozygotes and homozygotes were 1.7 (95% CI: 1.0-2.66) and 1.8 (95% CI: 1.0-3.3) respectively. The difference was accounted for by an over-representation of A allele in non-HNPCC familial cases (P=0.007). Over-representation of the CCND1 A allele was also seen in sporadic colorectal cancer cases compared to controls but this did not attain statistical significance (P=0.08). No significant differences between the frequency of CDH1 -160A/C genotypes in familial, sporadic colorectal cancer cases and controls were seen, although a possible association between the low expressing A allele and right-sided tumours was detected in familial cases.

Topics: Cadherins; Case-Control Studies; Colorectal Neoplasms; Cyclin D1; DNA Mutational Analysis; DNA Primers; DNA, Neoplasm; Female; Genetic Predisposition to Disease; Genotype; Humans; Male; Middle Aged; Polymerase Chain Reaction; Polymorphism, Genetic; Risk Factors

Colorectal cancer (CRC) is a serious threat to human health and is drug-resistant. Circular RNA _0004585 (circ_0004585) has been shown to be expressed in CRC, but whether it plays a role in CRC with chemoresistance remains unknown. Therefore, this study aimed to investigate the potential role of circ_0004585 in CRC with 5-fluorouracil (5-FU) resistance.. The expression of related genes was detected by quantitative real-time polymerase chain reaction (qRT-PCR), and the protein expressions of cleaved caspase-3, cleaved caspase-9, and cyclin D1 (CCND1) were detected by western blot. Cell functions were identified using CCK-8, colony formation, flow cytometry, tube formation and transwell assays. The putative relationships between miR-874-3p and circ_0004585 or CCND1 were validated by dual-luciferase reporter assays. Animal experiments were conducted to verify the effect of circ_0004585 on 5-FU resistance in vivo.. Circ_0004585 was highly expressed in CRC tissues and cells, particularly in 5-FU-resistant CRC tissues and cells. Circ_0004585 knockdown enhanced 5-FU sensitivity to further inhibit CRC cell viability, colony formation, cell migration and invasion, and accelerate cell apoptosis. MiR-874-3p was the target of circ_0004585, and miR-874-3p depletion partially recovered the malignant behaviors of 5-FU-resistant CRC cells that were blocked by silencing of circ_0004585. In addition, CCND1 was the target of miR-874-3p, and overexpression of CCND1 was able to restore the malignant effects of 5-FU-resistant CRC cells that were repressed by miR-874-3p enrichment. Animal experiments confirmed that circ_0004585 knockdown inhibited the growth of CRC tumors and enhanced 5-FU sensitivity in vivo.. Circ_0004585 promotes the development of CRC and increases 5-FU resistance in CRC through the miR-874-3p/CCND1 axis. These results suggest that circ_0004585 may be a therapeutic target for 5-FU-ressitant CRC.

Topics: Animals; Cell Line, Tumor; Cell Proliferation; Colorectal Neoplasms; Cyclin D1; Fluorouracil; Humans; MicroRNAs; RNA, Circular

The main strategy of cancer cells for survival is uncontrolled cell division and escape from apoptosis. The use of anticancer agents inducing the production of reactive oxygen species (ROS) and controlling cell division might be a therapeutic approach to eradicate cancer cells. Herein, we examined the therapeutic effects of Auraptene on CT26 cells as well as on a mouse model of colorectal cancer (CRC). The spheroid assay was also conducted to analyze the anti-proliferative activity of Auraptene. We also assessed the in vitro analysis of ROS generation. The impact of Auraptene on oxidant/antioxidant markers, as well as the mRNA expression of Bax, Bcl-2, Nrf2, Cyclin D1, and Survivin genes, was evaluated by qPCR in tumor samples. As a result, Auraptene significantly reduced the size of CT26 spheroids at a dose of 200 µM. After 12 h, ROS levels were significantly elevated in CT26 cells. The administration of Auraptene induced apoptosis and the cell cycle arrest by modulating Bax, Bcl-2, Nrf2, Cyclin D1, and Survivin mRNA levels. Furthermore, our results demonstrated that Auraptene suppressed CAT, GSH (reduced Glutathione), and FRAP while increasing MDA in tissue homogenates which in turn could raise oxidative stress and stimulate apoptosis. Therefore, Auraptene may act as a powerful adjuvant therapy in CRC since it triggers apoptosis and cell cycle.

Topics: Animals; Apoptosis; bcl-2-Associated X Protein; Cell Proliferation; Colorectal Neoplasms; Coumarins; Cyclin D1; Disease Models, Animal; Mice; NF-E2-Related Factor 2; Oxidative Stress; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species; Survivin

Colorectal cancer (CRC) is a common disease threatening human lives worldwide, and vitamin D receptor (VDR) contributes protective roles in this disease. However, the molecular mechanisms underlying VDR protection in CRC progression require further investigation.. In this study, we statistically analyzed the relationship between VDR expression and CRC development in patients and detected invasion and apoptosis in CRC cells with VDR overexpression and interference. We also detected the expression of key genes involved in Wnt/β-catenin signalling (β-catenin, lymphoid enhancer factor (LEF)-1 and cyclin D1) in SW480 cells and nude mice injected with VDR-overexpressing SW480 cells and observed tumour development. Additionally, we performed Co-immunoprecipitation (Co-IP) and glutathione-S-transferase (GST) pull-down assays to identify the protein interactions of VDR with β-catenin, dual luciferase (LUC) and chromatin immunoprecipitation (ChIP) to detect the activation of LEF-1 by VDR.. The VDR level was closely related to the development and prognosis of CRC patients. VDR overexpression inhibited invasion but promoted apoptosis in cancer cells. β-catenin shRNA contributed oppositely to cancer cell activity with VDR shRNA. Additionally, VDR interacted with β-catenin at the protein level and blocked its nuclear accumulation. VDR regulated the expression of β-catenin, cyclin D1 and LEF-1 and directly activated LEF-1 transcription in vitro. Furthermore, nude mice injected with VDR-overexpressing SW480 cells revealed suppression of tumour growth and decreased expression of β-catenin, cyclin D1 and LEF-1.. This study indicated that VDR protected against CRC disease in humans by inhibiting Wnt/β-catenin signalling to control cancer cell invasion and apoptosis, providing new evidence to explore VDR biomarkers or agonists for CRC patient diagnosis and treatment.

Topics: Animals; beta Catenin; Cell Line, Tumor; Cell Proliferation; Colorectal Neoplasms; Cyclin D1; Gene Expression Regulation, Neoplastic; Humans; Mice; Mice, Nude; Receptors, Calcitriol; RNA, Small Interfering; Wnt Signaling Pathway

Colorectal cancer (CRC) is the most common malignant tumor of the gastrointestinal tract with unfavorable prognosis. Therefore, novel biomarkers that may be used for new diagnostic strategies and drug-targeting therapy should be developed.. To investigate the expression of miR-29b in CRC and its association with ETV4 and cyclin D1 expression. Moreover, the current work aims to investigate the association between them and the clinicopathological features of CRC.. The expression of miR-29b and ETV4 (by qRT-PCR) and ETV4 and cyclin D1 (immunohistochemistry) was investigated in 65 cases of colon cancer and surrounding healthy tissues.. MiR-29b down-regulated and ETV4 and Cyclin D1 up-regulated significantly in colon cancer tissues compared to normal nearby colonic tissues. In addition, significant associations between ETV4 and cyclin D1 expressions and progressive stage and lymph node (LN) metastasis (P< 0.001 for each) were found. Furthermore, there was a negative correlation between miR-29b gene expression and ETV4 gene expression (r=-0.298, P<0.016).. Down-regulation of miR-29b and over-expression of ETV4 and cyclin D1 may be utilized as early diagnostic marker for development of colon cancer. ETV4 and cyclin D1 correlate with poor prognostic indicators and considered as a possible target for therapy in colon cancer.

Topics: Biomarkers, Tumor; Colonic Neoplasms; Colorectal Neoplasms; Cyclin D1; Gene Expression Regulation, Neoplastic; Humans; Lymphatic Metastasis; MicroRNAs; Prognosis; Proto-Oncogene Proteins c-ets

The high prevalence of colorectal cancer (CRC) and its leading death causing rate have placed a considerable burden on patients and healthcare providers. There is a need for a therapy that has fewer adverse effects and greater efficiency. Zearalenone (ZEA), an estrogenic mycotoxin, has been demonstrated to exert apoptotic properties when administrated in higher doses. However, it is unclear whether such apoptotic effect remains valid in an in vivo setting. The current study aimed to investigate the effect of ZEA on CRC and its underlying mechanisms in the azoxymethane/ dextran sodium sulfate (AOM/DSS) model. Our results revealed that ZEA significantly lowered the total number of tumours, colon weight, colonic crypt depth, collagen fibrosis and spleen weight. ZEA suppressed Ras/Raf/ERK/cyclin D1 pathway, increasing the expression of apoptosis parker, cleaved caspase 3, while decreasing the expression of proliferative marker, Ki67 and cyclin D1. The gut microbiota composition in ZEA group showed higher stability and lower vulnerability in the microbial community when compared to AOM/DSS group. ZEA increased the abundance of short chain fatty acids (SCFAs) producing bacteria unidentified Ruminococcaceae, Parabacteroidies and Blautia, as well as the faecal acetate content. Notably, unidentified Ruminococcaceae and Parabacteroidies were substantially correlated with the decrease in tumour count. Overall, ZEA demonstrated a promising inhibitory effect on colorectal tumorigenesis and exhibited the potential for further development as a CRC treatment.

Topics: Animals; Azoxymethane; Bacteria; Carcinogenesis; Cell Transformation, Neoplastic; Colitis; Colorectal Neoplasms; Cyclin D1; Dextran Sulfate; Disease Models, Animal; Humans; MAP Kinase Signaling System; Mice; Mice, Inbred C57BL; Zearalenone

The mechanisms underlying the transition from colitis-associated inflammation to carcinogenesis and the cell origin of cancer formation are still unclear. The azoxymethane (AOM)/dextran sodium sulfate (DSS) mouse model reproduces human colitis-associated colorectal cancer. To elucidate the mechanisms of cancer development and dynamics of the linker threonine-phosphorylated Smad2/3 (pSmad2/3L-Thr)-positive cells, we explored the early stages of colitis-associated colorectal cancer in AOM/DSS mice. The AOM/DSS mice were sacrificed at 4 to 6 weeks following AOM administration. To analyze the initial lesions, immunofluorescence staining for the following markers was performed: β-catenin, Ki67, CDK4, Sox9, Bmi1, cyclin D1, and pSmad2/3L-Thr. Micro-neoplastic lesions were flat and unrecognizable, and the uni-cryptal ones were either open to the surfaces or hidden within the mucosae. These neoplastic cells overexpressed β-catenin, Sox9, Ki67, and Cyclin D1 and had large basophilic nuclei in the immature atypical cells. In both the lesions, pSmad2/3L-Thr-positive cells were scattered and showed immunohistochemical co-localization with β-catenin, CDK4, and Bmi1 but never with Ki67. More β-catenin-positive neoplastic cells of both lesions were detected at the top compared to the base or center of the mucosae. We confirmed initial lesions in the colitis-associated colorectal cancer model mice and observed results that suggest that pSmad2/3L-Thr is a biomarker for tissue stem cells and cancer stem cells.

Topics: Animals; Azoxymethane; beta Catenin; Colitis; Colitis-Associated Neoplasms; Colorectal Neoplasms; Cyclin D1; Dextran Sulfate; Disease Models, Animal; Humans; Ki-67 Antigen; Mice; Mice, Inbred C57BL; Neoplastic Stem Cells

Colorectal cancer (CRC) is a commonly seen malignant tumor manifesting itself in the digestive tract, but it remains unclear what is the molecular mechanism behind its occurrence and development, which can have a significant impact on the clinical diagnosis and treatment of CRC. According to some studies, microRNA (miRNA) plays an essential role in the occurrence and development of cancer. In spite of this, there are still many miRNAs that play an important role in the progression of CRC but have yet to be reported. In our research, it was found out that the expression of mir-4746 is significantly down-regulated in CRC tissues and cells, and that its expression level is closely associated with the tumor size and prognosis of clinical patients. As revealed by function and mechanism experiments, targeting CCND1 mRNA 3'-UTR, mir-4746 can promote the degradation of CCND1 mRNA, thus reducing the protein level of CCND1, leading to cell G0-G1 phase arrest, and ultimately inhibiting the proliferation of CRC cells. For the first time, our study reported the biological functions of mir-4746 and its preliminary mechanism of action, in addition to demonstrating that mir-4746 can be applied as both a potential prognostic marker and the therapeutic target for CRC.

Topics: 3' Untranslated Regions; Animals; Apoptosis; Biomarkers, Tumor; Cell Line, Tumor; Cell Proliferation; Colorectal Neoplasms; Cyclin D1; Disease Progression; Down-Regulation; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; In Vitro Techniques; Mice; Mice, Inbred BALB C; MicroRNAs

Colorectal adenosquamous carcinoma (ASC) is exceedingly rare, comprising less than 0.1% of all colorectal malignancies, and is characterized by the admixture of glandular and squamous components. Due to its rarity, immunohistochemical and biological profiles have not been well investigated. The clinico-pathologic features of 29 cases of primary colorectal adenosquamous carcinomas, including four synchronous metastases, as well as the immunohistochemical expression of keratin 20, CDX2, keratin 34βE12, keratin 5/6, p63, p40, β-Catenin, Cyclin D1 and mismatch repair protein (MMR) expression in both squamous and glandular components are described. All ASCs showed aggressive clinico-pathologic features; all cases showed at least one aggressive pathologic characteristic (poorly differentiated, vascular invasion, infiltrative growth pattern) and 69% of cases were either stage III or IV. The squamous component was keratin 34βE12 positive in all cases and keratin 5/6 positive in 27 cases, while only 7 cases showed p63 and/or p40 expression. β-Catenin and Cyclin D1 showed different expression, with nuclear staining of Cyclin D1 in the squamous component of all cases (both primary and metastatic lesions) and nuclear staining of β-Catenin predominantly in the glandular component. All but one case showed proficient MMR profile. Sixteen patients (64%) died of their disease with median survival of 10 months. ASC show aggressive clinical outcome and aggressive pathologic characteristics. A peculiar keratin 34βE12 positive profile in the squamous component is seen differing from squamous cell carcinoma and non-intestinal ASC. The staining patterns for β-Catenin and Cyclin D1 between components, supports a possible divergent clonal evolution of the neoplasm.

Topics: beta Catenin; Carcinoma, Adenosquamous; Carcinoma, Squamous Cell; Colorectal Neoplasms; Cyclin D1; Humans; Keratin-5

colorectal cancer (CRC) is a common cancer with high mortality. This study aimed to investigate the role of microRNA (miR)-132-3p on proliferation, invasion and migration of CRC cells.. qRT-PCR and Western blot analyses were used to determine the expression of miR-132-3p and forking box (FOX) protein 2 (FOXP2) in CRC cell line CACO-2. The expression of miR-132-3p and FOX was regulated using miR inhibitor and siRNA, and the viability and migration ability of the transfected cells were assessed. Cell cycle dependent kinase (CDK) 1, cyclin D1, matrix metalloproteinase (MMP)-2 and MMP-9 were detected using Western blots. The dual luciferase reporter gene assays were used to verify the targeting of miR-132-3p to FOXP2.. Compared with control cells, FOXP2 and miR-132-3p expressions were decreased or increased significantly (P<0.05), respectively in CACO-2 cells. Up-regulation of miR-132-3p effectively inhibited the proliferation, migration and invasion of CACO-2 cells, and suppressed the expression of FORX2, cyclin-dependent kinase 1 (CDK1), cyclin D1, MMP-2 and MMP-9. Luciferase reporter gene assays reveled that FOXP2 expression was negatively regulated by miR-132-3p. Knockdown of FOXP2 using siRNA significantly reduced the proliferation and migration of CACO-2 cells, down-regulated the expression FOXP2 as well as CDK1, cyclin D1, MMP-2 and MMP-9. Since FOXP2 is targeted by miR-132-3p, it is likely that miR-132-3p-mediated reduction of proliferation and migration of CACO-2 cells was achieved via reduced translation of FOXP2 mRNA.. miR-132-3p inhibits the proliferation, migration and invasion of CRC cells. This is likely achieved via negative regulation of the targeted FOXP2 expression. This role may be further explored for therapeutic applications in CRC.

Topics: Caco-2 Cells; Cell Line, Tumor; Cell Movement; Cell Proliferation; Colorectal Neoplasms; Cyclin D1; Forkhead Transcription Factors; Gene Expression Regulation, Neoplastic; Humans; Luciferases; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; MicroRNAs; RNA, Small Interfering

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

The aim of this study was to analyze the effect of avidin treatment on cell viability, proliferation and cyclin D1 expression in colorectal cancer cells HT-29.. Colorectal cancer cell line HT-29 incubated with 50, 100, 150, and 200 μg/mL of avidin concentration during 24, 48, and 72 hours, then the cell viability and proliferation   were analyzed. Each avidin concentration was conducted together with HT-29 cell line without avidin treatment as a control group. The cell viability was measured by MTS assay and the proliferation was measured by BrdU (5-bromo-2'-deoxyuridine) cell proliferation assay. According to cell viability and proliferation result, we determined the 100 μg/mL avidin concentration for analyzing mRNA and protein of cyclin D1.. We demonstrated that the viability and proliferation of HT-29 cells were significantly decreased in all concentration of avidin treatment compared to control.   The cell proliferation showed larger reduction in avidin treatment rather than cell viability. This proves avidin could inhibit proliferation of colorectal cancer cell HT-29 quite well. The expression of cyclin D1, both mRNA and protein, was also significantly decreased after the avidin treatment group compared to control group, it supports the suppression of proliferation result.. We concluded that avidin treatment could decrease cell viability and proliferation, accompanied by suppression of cyclin D1 expression in colorectal cells HT-29.

Topics: Avidin; Bromodeoxyuridine; Cell Proliferation; Colorectal Neoplasms; Cyclin D1; HT29 Cells; Humans; RNA, Messenger

Metastasis is the leading cause of death in patients with colorectal cancer (CRC). The 5-year survival rate of CRC patients in whom the cancer has spread to distant sites is 13.5%. The most common sites of CRC metastasis are liver and lung. The principal therapies for CRC metastatic disease are surgery, but its benefits are limited.. This study aimed to reveal the regulatory mechanism of berberine on secondary homing of CRC cells to form metastatic focus. This was more valuable than the previous direct study of the migration and metastasis characteristics of CRC cells.. In this study, we used the functional enrichment analysis of differentially expressed genes after berberine treatment and investigated co-expression modules related with CRC metastasis by WGCNA. PPI and survival analyses of significant modules were also conducted. The biological functions of berberine in CRC lung and liver metastasis were investigated by a series of in vitro and in vivo experiments: MTT, colony formation and mouse tail vein injection. And we scanned through the entire extracellular domain of HEY2 protein for autodocking analysis with berberine.. We found the differentially expressed genes (DEGs) after berberine treatment were related with cancer progression and metastasis related pathways. Through WGCNA analysis, four cancer progression and metastasis related modules were detected. After PPI and survival analysis, we identified and validated HEY2 as a hub gene, high expression and poor survival at the metastatic stage. Functionally, berberine inhibited the survival, invasion and migration of CRC cells in vitro and in vivo. Mechanistically, berberine treatment down-regulated the expression of HEY2, metastasis related protein E-cadherin, β-catenin and Cyclin D1 during Mesenchymal epithelial transformation (MET). Berberine and HEY2 showed a significant interaction, and berberine binded to HEY2 protein at the residue HIS-99 interface with a hydrogen-bond distance of 1.9A.. We revealed that berberine could significantly inhibit the expression of hub gene HEY2 and metastasis related proteins E-cadherin and β-catenin and Cyclin D1 during MET in CRC lung and liver metastases. In total, HEY2 was a promising candidate biomarker for prognosis and molecular characteristics in CRC metastasis.

Topics: Animals; Basic Helix-Loop-Helix Transcription Factors; Berberine; beta Catenin; Cadherins; Cell Line, Tumor; Cell Movement; Colorectal Neoplasms; Cyclin D1; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Liver Neoplasms; Lung; Mice; Repressor Proteins

Although cancer immunotherapies are effective for advanced-stage cancers, there are no clinically approved immunotherapies for colon cancers (CRCs). Therefore, there is a high demand for the development of novel therapies. Extracellular adenosine-mediated signaling is considered a promising target for advanced-stage cancers that are nonresponsive to programmed death 1 (PD-1)-/programmed death-ligand 1 (PD-L1)-targeted immunotherapies. In this study, we aimed to elucidate novel tumorigenic mechanisms of extracellular adenosine.. To investigate the effects of extracellular adenosine on tumor-associated macrophages, peripheral blood-derived human macrophages were treated with adenosine and analyzed using flow cytometry and Western blot. Changes in adenosine-treated macrophages were further assessed using multi-omics analysis, including total RNA sequencing and proteomics. Colon cancer mouse models were used to measure the therapeutic efficacy of AB680 and palbociclib. We also used tissue microarrays of patients with CRC, to evaluate their clinical relevance.. Extracellular adenosine-mediated reduction of cyclin D1 (CCND1) was found to be critical for the regulation of immune checkpoint molecules and PD-L1 levels in human macrophages, indicating that post-translational modification of PD-L1 is affected by adenosine. A potent CD73 selective inhibitor, AB680, reversed the effects of adenosine on CCND1 and PD-L1. This result strongly suggests that AB680 is a combinatory therapeutic option to overcome the undesired side effects of the cyclin-dependent kinase 4/6 inhibitor, palbociclib, which increases PD-L1 expression in tumors. Because palbociclib is undergoing clinical trials for metastatic CRC in combination with cetuximab (clinical trial number: NCT03446157), we validated that the combination of AB680 and palbociclib significantly improved anti-tumor efficacy in CRC animal models, thereby highlighting it as a novel immunotherapeutic strategy. We further assessed whether the level of CCND1 in tumor-associated macrophages was indeed reduced in tumor sections obtained from patients with CRC, for evaluating the clinical relevance of this strategy.. In this study, we demonstrated that a novel combination therapy of AB680 and palbociclib may be advantageous for the treatment of CRC.

Topics: Adenosine; Animals; B7-H1 Antigen; Cetuximab; Colorectal Neoplasms; Cyclin D1; Cyclin-Dependent Kinase 4; Humans; Immune Checkpoint Proteins; Mice; Programmed Cell Death 1 Receptor

The human deafness, autosomal dominant 5 gene (DFNA5), a newly discovered executor of pyroptosis, has been strongly implicated in the tumorigenesis of several human cancers. However, an understanding of the functional role of DFNA5 in the development and progression of colorectal cancer (CRC) is limited. In this study, we demonstrated that DFNA5 was downregulated in CRC tissues. Ectopic expression of DFNA5 inhibited tumor cell growth in vitro, retarded tumor formation in vivo, and blocked a cell-cycle transition from the G0/G1 to the S phase, whereas a DFNA5 knockdown promoted cell proliferation. Western blotting showed that the levels of cell cycle-related proteins, including cyclin D1, cyclin E, CDK2, and p21, were accordingly altered upon DFNA5 overexpression or DFNA5 knockdown. Mechanistic studies indicated that DFNA5 exerted its tumor suppressor functions by antagonizing mTORC1/2 signaling via upregulation of DEPTOR. In addition, blockage of mTORC1/2 signaling by Torin-1 abolished the accelerative proliferation by DFNA5 knockdown. In conclusion, these results indicated that DFNA5 inhibits the proliferation and tumor formation of colon cancer cells by suppressing mTORC1/2 signaling.

Topics: Cell Line, Tumor; Cell Proliferation; Colonic Neoplasms; Colorectal Neoplasms; Cyclin D1; Cyclin E; Gene Expression Regulation, Neoplastic; Hearing Loss, Sensorineural; Humans; Intracellular Signaling Peptides and Proteins; Mechanistic Target of Rapamycin Complex 1; Pore Forming Cytotoxic Proteins; Signal Transduction; Up-Regulation

Colorectal cancer is related to ulcerative colitis. This study aimed to investigate the effects of aspirin on non-specific inflammation developing into cancer.. Ulcerative colitis model was generated by administrating azoxymethane/dextran sulfate sodium to mice. Weight, tumor size/ amount, and intestinal mucositis scores were analyzed. Inflammatory cell infiltration and atypical hyperplasia were determined with hematoxylin-eosin staining. Immunohistochemical assay was used to detect the proliferating cell nuclear antigen. Interleukin-6 and interleukin-10 were detected using enzyme-linked immunosorbent assay. Signal transducer and activator of transcription 3, phosphorylated-STAT3, cyclin D1, and suppressor of cytokine signaling 3 were examined with western blotting.. Aspirin remarkably decreased tumor size/amount compared to those of the ulcerative colitis model group (P < .05). Interleukin-6 was increased and interleukin-10 was decreased in mice of ulcerative colitis model group compared with the control group (P < .05). Aspirin markedly reduced interleukin-6 and enhanced interleukin-10 compared to the ulcerative colitis model group (P < .05) induced Azoxymethane/dextran sulfate sodium inflammation (3 weeks) and atypical hyperplasia (8 weeks). Aspirin predominantly inhibited the "inflammation-atypical hyperplasia-cancer" process and alleviated inflammatory cell infiltration of mice in the ulcerative colitis model group. Aspirin promoted apoptosis and alleviated proliferating cell nuclear antigen of atypical hyperplastic intestinal mucosal cells at 8 weeks post-modeling. The expression of phosphorylated-STAT3, signal transducer and activator of transcription 3, cyclin D1, and suppressor of cytokine signaling 3 was significantly increased in mice of ulcerative colitis model group compared to the control group (P < .05). Aspirin remarkably decreased phosphorylated-STAT3, signal transducer and activator of transcription, and cyclin D1 expression compared with ulcerative colitis model group (P < .05).. Aspirin inhibited carcinogenesis of intestinal mucosal cells in the ulcerative colitis model by inhibiting the interleukin-6/ Janus kinase/signal transducer and activator of transcription 3 signaling pathway and promoted apoptosis, thereby suppressing proliferation.

Topics: Animals; Apoptosis; Aspirin; Azoxymethane; Carcinogenesis; Cell Proliferation; Colitis, Ulcerative; Colorectal Neoplasms; Cyclin D1; Dextran Sulfate; Hyperplasia; Inflammation; Interleukin-10; Interleukin-6; Janus Kinases; Mice; Proliferating Cell Nuclear Antigen; Signal Transduction; STAT3 Transcription Factor

Portulaca oleracea is a known medicinal plant with antioxidant, anti-inflammatory, and anticancer activities, and it may also function an important role in colorectal cancer (CRC).. We probed into study the critical function of Portulaca oleracea extract (POE) in CRC and the related downstream factors.. Azoxymethane (AOM) and dextransodiumsulfate (DSS) were used to induce mouse models of CRC, which were then administered different doses of POE to evaluate the therapeutic effects of POE on CRC. Diversity, abundance, and function of gut microbiota were analyzed. Moreover, the potential molecular targets of POE inhibiting CRC development were determined. Expression of c-Myc and cyclin D1 as well as CRC cell proliferation and apoptosis was detected.. POE treatment inhibited AOM/DSS-induced CRC development in mice and ameliorated gut microbial imbalance. Bioinformatic analysis revealed marked differences in the gut microbiota between CRC samples and normal samples and that 20 differential microbiota may be involved in CRC development through the Wnt signaling pathway. Additionally, c-Myc and cyclin D1 were identified to be the key downstream target genes of the Wnt/β-catenin signaling pathway. In vitro data revealed that POE played a suppressive role in the proliferation of CRC cells by reducing the expression of c-Myc and cyclin D1 and inactivating the Wnt/β-catenin signaling pathway.. This study underlines that POE reduces gut microbiota imbalance and inhibits CRC development and progression via inactivation of the Wnt/β-catenin signaling pathway and downregulation of c-Myc and cyclin D1 expression, which is expected to be a potential biomarker for CRC.

Topics: Animals; Azoxymethane; beta Catenin; Cell Line, Tumor; Cell Proliferation; Colorectal Neoplasms; Cyclin D1; Gastrointestinal Microbiome; Gene Expression Regulation, Neoplastic; Mice; Portulaca; Wnt Signaling Pathway

Exosomes derived from mesenchymal stem cells (MSCs) are mostly responsible for the therapeutic effects of MSCs. To show the therapeutic effects of the human bone marrow MSC-derived exosomes (MSC-Exos) on colorectal cancer (CRC) and explore the molecular cross-talks between them, CRC cells were treated with the MSC-Exos. We found that MSC-Exos were enriched with miR-100 and miR-143, which effectively downregulated mTOR, Cyclin D1, K-RAS, HK2 while upregulated p-27 expression. All these effects were reversed by concurrent treatment with MSC-Exos and antagomiR-100, confirming that they were caused by exosomal transfer of miR-100 into recipient CRC cells. Moreover, exosomal miR-100 promoted endogenous miR-143 expression. The flow cytometry, MTT and trypan blue assays revealed that MSC-Exos could efficiently suppress proliferation and induce apoptosis of the CRC cells. Furthermore, wound healing, transwell migration and invasion assays confirmed their inhibitory effects on the migration and invasiveness of SW480 cells. We further confirmed these effects by analyzing the expression levels of epithelial to mesenchymal transition (EMT) factors and metastasis-related genes. Results showed that MSC-Exos significantly suppressed the expression of MMP2 and MMP9 (metastasis-related genes), SNAIL and TWIST (EMT-inducing transcription factors), Vimentin and N-cadherin (mesenchymal cell markers), whereas E-cadherin (epithelial cell marker) was remarkably up-regulated. Collectively, our data indicated that MSC-Exos could suppress proliferation, migration, invasion and metastasis while inducing the apoptosis of the CRC cells via miR-100/mTOR/miR-143 axis. Our findings highlight that MSC-Exo treatment as well as miR-100 restoration might be considered as potential therapeutic strategies for CRC.

Topics: Antagomirs; Cadherins; Cell Movement; Cell Proliferation; Colorectal Neoplasms; Cyclin D1; Epithelial-Mesenchymal Transition; Exosomes; Humans; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; MicroRNAs; TOR Serine-Threonine Kinases; Transcription Factors; Trypan Blue; Vimentin

Long non-coding RNAs modulate tumor occurrence through different molecular mechanisms. It had been reported that HNF1A-AS1 (HNF1A Antisense RNA 1) was differently expressed in multiple tumors. The role of HNF1A-AS1 in colorectal cancer was less analyzed, and the mechanism of regulating the cell cycle has not been completely elucidated.. Differentially expressed lncRNAs were screened out from the TCGA database. HNF1A-AS1 was examined in CRC clinical samples and cell lines by RT-qPCR. CCK8 assay, colony formation assay, flow cytometry, transwell assays, tube forming assay and vivo experiments were performed to study the function of HNF1A-AS1 in CRC tumor progression. Bioinformatic analysis, luciferase report assay, RNA pull-down and RIP assays were carried out to explore proteins binding HNF1A-AS1 and the potential downstream targets.

Topics: Apoptosis Regulatory Proteins; Cell Cycle; Cell Line, Tumor; Cell Movement; Cell Proliferation; Colorectal Neoplasms; Cyclin D1; Gene Expression Regulation, Neoplastic; Hepatocyte Nuclear Factor 1-alpha; Humans; Methyltransferases; MicroRNAs; RNA-Binding Proteins; RNA, Long Noncoding; RNA, Messenger

Engulfment and cell motility 1 (ELMO1) plays a crucial role in the process of migration, chemotaxis, and metastasis of tumor cells. ELMO1 has been implicated in the pathogenesis of various cancers. However, the distinct function of ELMO1 in colorectal cancer (CRC) is unclear. We determined whether ELMO1 affects the oncogenic behavior of CRC cells and investigated its prognostic value in CRC patients.. We investigated the impact of ELMO1 on tumor cell behavior using small interference RNA (siRNA) in CRC cell lines, including SW480 and DLD1. The expression of ELMO1 was investigated by reverse transcription-polymerase chain reaction (RT-PCR), immunohistochemistry, and enzyme-linked immunosorbent assay (ELISA) in cancer tissues and sera obtained from CRC patients.. ELMO1 knockdown suppressed tumor cell proliferation in SW480 and DLD1 cells. ELMO1 knockdown-induced apoptosis through up-regulation of caspase-3, -7, and PARP activities and down-regulation of the anti-apoptotic Mcl-1 protein. ELMO1 knockdown-induced cell-cycle arrest by decreasing cyclin D1, cyclin-dependent kinase 2, 4 and 6, and the 25C cell division cycle (CDC25C). ELMO1 knockdown suppressed tumor cell invasion and migration. The expression of E-cadherin was increased, while that of Vimentin and Claudin 1 decreased following ELMO1 knockdown. The phosphorylation levels of PDK1, Akt, and GSK-3β and were down-regulated after ELMO1 knockdown. The expression of ELMO1 was found up-regulated in cancer tissues and sera taken from CRC patients. ELMO1 expression was significantly associated with tumor stage, lymph node metastasis, distant metastases, and poor survival.. ELMO1 mediates tumor progression by increasing tumor cell motility and inhibiting apoptosis in human CRC.

Topics: Cadherins; Caspase 3; Cell Line, Tumor; Cell Movement; Cell Proliferation; Claudin-1; Colorectal Neoplasms; Cyclin D1; Cyclin-Dependent Kinase 2; Gene Expression Regulation, Neoplastic; Glycogen Synthase Kinase 3 beta; Humans; Myeloid Cell Leukemia Sequence 1 Protein; Poly(ADP-ribose) Polymerase Inhibitors; Prognosis; Proto-Oncogene Proteins c-akt; RNA, Small Interfering; Vimentin

CXCR4 plays an important role in colorectal cancer (CRC) development and metastasis. Some previous studies have indicated CXCR4 as a therapeutic target in cancer. CXCR4 is known as a direct target of miR-146a. The present study aimed to investigate how exogenous induction of miR-146a affects CXCR4 gene and protein expression and also proliferation, apoptosis and migration of CRC cells. Transfection of Caco-2 and SW480 cells by a synthetic miR-146a mimic led to downregulation of CXCR4 expression at both gene and protein levels. It also downregulated expression of several miR-146a targets, including GSK3B, IRAK1, TRAF6, AKT2, SMAD4, EGFR and NFKB1, mostly in SW480 cells. Overexpression of miR-146a resulted in a partial cell cycle arrest in the both cell lines, while the apoptotic rate was also decreased. In regards to epithelial-mesenchymal transition factors, VIM was downregulated in the both cell lines, but SNAI1 was upregulated in Caco-2 cells. The wound closure assay showed a reduction in cell migration in SW480 cells, but an opposite effect was detected in Caco-2 cells following transfection with miR-146a mimic. Therefore, our results are indicating that overexpression of miR-146a, despite downregulation of oncogenic CXCR4, may not lead to a universal tumor suppressive effect in all CRC cells, and this is possibly due to differences in miR-146a effects on signaling pathways in each cell type. Selection of miR-146a for tumor suppression requires enough details regarding the signaling profile of cancer cells otherwise it may produce unexpected outcome.

Topics: Apoptosis; Cell Cycle; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cell Survival; Colorectal Neoplasms; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Down-Regulation; Epithelial-Mesenchymal Transition; ErbB Receptors; Gene Expression Regulation, Neoplastic; Humans; MicroRNAs; NF-kappa B; Receptors, CXCR4; RNA, Messenger; Signal Transduction; Toll-Like Receptors; Wound Healing

The microRNA miR-130a-3p (miR-130a-3p) has anti-tumor activity against numerous cancer types. Further, miR-130a-3p may target Wnt signaling, which is a critical pathway regulating tumorigenesis. Functions of miR-130a-3p in colorectal cancer (CRC) and contributions of Wnt1 pathway modulation, however, have not been examined, hence the exploration on these two aspects. In this study, in comparison with normal controls, both CRC tissue and multiple CRC cell lines showed downregulated miR-130a-3p. MiR-130a-3p overexpression contributed to a decrease in CRC cell proliferation. Additionally, its overexpression also caused reduced expression of WNT Family Member 1 (WNT1) and downstream WNT pathway factors c-myc and cyclin D1. Dual-luciferase assay revealed WNT1 as a direct target of miR-130a-3p, and further the inhibitory effect of miR-130a-3p on c-myc and cyclin D1 was proved to be reversed by overexpressed WNT1. Collectively, miR-130a-3p inhibits CRC growth by directly targeting WNT1, and miR-130a-3p and WNT1 pathway-associated factors are defined as potential targets for CRC treatment.

Topics: 3' Untranslated Regions; Animals; Cell Line, Tumor; Cell Movement; Cell Proliferation; Colorectal Neoplasms; Cyclin D1; Down-Regulation; Female; Gene Expression Regulation, Neoplastic; HCT116 Cells; Humans; Mice; MicroRNAs; Neoplasm Transplantation; Proto-Oncogene Proteins c-myc; Wnt1 Protein

Topics: Anticarcinogenic Agents; Antiviral Agents; Atlases as Topic; Colorectal Neoplasms; COVID-19; COVID-19 Drug Treatment; Cyclin D1; ErbB Receptors; Gene Expression Regulation, Neoplastic; Genistein; Humans; Interleukin-1alpha; Interleukin-2; Janus Kinases; Metabolic Networks and Pathways; Molecular Targeted Therapy; Multigene Family; Network Pharmacology; Pharmacogenetics; Phosphatidylinositol 3-Kinases; Phytoestrogens; PPAR gamma; Proto-Oncogene Proteins c-akt; Receptors, Interleukin-6; SARS-CoV-2; Signal Transduction; STAT Transcription Factors

Mitochondria-localized sirtuin 4 (SIRT4) is associated with malignant phenotypes in colorectal cancer (CRC). However, the molecular mechanisms that drive SIRT4-mediated carcinogenesis are unclear. Initially, we confirmed expression of SIRT4 in CRC through public database and in CRC patient tissues using quantitative real-time reverse transcription PCR. We established HCT116 colorectal cells that overexpressed SIRT4 and HT29 cells were transfected with plasmids bearing a small interfering RNA construct to silence SIRT4. Assays to determine the malignant phenotypes (proliferation, invasion and migration) were performed. Xenograft in vivo models were also constructed. A protein interactome network was built using differentially expressed proteins identified using the liquid chromatography/tandem mass spectrophotometry, the findings of which were confirmed using co-immunoprecipitation, western blotting and phenotype rescue experiments. Decreased SIRT4 expression was associated with malignant phenotypes in vitro and in vivo. The ribosomal biogenesis pathway was enriched in the interactome network. SIRT4 suppression activated glutaminase, thereby initiating AKT activation. Our research provided novel insights into the molecular mechanisms underlying CRC, and identified that SIRT4 exerts its antitumor activity in CRC possibly dependent on glutaminase to inhibit proliferation, migration and invasion via the AKT/GSK3β/CyclinD1 pathway.

Topics: Animals; Carcinogenesis; Cell Movement; Cell Proliferation; Colectomy; Colon; Colorectal Neoplasms; Cyclin D1; Female; Gene Knockdown Techniques; Glutaminase; Glycogen Synthase Kinase 3 beta; HCT116 Cells; HT29 Cells; Humans; Mice; Mitochondrial Proteins; Neoplasm Invasiveness; Protein Interaction Mapping; Protein Interaction Maps; Proto-Oncogene Proteins c-akt; Signal Transduction; Sirtuins; Tumor Suppressor Proteins; Xenograft Model Antitumor Assays

Sesamin, a lignan compound, exhibits a variety of biological activities and possesses potent anticancer properties on some human cancers. However, its effect on human colorectal cancer (CRC) remains to be elucidated. To investigate the effects of sesamin on CRC cells and further to explore the mechanisms, cell viability, cell cycle and apoptosis assays were performed in this study. We found that sesamin had a selective antiproliferation of CRC cell line HCT116 in a dose- and time-dependent manner, but no obvious effect on human normal colorectal mucosa epithelial cell FHC. Further study showed that sesamin-induced cell cycle arrest and decreased the expression of Cyclin D1 significantly and dose-dependently in HCT116 cells. Moreover, sesamin dose-dependently triggered apoptosis of HCT116 but not FHC, and promoted the expression levels of proapoptotic biomarkers Bax, cleaved caspase-3 and cleaved PARP-1 and inhibited the expression of antiapoptotic biomarker Bcl-2. Western blot analysis was used to reveal the possible signaling pathways, and we found that sesamin upregulated the phosphorylation expression levels of C-Jun N-terminal kinase (JNK) and p38 except ERK1/2 in a dose-dependent way in both HCT116 and another CRC cell line SW480. Moreover, we found that the apoptosis effect induced by sesamin was partially eliminated by inhibiting JNK or p38 activation. Finally, we showed that sesamin effectively reduced the growth of xenograft tumors derived from cell lines with limited toxicity. Taken together, the potential ability of sesamin to induce cell cycle arrest and apoptosis was shown to be via the p38 and JNK mitogen-activated protein kinase signaling pathways, which may be one of the mechanisms of the anticancer activity of this low-toxic agent.

Topics: Animals; Antioxidants; Apoptosis; Cell Cycle; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Survival; Colorectal Neoplasms; Cyclin D1; Dioxoles; Dose-Response Relationship, Drug; Humans; JNK Mitogen-Activated Protein Kinases; Lignans; Mice; Mice, Inbred BALB C; p38 Mitogen-Activated Protein Kinases; Signal Transduction; Time Factors; Up-Regulation

Anticancer bioactive peptide (ACBP), a novel bioactive peptide isolated from spleens of goats immunized with tumor extracts in our lab, can inhibit the proliferation of CRC in vitro and vivo. However, it remains unclear how the proliferation of CRC is inhibited by ACBP at the molecular level. Here, we provide evidences showing that ACBP significantly inhibits the expression of Wnt/β-catenin related genes (cyclin D1, met and c-myc) through pharmacotranscriptomic and qRT-PCR analysis in CRCs. Active β-catenin, a key protein within Wnt pathway, was compromised remarkably by ACBP in three CRCs, including HCT116, RKO and HT29. Thus nuclear accumulation of active β-catenin was retarded and finally lead to the decreased expression of oncogenes cyclin D1, met, and c-myc. In addition, we proved that active β-catenin reduction was mainly due to the inhibition of phospho-LRP6 and stimulation of phospho-β-catenin by ACBP. Based on the detection of Met and C-Myc in CRC tumor tissue without prior radiotherapy or chemotherapy, our results demonstrated that ACBP can act as a promising anticancer agent for CRC by targeting Wnt/β-catenin pathway, especially active β-catenin.

Topics: Antineoplastic Agents; beta Catenin; Cell Movement; Cell Proliferation; Colorectal Neoplasms; Cyclin D1; Gene Expression Regulation, Neoplastic; HCT116 Cells; HT29 Cells; Humans; Low Density Lipoprotein Receptor-Related Protein-6; Neoplasm Invasiveness; Peptides; Phosphorylation; Proto-Oncogene Proteins c-met; Proto-Oncogene Proteins c-myc; Wnt Signaling Pathway

A xanthophyll of fucoxanthin (Fx) is a potential chemopreventive agent. Familial adenomatous polyposis (FAP) is an inherited disease that is associated with a high risk of developing colorectal cancer. However, it remains unclear whether Fx can modify colorectal tumorigenesis in Apc. We investigated the chemopreventive effect of Fx in dextran sodium sulfate (DSS)-treated Apc. Administration of Fx in the diet for 5 weeks significantly suppressed the number of colorectal adenocarcinomas in DSS-treated male Apc. Fx possesses chemopreventive potential against progression of colorectal carcinogenesis in Apc

Topics: Adenomatous Polyposis Coli; Animals; Anticarcinogenic Agents; Colorectal Neoplasms; Cyclin D1; Dextran Sulfate; Disease Models, Animal; Male; Mice; Xanthophylls

The crypt-villus axis represents the essential unit of the small intestine, which integrity and functions are fundamental to assure tissue and whole-body homeostasis. Disruption of pathways regulating the fine balance between proliferation and differentiation results in diseases development. Nowadays, it is well established that microRNAs (miRNAs) play a crucial role in the homeostasis maintenance and perturbation of their levels may promote tumor development. Here, by using microarray technology, we analysed the miRNAs differentially expressed between the crypt and the villus in mice ileum. The emerged miRNAs were further validated by Real Time qPCR in mouse model (ApcMin/+), human cell lines and human tissue samples (FAP) of colorectal cancer (CRC). Our results indicated that miRNAs more expressed in the villi compartment are negatively regulated in tumor specimens, thus suggesting a close association between these microRNAs and the differentiation process. Particularly, from our analysis let-7e appeared to be a promising target for possible future therapies and a valuable marker for tumor staging, being upregulated in differentiated cells and downregulated in early-stage colonic adenoma samples.

Topics: Adenoma; Adenomatous Polyposis Coli; Adenomatous Polyposis Coli Protein; Animals; Cell Line, Tumor; Colorectal Neoplasms; Cyclin D1; Down-Regulation; Humans; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; MicroRNAs; Proto-Oncogene Proteins c-myc

5-Fluorouracil (5-FU) resistance is an urgent problem of colorectal cancer (CRC) chemotherapy that needs to be resolved. To investigate 5-FU-associated lncRNAs for CRC might be of great significant. LncRNA ENSG00000254615 was detected by RNA-sequencing. ENSG00000254615 were detected highly expressed in 5-FU-sensitive CRC cells and tissue specimens, and inhibited cell proliferation and attenuated 5-FU resistance

Topics: Animals; Antimetabolites, Antineoplastic; Cell Line, Tumor; Cell Proliferation; Colorectal Neoplasms; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Drug Resistance, Neoplasm; Fluorouracil; Gene Expression Regulation, Neoplastic; HCT116 Cells; HEK293 Cells; Humans; Mice, Inbred BALB C; Mice, Nude; RNA, Long Noncoding; Tumor Burden; Xenograft Model Antitumor Assays

Long noncoding RNA (lncRNA) CDKN2B‑antisense RNA 1 (AS1) functions as a tumor oncogene in numerous cancers. However, the roles and mechanism of CDKN2B‑AS1 in colorectal cancer (CRC) have not been explored. The present study aimed to investigate whether and how CDKN2B‑AS1 contributes to CRC progression. The data revealed that CDKN2B‑AS1 expression was upregulated in CRC tissues. Loss‑of‑function assays demonstrated that CDKN2B‑AS1 in CRC modulated cell proliferation and apoptosis, which was mediated by cyclin D1, cyclin‑dependent kinase (CDK) 4, p‑Rb, caspase‑9 and caspase‑3. Bioinformatics analysis and luciferase reporter assays indicated direct binding of microRNA (miR)‑28‑5p to CDKN2B‑AS1. Moreover, the results herein revealed that the expression of miR‑28‑5p was negatively correlated with that of CDKN2B‑AS1 in CRC tissue. Moreover, CDKN2B‑AS1 acted as a miR‑28‑5p competing endogenous RNA (ceRNA) to target and regulate the expression of URGCP. These findings indicated that CDKN2B‑AS1 plays roles in CRC progression, providing a potential therapeutic target or novel diagnostic biomarker for CRC.

Topics: Adult; Aged; Apoptosis; Caspases; Cell Line, Tumor; Cell Proliferation; Colorectal Neoplasms; Cyclin D1; Disease Progression; Female; Gene Expression Regulation, Neoplastic; Humans; Loss of Function Mutation; Male; MicroRNAs; Middle Aged; RNA, Long Noncoding; Up-Regulation

MicroRNA (miR)‑628‑5p serves as an antitumor gene in a variety of cancers; however, the role of miR‑628‑5p in colorectal cancer remains largely unclear. The purpose of this study was to investigate the role and mechanism of miR‑628‑5p in colorectal cancer. Reverse transcription‑quantitative PCR (RT‑qPCR), colony formation assays and flow cytometric analysis were used to determine the expression levels of miR‑628‑5p in colorectal cancer tissues and cell lines, and the proliferative ability of colorectal cancer cells. TargetScan version 7.2 and dual‑luciferase reporter assay were performed to predict and confirm miR‑628‑5p target genes. The expression levels of cyclin D1 (CCND1) and related genes were determined using RT‑qPCR or/and western blotting analysis. miR‑628‑5p mimics and CCND1 plasmids were used to overexpress miR‑628‑5p and CCND1; it was demonstrated that the expression levels of miR‑628‑5p were significantly downregulated in colorectal cancer tissues and cell lines. miR‑628‑5p mimic‑transfected cells inhibited the proliferation and induced apoptosis of HT‑29 cells. CCND1, a downstream effector of miR‑628‑5p, promoted the proliferation and suppressed apoptosis of HT‑29 cells, and the effects were reversed by miR‑628‑5p mimics. In conclusion, the present study suggested that colorectal cancer progression may be regulated through the miR‑628‑5p/CCND1 axis, and miR‑628‑5p could be used as a potential diagnostic and prognostic biomarker for colorectal cancer.

Topics: 3' Untranslated Regions; Apoptosis; Cell Line, Tumor; Cell Proliferation; Colorectal Neoplasms; Cyclin D1; Gene Expression Regulation, Neoplastic; Humans; MicroRNAs; RNA Interference

CircPRTM5 is associated with cell proliferation and migration in many kinds of malignancies. However, the functions and mechanisms of CircPRTM5 in CRC progression remain unclear. We explored the role and the mechanisms of CircPRTM5 in the development of CRC. Tissues of CRC patients and matched adjacent non-tumour tissues were collected to evaluate the expression of CircPRTM5. The expression of CircPRTM5 in CRC tissues was significantly higher than that in adjacent tissues. The biological functions of CircPRTM5 in CRC were determined by overexpression and down-regulation of CircPRTM5 in CRC cells in vitro and in vivo. The results indicate that knockdown of CircPRTM5 can significantly inhibit the proliferation of CRC cells. The potential mechanisms of CircPRTM5 in CRC development were identified by RT-qPCR, Western blotting analysis and luciferase reporter assay. CircPRTM5 competitively regulates the expression of E2F3 by capillary adsorption of miR-377. CircPRMT5 regulates CRC proliferation by regulating the expression of E2F3, which affects the expression of the cell cycle-associated proteins cyclinD1 and CDK2. CircPRTM5 exerts critical regulatory role in CRC progression by sponging miR-377 to induce E2F3 expression.

Topics: Animals; Cell Line, Tumor; Cell Movement; Cell Proliferation; Colorectal Neoplasms; Cyclin D1; Cyclin-Dependent Kinase 2; E2F3 Transcription Factor; Female; Gene Expression Regulation, Neoplastic; HCT116 Cells; HT29 Cells; Humans; Male; Mice; Mice, Inbred BALB C; Mice, Nude; MicroRNAs; Middle Aged; RNA, Circular

Colorectal cancer (CRC) is one of the most frequent neoplasms worldwide, and up to 15% have a family history. Lynch syndrome (LS) is a hereditary cause of CRC and gastric (GC). Individuals with LS have mutations in mismatch genes repair. p53, cyclin D1, β-catenin, APC and c-myc proteins are involved in the cell cycle and carcinogenesis.. To study the expression of p53, Cyclin D1, β-catenin, APC and c-myc proteins in patients with CRC and GC with at least one of the Bethesda positive criteria. Compare the expression of these proteins with the presence or absence of expression of the DNA repair proteins.. We included 70 individuals with CRC or GC with at least one of the Bethesda positive criteria. Protein expression of MLH1, MSH2, MSH6, PMS2, p53, cyclin D1, β-catenin, APC and c-myc were analized by immunohistochemistry tumours tissues.. Deficient expression of MLH1, MSH2, MSH6 and PMS2 were respectively 38.7%; 17.7%; 26.22% and 48.38%. We found a negative association between deficiency of PMS2 and age, and positive association between PMS2 deficiency and APC positive. The positive imunoexpression of APC increases by 4 times the chance of having deficiency of PMS2.. Patients with loss of expression of PMS2 had a higher risk of mutation or deletion of APC and tumours with positive immunoexpression of cyclin D1 had an increased risk of loss of expression of MSH2. These results suggest that tumours with loss of expression of DNA repair proteins had a higher loss of cell control cycle.
.

Topics: Adenomatous Polyposis Coli Protein; beta Catenin; Biomarkers, Tumor; Brazil; Colorectal Neoplasms; Colorectal Neoplasms, Hereditary Nonpolyposis; Cyclin D1; DNA Repair Enzymes; Female; Follow-Up Studies; Humans; Male; Middle Aged; Prognosis; Proto-Oncogene Proteins c-myc; Retrospective Studies; Stomach Neoplasms; Tumor Suppressor Protein p53

Emerging evidence indicates that DJ-1 is highly expressed in different cancers. It modulates cancer progression, including cell proliferation, cell apoptosis, invasion, and metastasis. However, its role in colorectal cancer (CRC) remains poorly defined. The current study noted increased DJ-1 expression in CRC tumor tissue and found that its expression was closely related to clinical-pathological features. Similarly, DJ-1 increased in CRC cells (SW480, HT-29, Caco-2, LoVo, HCT116, and SW620), and especially in SW480 and HCT116 cells. Functional analyses indicated that overexpression of DJ-1 promoted CRC cell invasion, migration, and proliferation in vitro and in vivo. Mechanistic studies indicated that DJ-1 increased in CRC cell lines, activated specific protein cyclin-D1, and modulated the MDM2/p53 signaling pathway by regulating the levels of the downstream factors Bax, Caspase-3, and Bcl-2, which are related to the cell cycle and apoptosis. Conversely, knockdown of DJ-1 upregulated p53 expression by disrupting the interaction between p53 and MDM2 and inhibiting CRC cell proliferation, revealing the pro-oncogenic mechanism of DJ-1 in CRC. In conclusion, the current findings provide compelling evidence that DJ-1 might be a promoter of CRC cell invasion, proliferation, and migration via the cyclin-D1/MDM2-p53 signaling pathway. Findings also suggest its potential role as a postoperative adjuvant therapy for patients with CRC.

Topics: Aged; Antineoplastic Agents; Apoptosis; Cell Line, Tumor; Cell Movement; Cell Proliferation; Chemotherapy, Adjuvant; Colectomy; Colon; Colorectal Neoplasms; Cyclin D1; Female; Gene Knockdown Techniques; Humans; Kaplan-Meier Estimate; Male; Middle Aged; Neoplasm Invasiveness; Protein Deglycase DJ-1; Proto-Oncogene Proteins c-mdm2; Signal Transduction; Tumor Suppressor Protein p53; Up-Regulation; Xenograft Model Antitumor Assays

Topics: Animals; Azoxymethane; Carcinogenesis; Caspase 3; Colon; Colonic Neoplasms; Colorectal Neoplasms; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Cyclooxygenase 2; Cytokines; Dextran Sulfate; Dietary Carbohydrates; Disease Models, Animal; Edible Grain; Interleukin-1beta; Interleukin-6; Male; Mice; Mice, Inbred C57BL; Nitric Oxide Synthase Type II; RNA, Messenger; Tumor Necrosis Factors; Tumor Suppressor Protein p53

Solanum nigrum L. (Longkui) is one the most widely used anticancer herbs in traditional Chinese medicine. α‑Solanine is an important ingredient of S. nigrum L. and has demonstrated anticancer properties in various types of cancer. However, the effects of α‑solanine on colorectal cancer remain elusive. The aim of the present study was to assess the effects of α‑solanine on human colorectal cancer cells. The results demonstrated that α‑solanine inhibited the proliferation of RKO cells in a dose‑ and time‑dependent manner. In addition, α‑solanine arrested the cell cycle at the G0/G1 phase and suppressed the expression levels of cyclin D1 and cyclin‑dependent kinase 2 in RKO cells. α‑Solanine induced apoptosis of RKO cells, as indicated by morphological changes and positive Annexin‑FITC/propidium iodide staining. Additionally, α‑solanine activated caspase‑3, ‑8 and ‑9 in RKO cells, which contributed to α‑solanine‑induced apoptosis. α‑Solanine also increased the generation of reactive oxygen species, which contributed to caspase activation and induction of apoptosis. α‑Solanine inhibited the migration, invasion and adhesion of RKO cells, as well as the expression levels and activity of matrix metalloproteinase (MMP)‑2 and MMP‑9. In addition, α‑solanine inhibited cell proliferation, activated caspase‑3, ‑8 and ‑9, induced apoptosis, and inhibited the migration and invasion of HCT‑116 cells. Furthermore, α‑solanine inhibited tumor growth and induced apoptosis in vivo. These findings demonstrated that α‑solanine effectively suppressed the growth and metastatic potential of human colorectal cancer.

Topics: Animals; Antineoplastic Agents; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Movement; Cell Proliferation; Colorectal Neoplasms; Cyclin D1; Cyclin-Dependent Kinase 2; Dose-Response Relationship, Drug; Down-Regulation; Gene Expression Regulation, Neoplastic; HCT116 Cells; Humans; Male; Mice; Neoplasm Metastasis; Solanine; Time Factors; Xenograft Model Antitumor Assays

Circular RNAs (circRNAs), non-coding RNAs generated by precursor mRNA back-splicing of exons, have been reported to fulfill multiple roles in cancer. However, the role of quite a lot circRNAs in colorectal cancer (CRC) remains mostly unknown. Herein, we explored the expression profiles of circRNAs in 5 paired samples of CRC patients by microarray and noted a circRNA, hsa_circ_0005615 (circ5615), was significantly upregulated in CRC tissues. Circ5615 was derived from exon 2 of NFATC3 and its upregulation was tightly correlated with higher T stage and poor prognosis in CRC patients. Studies in vitro and in vivo demonstrated that knockdown of circ5615 in cancer cells inhibited proliferation and cell cycle acceleration, while overexpression promoted malignant phenotypes. Mechanistically, RNA immunoprecipitation, biotin-coupled probe pull-down and luciferase reporter assays revealed circ5615 effectively bound to miR-149-5p and might play a role like miR-149-5p sponge. Additionally, tankyrase (TNKS), regulator of β-catenin stabilization, was identified as circ5615 downstream and the potential miR-149-5p targets by RNA-seq and bioinformatics analysis. We further verified the upregulation of β-catenin and cyclin D1 induced by circ5615. Our results indicated that circ5615 exerted oncogenic function as competing endogenous RNA (ceRNA) of miR-149-5p to release TNKS and activated Wnt/β-catenin pathway.

Topics: Animals; beta Catenin; Cell Cycle; Cell Proliferation; Colorectal Neoplasms; Cyclin D1; Disease Progression; Gene Expression Regulation, Neoplastic; HCT116 Cells; HT29 Cells; Humans; Male; Mice, Inbred BALB C; Mice, Nude; MicroRNAs; RNA, Circular; Tankyrases; Tumor Burden; Up-Regulation; Wnt Signaling Pathway

Proton pump inhibitors (PPIs) are administered commonly to aged people; however, their effect on colorectal cancer (CRC) has still not been fully elucidated. Here, we examined the effect of PPIs and consequent alkalization on CRC cells. PPI administration alkalized the fecal pH and increased serum gastrin concentration. PPI and pH8 treatment (alkalization) of CMT93 mouse colon cancer cells inhibited cell growth and invasion, increased oxidative stress and apoptosis, and decreased mitochondrial volume and protein levels of cyclin D1 and phosphorylated extracellular signal-regulated kinase (pERK) 1/2. In contrast, gastrin treatment enhanced growth and invasion, decreased oxidative stress and apoptosis, and increased mitochondrial volume and cyclin D1 and pERK1/2 levels. Concurrent treatment with a PPI, pH8, and gastrin increased aldehyde dehydrogenase activity and also enhanced liver metastasis in the BALB/c strain of mice. PPI administration was associated with

Topics: Animals; Apoptosis; Cell Line, Tumor; Cell Proliferation; Clostridium perfringens; Colorectal Neoplasms; Cyclin D1; Enterotoxins; Extracellular Signal-Regulated MAP Kinases; Feces; Gastrins; Hydrogen-Ion Concentration; Liver Neoplasms; Male; Mice; Mice, Inbred BALB C; Mitochondria; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasm Transplantation; Oxidative Stress; Proton Pump Inhibitors

Paeonol is a simple phenolic compound isolated from herbal root bark, which has been reported to possess numerous biological and pharmacological characteristics, including a desirable anti‑tumor effect. To date, the effect of paeonol against colorectal cancer (CRC) cells is yet to be fully elucidated. Therefore, the present study aimed to identify the underlying mechanism via which paeonol exerts its anti‑tumor activity on HCT116 cells. After incubation with various concentrations of paeonol (7.8125, 15.625, 31.25, 62.5, 125, 250 and 500 µg/ml), the inhibitory effect of paeonol on cell viability was assessed using a Cell Counting Kit‑8 assay. Cell apoptosis and cell cycle distribution were measured using flow cytometry. Moreover, caspase activity was measured using a colorimetric caspase assay. Luciferase assay was also used to determine the β‑catenin‑mediated transcriptional activity of T‑cell specific transcription factor/lymphoid‑enhancer binding factor (TCF/LEF), and western blotting analysis was performed to measure the related expression of proteins. The results indicated that paeonol exhibited a notable effect against HCT116 cells by inducing G0/G1‑phase arrest, as demonstrated by downregulation of the cell cycle regulators cyclin‑dependent kinase 4 and cyclin D1 and upregulation of p21Cip1 in a dose‑dependent manner. Furthermore, paeonol dose‑dependently induced cell apoptosis, accompanied by an increase in the Bax/Bcl‑2 ratio, release of cytochrome c and further activation of caspases. Paeonol also dose‑dependently blocked the activation of the Wnt/β‑catenin signaling pathway by suppressing the expression of β‑catenin, resulting in a decrease in β‑catenin‑mediated activity of TCF/LEF and downregulation of downstream target genes, including cyclin D1, survivin and c‑Myc. Therefore, the present results suggested that paeonol exerted its anti‑tumor effects on CRC cells, including the inhibition of cell proliferation, induction of cell cycle arrest and initiation of apoptosis, at least partly by suppressing the Wnt/β‑catenin pathway, which may offer a promising therapeutic strategy for CRC.

Topics: Acetophenones; Apoptosis; Blotting, Western; Cell Cycle; Cell Cycle Checkpoints; Cell Proliferation; Colorectal Neoplasms; Cyclin D1; Cyclin-Dependent Kinase 4; G1 Phase; HCT116 Cells; Humans; Resting Phase, Cell Cycle; Survivin; Wnt Signaling Pathway

Colorectal cancer (CRC) is one of the most common malignancies in the world. It has been reported that the abnormal expression of long noncoding RNA HOXD-AS1 promotes the development of CRC, while the mechanism is still unclear. The aim of this study is to investigate the effects of HOXD-AS1 on proliferation, migration, and invasion in CRC and explore the underlying mechanism.. Quantitative real-time polymerase chain reaction was used to detect the expression levels of HOXD-AS1, miR-526b-3p, and cyclin D1 (CCND1) in CRC tissues and cells. Dual-luciferase reporter assay was applied to examine the interaction between miR-526b-3p and HOXD-AS1 or CCND1. In addition, cell proliferation ability was assessed by Cell Counting Kit-8 assay. Cell migration and invasion abilities were determined using transwell assay. Furthermore, Western blot assay was conducted to measure the protein expression of CCND1.. HOXD-AS1 was highly expressed in CRC, and high expression of HOXD-AS1 was related to the poor prognosis of patients with CRC. MiR-526b-3p could be targeted by HOXD-AS1. Function experiment results revealed that miR-526b-3p inhibitor could reverse the suppressive effect of HOXD-AS1 knockdown on the proliferation, migration, and invasion of CRC cells. Moreover, CCND1 was a target of miR-526b-3p, and its overexpression could reverse the inhibitory effect of miR-526b-3p overexpression on the proliferation, migration, and invasion of CRC cells. In addition, CCND1 overexpression reversed the suppressive effect of HOXD-AS1 knockdown on the proliferation, migration, and invasion of CRC.. HOXD-AS1 upregulated the expression of CCND1 to promote the proliferation, migration, and invasion of CRC through targeting miR-526b-3p. This provided a new theoretical basis for clinical anticancer research of CRC.

Topics: Cell Line, Tumor; Cell Movement; Cell Proliferation; Colorectal Neoplasms; Cyclin D1; Female; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Humans; Male; MicroRNAs; Middle Aged; Neoplasm Invasiveness; RNA, Long Noncoding

Rab31, a Rab5 subfamily member, has emerged as a modulator of membrane trafficking. Our study serves to clarify the role and mechanism of Rab31 in colorectal carcinoma (CRC) pathogenesis.. The differential expression of Rab31 was examined in paired normal and cancerous colonic tissues by quantitative PCR, western blot and immunochemistry. The prognostic significance of Rab31 was analysed by univariate and multivariate survival analyses. We also investigated the effects of Rab31 on tumour growth in vitro.. We observed that Rab31, which is related to histological differentiation in CRC, was markedly overexpressed in CRC cells. Moreover, patients who showed higher Rab31 levels had a shortened survival period relative to those with low Rab31 levels. Rab31 knockdown significantly downregulated cyclin D1, p-mTOR, and p-p70S6K expression. Moreover, the expression of Rab31-induced p-p70S6K was almost inhibited by rapamycin, a well-established inhibitor of mTOR. Similarly, rapamycin also significantly decreased the stimulatory effect of Rab31 on the expression of cyclin D1.. These findings suggested that Rab31 enhanced proliferation, promoted cell cycle progression, and inhibited apoptosis of colorectal carcinoma cells through the mTOR pathway.

Topics: Aged; Apoptosis; Blotting, Western; Caco-2 Cells; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Colorectal Neoplasms; Cyclin D1; Female; HCT116 Cells; Humans; Male; Middle Aged; Prognosis; Proportional Hazards Models; rab GTP-Binding Proteins; Real-Time Polymerase Chain Reaction; Ribosomal Protein S6 Kinases, 70-kDa; Signal Transduction; Survival Analysis; TOR Serine-Threonine Kinases; Up-Regulation

A. oxyphylla extract is known to possess a wide range of pharmacological activites. However, the molecular mechanism of A. oxyphylla and its bioactive compound nootkatone in colorectal cancer is unknown.. Our study aims to examine the role of A. oxyphylla and its bioactive compound nootkatone, in tumor suppression using several in vitro assays.. Both A. oxyphylla extract and nootkatone exhibited antiproliferative activity in colorectal cancer cells. A. oxyphylla displayed antioxidant activity in colorectal cancer cells, likely mediated via induction of HO-1. Furthermore, expression of pro-apoptotic protein NAG-1 and cell proliferative protein cyclin D1 were increased and decreased respectively in the presence of A. oxyphylla. When examined for anticancer activity, nootkatone treatment resulted in the reduction of colony and spheroid formation. Correspondingly, nootkatone also led to increased NAG-1 expression and decreased cyclin D1 expression. The mechanism by which nootkatone suppresses cyclin D1 involves protein level regulation, whereas nootkatone increases NAG-1 expression at the transcriptional level. In addition to having PPARγ binding activity, nootkatone also increases EGR-1 expression which ultimately results in enhanced NAG-1 promoter activity.. In summary, our findings suggest that nootkatone is an anti-tumorigenic compound harboring antiproliferative and pro-apoptotic activity.

Topics: Alpinia; Apoptosis; Cell Line, Tumor; Cell Proliferation; Colorectal Neoplasms; Cyclin D1; Early Growth Response Protein 1; Gene Expression Regulation, Neoplastic; Growth Differentiation Factor 15; Heme Oxygenase-1; Humans; Plant Extracts; Polycyclic Sesquiterpenes; PPAR gamma; Promoter Regions, Genetic; Sesquiterpenes

Upregulation of histone methyltransferase SET domain bifurcated 1 (SETDB1) is associated with poor prognosis in cancer patients. However, the mechanism of oncogenicity of SETDB1 in cancer is hitherto unknown. Here, we show that SETDB1 is upregulated in human colorectal cancer (CRC) where its level correlates with poor clinical outcome. Ectopic SETDB1 promotes CRC cell proliferation, whereas SETDB1 attenuation inhibits this process. Flow cytometry reveals that SETDB1 promotes proliferation by driving the CRC cell cycle from G0/G1 phase to S phase. Mechanistically, SETDB1 binds directly to the STAT1 promoter region resulting in increased STAT1 expression. Functional characterization reveals that STAT1-CCND1/CDK6 axis is a downstream effector of SETDB1-mediated CRC cell proliferation. Furthermore, SETDB1 upregulation is sufficient to accelerate in vivo proliferation in xenograft animal model. Taken together, our results provide insight into the upregulation of SETDB1 within CRC and can lead to novel treatment strategies targeting this cell proliferation-promoting gene.

Topics: Animals; Apoptosis; Biomarkers, Tumor; Cell Movement; Cell Proliferation; Colorectal Neoplasms; Cyclin D1; Cyclin-Dependent Kinase 6; Gene Expression Regulation, Neoplastic; Histone-Lysine N-Methyltransferase; Humans; Male; Mice; Mice, Inbred BALB C; Mice, Nude; STAT1 Transcription Factor; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

Background and study aim: Cyclin D1 is a key regulatory protein in the cell cycle and is over-expressed in\ many tumors, including endometrial, thyroid, urothelial, breast, brain gliomas, and esophageal cancers. The main\ aim of the present study is to examine the expression pattern of cyclin D1 and its correlation with the different\ clinicopathological features in patients with colorectal camcer (CRC) from the Madinah region of Saudi Arabia.\ Patients and methods: The archival tumor blocks were analyzed using immunohistochemistry for Cyclin D1\ over-expression in 324 CRC patients diagnosed from January 2006 to December 2017, at the Department of Pathology,\ King Fahad Hospital, Madinah, Saudi Arabia. Results: Cyclin D1 over-expression was absent in normal mucosa, while\ 15% cases of adenoma showed its over-expression. In CRC, Cyclin D1 was expressed at high levels in 24.1% of case.\ No significant correlation was observed between Cyclin D1 over-expression and age, gender, tumor size, type and\ location. However, Cyclin D1 over-expression exhibited a significant correlation with tumor differentiation (p=0.04),\ lymph node involvement (p=0.001), lymphovascular invasion (p=0.001), distant metastasis (p=0.006) and AJCC staging\ (p=0.001). The Kaplan-Meir analysis revealed a shorter period of survival with Cyclin D1 over-expression (p=0.000).\ The Cox-regression model analysis showed that Cyclin D1 over-expression was an independent prognostic marker\ in CRC (p=0.000). Conclusion: Cyclin D1 over-expression increases during normal-adenoma-carcinoma sequence.\ The significant association observed between Cyclin D1 over-expression, advanced tumor stage and short survival\ period clearly suggest the role of Cyclin D1 in the carcinogenesis and progression of CRC.

Topics: Adenoma; Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Case-Control Studies; Colorectal Neoplasms; Cyclin D1; Female; Follow-Up Studies; Humans; Lymphatic Metastasis; Male; Middle Aged; Neoplasm Invasiveness; Prognosis; Retrospective Studies; Saudi Arabia; Survival Rate; Young Adult

For treatment of metastatic colorectal cancer, the dynamics of tumor growth is an important factor for treatment decision. However, it is difficult to evaluate the dynamics of tumor growth, especially those of synchronous metastatic diseases. This study aimed to find some indicators related to tumor proliferation to judge the dynamics of tumor progression. The pathological reports and clinical data of 1205 patients with metastatic colorectal cancer were retrospectively reviewed; 75 patients with known relapse time after radical resection were included, and the expression of proliferation-associated proteins was detected by immunohistochemistry. Relapse-free time (RFT) from radical resection to relapse was obtained to analyze the relationship with expression of these factors. Kaplan-Meier univariate analysis showed that the overexpression of cyclin D1 and epidermal growth factor receptor (EGFR) and late pathological stage after surgery indicated shorter RFT. Multivariate analysis showed that EGFR and the stage were independent predictors of RFT. Expression of EGFR and cyclin D1 and the pathological stage were included as combination risk factors for RFT analysis; more risk factors were correlated with shorter RFT. EGFR and cyclin D1 seemed to be indicators of the dynamics of tumor growth, and overexpression of those molecules may suggest rapid growth and poor prognosis.

Topics: Adenocarcinoma; Adenocarcinoma, Mucinous; Biomarkers, Tumor; Cell Proliferation; Colorectal Neoplasms; Cyclin D1; ErbB Receptors; Female; Follow-Up Studies; Humans; Male; Middle Aged; Neoplasm Recurrence, Local; Prognosis; Survival Rate

Wnt signaling contributes to the development of colorectal cancer (CRC). We studied interactions between lysine demethylase 4D (KDM4D or JMJD2D) and β-catenin, a mediator of Wnt signaling, in CRC cell lines and the effects on tumor formation in mice.. We obtained colorectal tumor specimens and surrounding nontumor colon tissues (controls) from patients undergoing surgery in China; levels of JMJD2D were measured by immunohistochemical or immunoblot analysis. JMJD2D expression was knocked down in CRC (CT26, HCT116, and SW480 cells) using small hairpin RNAs, and cells were analyzed with viability, flow cytometry, colony formation, and transwell migration and invasion assays. Cells were also grown as tumor xenografts in nude mice or injected into tail veins or spleens of mice, and metastases were measured. We performed promoter activity, co-immunoprecipitation, and chromatin immunoprecipitation assays. We also performed studies with Apc. Levels of JMJD2D were significantly higher in human colorectal tumors than in control tissues and correlated with levels of proliferating cell nuclear antigen. JMJD2D knockdown reduced CRC cell proliferation, migration, and invasion, as well as growth of xenograft tumors and formation of metastases in mice. JMJD2D was required for expression of β-catenin in CRC cell lines; ectopic expression of JMJD2D increased the promoter activities of genes regulated by β-catenin (MYC, CCND1, MMP2, and MMP9). We found that JMJD2D and β-catenin interacted physically and that JMJD2D demethylated H3K9me3 at promoters of β-catenin target genes. JMJD2D-knockout mice developed fewer colitis-associated colorectal tumors than control mice, and their tumor tissues had lower levels of β-catenin, MYC, cyclin D1, and proliferating cell nuclear antigen than tumors from control mice. Apc. Levels of the histone demethylase JMJD2D are increased in human colorectal tumors compared with nontumor colon tissues. JMJD2D interacts with β-catenin to activate transcription of its target genes and promote CRC cell proliferation, migration, and invasion, as well as formation of colorectal tumors in mice.

Topics: Animals; beta Catenin; Cell Movement; Cell Proliferation; Cell Survival; Chloroquinolinols; Colorectal Neoplasms; Cyclin D1; Female; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; HCT116 Cells; Histones; Humans; Jumonji Domain-Containing Histone Demethylases; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Methylation; Mice; Mice, Knockout; Mice, Nude; Neoplasm Metastasis; Neoplasm Transplantation; Proliferating Cell Nuclear Antigen; Proto-Oncogene Proteins c-myc; Transcription, Genetic; Tumor Stem Cell Assay; Wnt Signaling Pathway

Colorectal cancer (CRC) is among the leading causes of cancer-related mortality worldwide. Compelling evidence suggests that long non-coding RNA (lncRNAs) can control carcinogenesis by regulating various aspects of cell biology. However, limited number of CRC-related lncRNAs has been well characterized. This study was undertaken to investigate the expression pattern of the novel lncRNA-CCHE1 in CRC patients and to examine its correlation with clinicopathological features, ERK/COX-2 pathway and some cell proliferation markers in order to gain biological insights on its role in CRC pathogenesis. Colon cancer specimens with their adjacent non-cancerous tissues were taken from 60 patients with primary CRC. LncRNA-CCHE1 relative expression was assessed using quantitative real-time RT-PCR. P-ERK ½ and cyclin D1 levels were estimated by ELISA. COX-2 and proliferating cell nuclear antigen (PCNA) expression were assessed immunohistochemically. lncRNA-CCHE1 expression was upregulated in CRC tissues compared to adjacent non-cancerous tissues, and was significantly associated with larger tumor size, less differentiated histology, advanced dukes' stage, positive lymph node involvement and vascular invasion. It also showed a significant positive correlation with the expression of p-ERK1/2, COX-2 as well as cyclin D1and PCNA (as markers for cell proliferation). These findings signify that lncRNA-CCHE1 is a key oncogene possibly involved in CRC development and progression by modulating ERK/COX-2 pathway and cell proliferation activity. Our study also provides a rationale for potential use of lncRNA-CCHE1 as a novel prognostic marker, and opens the door for the development of lncRNA-CCHE1-directed therapeutic approaches for CRC patients.

Topics: Cell Line, Tumor; Colon; Colorectal Neoplasms; Cyclin D1; Cyclooxygenase 2; Female; Gene Expression Regulation, Neoplastic; Humans; Male; MAP Kinase Signaling System; Middle Aged; Phosphorylation; Prognosis; Proliferating Cell Nuclear Antigen; RNA, Long Noncoding

Epidemiological studies indicate that long‑term aspirin usage reduces the incidence of colorectal cancer (CRC) and may protect against other non‑CRC associated adenocarcinomas, including oesophageal cancer. A number of hypotheses have been proposed with respect to the molecular action of aspirin and other non‑steroidal anti‑inflammatory drugs in cancer development. The mechanism by which aspirin exhibits toxicity to CRC has been previously investigated by synthesising novel analogues and derivatives of aspirin in an effort to identify functionally significant moieties. Herein, an early effect of aspirin and aspirin‑like analogues against the SW480 CRC cell line was investigated, with a particular focus on critical molecules in the epidermal growth factor (EGF) pathway. The present authors proposed that aspirin, diaspirin and analogues, and diflunisal (a salicylic acid derivative) may rapidly perturb EGF and EGF receptor (EGFR) internalisation. Upon longer incubations, the diaspirins and thioaspirins may inhibit EGFR phosphorylation at Tyr1045 and Tyr1173. It was additionally demonstrated, using a qualitative approach, that EGF internalisation in the SW480 cell line may be directed to endosomes by fumaryldiaspirin using early endosome antigen 1 as an early endosomal marker and that EGF internalisation may also be perturbed in oesophageal cell lines, suggestive of an effect not only restricted to CRC cells. Taken together and in light of our previous findings that the aspirin‑like analogues can affect cyclin D1 expression and nuclear factor‑κB localisation, it was hypothesized that aspirin and aspirin analogues significantly and swiftly perturb the EGFR axis and that the protective activity of aspirin may in part be explained by perturbed EGFR internalisation and activation. These findings may also have implications in understanding the inhibitory effect of aspirin and salicylates on wound healing, given the critical role of EGF in the response to tissue trauma.

Topics: Aspirin; Cell Line, Tumor; Cell Proliferation; Cell Survival; Colorectal Neoplasms; Cyclin D1; Drug Screening Assays, Antitumor; EGF Family of Proteins; ErbB Receptors; Gene Expression Regulation, Neoplastic; Humans; NF-kappa B; Phosphorylation; Salicylates; Signal Transduction

Sageretia thea (S. thea) has been used as the medicinal plant for treating hepatitis and fevers in Korea and China. Recently, anticancer activity of S. thea has been reported, but the potential mechanism for the anti-cancer property of S. thea is still insufficient. Thus, we evaluated whether extracts from the leaves (STL) and branches (STB) of S. thea exert anticancer activity and elucidated its potential mechanism in SW480 cells.. MTT assay was performed for measuring cell viability. Western blot and RT-PCR were used for analyzing the level of protein and mRNA, respectively.. Treatment of STL or STB decreased the cell viability and induced apoptosis in SW480 cells. Decreased level of cyclin D1 protein was observed in SW480 cells treated with STL or STB, but no change in cyclin D1 mRNA level was observed with the treatment of STL or STB. MG132 blocked downregulation of cyclin D1 protein by STL or STB. Thr286 phosphorylation of cyclin D1 by STL or STB occurred faster than downregulation of cyclin D1 protein in SW480 cells. When SW480 cells were transfected with T286A-cyclin D1, cyclin D1 degradation by STL or STB did not occur. Inhibition of GSK3β and cyclin D1 nuclear export attenuated STL or STB-mediated cyclin D1 degradation. In addition, STL or STB increased HO-1 expression, and the inhibition of HO-1 attenuated the induction of apoptosis by STL or STB. HO-1 expression by STL or STB resulted from Nrf2 activation through ROS-dependent p38 activation.. These results indicate that STL or STB may induce GSK3β-dependent cyclin D1 degradation, and increase HO-1 expression through activating Nrf2 via ROS-dependent p38 activation, which resulted in the decrease of the viability in SW480 cells. These findings suggest that STL or STB may have great potential for the development of anti-cancer drug.

Topics: Antineoplastic Agents; Cell Line, Tumor; Cell Survival; Colorectal Neoplasms; Cyclin D1; Heme Oxygenase-1; Humans; Plant Extracts; Proteasome Endopeptidase Complex; Rhamnaceae

MicroRNA-9 (miR-9) has been reported to play a suppressive or promoting role according to cancer type. In this study, we investigated the effects of anoctamin-1 (ANO1) and miR-9 on colorectal cancer (CRC) cell proliferation, migration, and invasion and determined the underlying molecular mechanisms. Thirty-two paired CRC tissues and adjacent normal tissues were analyzed for ANO1 expression using quantitative real-time PCR (qRT-PCR). HCT116 cells were transiently transfected with miR-9 mimic, miR-9 inhibitor, or si-ANO1. Cell proliferation was determined by MTT, and flow cytometric analysis, while cell migration and invasion were assayed by trans-well migration and invasion assay in HCT116 cells. ANO1 was validated as a target of miR-9 using luciferase reporter assay and bioinformatics algorithms. We found that ANO1 expression was up-regulated in CRC tissues compared with adjacent normal tissues. ANO1 expression was associated with advanced tumor stage and lymph node metastasis, and there was an inverse relationship between miR-9 and ANO1 mRNA expression in CRC specimens, but no significant difference was found between miR-9 and ANO1 expression. ANO1 is a direct target of miR-9, and overexpression of miR-9 suppressed both mRNA and protein expression of ANO1 and inhibited cell proliferation, migration, and invasion of HCT116 cells. We also showed that overexpression of miR-9 suppressed expression of p-AKT, cyclin D1, and p-ERK in HCT116 cells. We conclude that miR-9 inhibits CRC cell proliferation, migration, and invasion by directly targeting ANO1, and miR-9/ANO1 could be a potential therapeutic target for CRC.

Topics: Anoctamin-1; Apoptosis; Base Sequence; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Movement; Cell Proliferation; Colorectal Neoplasms; Cyclin D1; Down-Regulation; Epithelial-Mesenchymal Transition; Extracellular Signal-Regulated MAP Kinases; Female; Gene Expression Regulation, Neoplastic; Humans; Lymphatic Metastasis; Male; MicroRNAs; Middle Aged; Neoplasm Invasiveness; Neoplasm Proteins; Neoplasm Staging; Phosphorylation; Proto-Oncogene Proteins c-akt; RNA, Messenger; Up-Regulation

Growing evidence has shown that long non-coding RNAs (lncRNAs) can serve as prospective markers for survival in patients with colorectal adenocarcinoma. In this study, we mainly focused on the potential roles of lncRNA- ZDHHC8P1 in the development process of colorectal cancer (CRC) via miR-34a.. Quantitative Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) was used to detect the expression of lncRNA ZDHHC8P1 in CRC patients and cell lines. The relationships between the expression level of ZDHHC8P1, patient clinicopathological parameters and overall survival (OS) were analyzed using the univariate Kaplan-Meier method. Proliferation ability was tested by Cell Counting Kit-8 (CCK-8) and cyclin D1 assay, through loss- and gain-of-function approaches. Moreover, the cycle distribution and wound-healing assay were used to measure the function of progression and metastasis in CRC cell lines. Furthermore, Luciferase activity proved the relationship between miR-34a and ZDHHC8P1.. We found that lncRNA-ZDHHC8P1 was highly expressed in heatmap of colon cancer with lncRNAtor. Additionally, the lncRNA ZDHHC8P1, an independent prognostic factor for overall survival, was increased in CRC and correlated positively with CRC progression. Most importantly, through loss- and gain-of-function approaches, we showed that ZDHHC8P1 promotes progression and metastasis in vitro. What's more, the ZDHHC8P1 expression level was reversely correlated with miR-34a expression in patients with CRC. Mechanistically, miR-34a was identified as an important ZDHHC8P1 functional target.. According to the results, this work uncovers a previously unappreciated ZDHHC8P1/miR-34a regulatory axis in controlling progression and metastasis of CRC and suggests that interfering with lncRNA-ZDHHC8P1 and miR-34a could be a viable approach to treat late-stage metastatic CRC patients.

Topics: Base Sequence; Cell Line; Cell Movement; Cell Proliferation; Colorectal Neoplasms; Cyclin D1; Disease Progression; Female; Gene Expression Regulation, Neoplastic; Humans; Kaplan-Meier Estimate; Lymphatic Metastasis; Male; MicroRNAs; Middle Aged; Prognosis; RNA Interference; RNA, Long Noncoding; RNA, Small Interfering; Sequence Alignment

Only 10%-20% of colorectal cancer (CRC) patients observe effective responses to 5-fluorouracil (5-FU) based chemo-treatment. We used real-time PCR array and Western blot analysis to examine the expression alteration of acetyltransferases and deacetylases in 5-FU resistant CRC cell lines as compared to their respective parental CRC cell lines. Unlike other acetyltransferases and deacetylases, we found that the expression of acetyltransferase P300/CBP-associated factor (PCAF) is consistently decreased in three 5-FU resistant CRC cell lines. Similarly, knockdown of PCAF in HCT116 CRC parental cell line also increases the resistance to 5-FU and attenuates 5-FU-induced apoptosis. Mechanistically, we demonstrated that increased binding of trimethylated histone H3K27 in the promoter region of PCAF attenuated its transcription in 5-FU resistant HCT116/5-FU cells. Decreased PCAF impairs the acetylation of p53 and attenuates the p53-dependent transcription of p21, which results in the increased cyclin D1 and phosphorylation of Retinoblastoma 1. Conversely, overexpression of PCAF in CRC cell lines increases p21 and their susceptibility to 5-FU in vitro and in vivo. However, knockdown of p21 abolishes the beneficial effects of PCAF overexpression on increasing the sensitivity of HCT116/5-FU cells to 5-FU. Also, the reduced intensity of PCAF immunostaining was observed in the precancerous lesion, and microarray data from the public database further demonstrated the association between PCAF down-regulation and poor survival outcome. Our data suggest that PCAF-mediated p53 acetylation is an essential regulatory mechanism for increasing the susceptibility of CRC to 5-FU.

Topics: Acetylation; Animals; Apoptosis; Cell Proliferation; Cell Survival; Colorectal Neoplasms; Cyclin D1; Drug Resistance, Neoplasm; E2F1 Transcription Factor; Epigenesis, Genetic; Fluorouracil; Gene Expression Regulation, Neoplastic; HCT116 Cells; Heterografts; Humans; Mice; p300-CBP Transcription Factors; Tumor Suppressor Protein p53

Cell division cycle associated 2 (CDCA2), upregulated in lung adenocarcinoma and oral squamous cell carcinoma, may be related to some malignant diseases. Nevertheless, its role in colorectal cancer (CRC) remains unknown.. CDCA2 expression was analyzed using The Cancer Genome Atlas (TCGA), quantitative real-time PCR (qRT-PCR), and immunohistochemistry. The impact of CDCA2 on cell proliferation was analyzed via loss- or gain-of-function assays. Furthermore, gene set enrichment analysis was conducted to explore the potential mechanism of CDCA2 in CRC. Lastly, the expression levels of CCND1 and AKT were measured in CRC cell lines.. Our study revealed that CDCA2 expression was associated with tumor progression. Through loss- or gain-of-function assays, we found that upregulation of CDCA2 promoted the proliferation of DLD-1 cells, however, downregulation of CDCA2 in SW480 cells restrained proliferative capacity both in vitro and in vivo. The results of flow cytometry showed that CDCA2 promoted cell cycle progression via upregulation of CCND1 in CRC cell lines. In the following experiments, we found that CDCA2 regulated CCND1 expression through activating the PI3K/AKT pathway, and confirmed this using a specific PI3K inhibitor (LY294002).. This study demonstrates that overexpression of CDCA2 might target CCND1 to promote CRC cell proliferation and tumorigenesis through activation of the PI3K/AKT pathway.

Topics: Animals; Carcinogenesis; Carrier Proteins; Cell Cycle Proteins; Cell Line, Tumor; Cell Proliferation; Chromones; Colorectal Neoplasms; Cyclin D1; Female; Humans; Male; Mice; Middle Aged; Morpholines; Nuclear Proteins; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Proto-Oncogene Proteins c-akt; RNA, Small Interfering; Signal Transduction

Tumor recurrence and metastasis decrease the survival rate of colorectal cancer (CRC) patients. Menadione reduces the numbers and incidences of 1,2-dimethylhydrazine induced colon tumors in mouse but the mechanism of anticancer activity of menadione in colorectal cancer is not very clear. Since Wnt signaling is constitutively active in CRC and it aggravates the epithelial mesenchymal transition (EMT), the regulation of EMT and Wnt signaling by menadione (vitamin K3) was investigated in CRC cells. Menadione showed cytotoxicity against human CRC cells (SW480 and SW620) and human primary colon cancer cells but was relatively ineffective against the cells from human normal colon (CRL-1790) and human primary colon epithelial cells. Menadione suppressed invasion, migration and epithelial-mesenchymal transition in human CRC cells by upregulating the expression of E-cadherin (CDH1), ZO-1 and downregulating that of N-cadherin (CDH2), Vimentin (VIM), ZEB1, MMP2 and MMP9. Menadione decreased TOPFlash/FOPFlash luciferase activity and expression of several downstream targets of Wnt signaling and coactivators such as β-catenin (CTNNB1), TCF7L2, Bcl9l, p300 (EP300) and cyclin D1 (CCND1) was suppressed. Menadione induced differentiation and increased apoptotic cell population in SubG0 phase of cell cycle in SW480 and SW620 cells. The ability of menadione to suppress EMT, migration, invasion, Wnt signaling, cell proliferation and induce Sub G0 arrest, highlights its potential to be considered for intensive preclinical and clinical investigation in CRC.

Topics: Cadherins; Cell Cycle Checkpoints; Cell Differentiation; Cell Line, Tumor; Cell Movement; Colorectal Neoplasms; Cyclin D1; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Humans; Matrix Metalloproteinase 2; Vitamin K 3; Wnt Signaling Pathway

Dysregulation of deubiquitinating enzymes (DUBs), which regulate the stability of key proteins, has been implicated in many human diseases, including cancers. Thus, DUBs can be considered as potential therapeutic targets for many diseases. Among them, USP4 has been proposed as a promising target for colon cancer drugs since USP4 controls the stability of β-catenin, a key factor in the Wnt signaling involved in the tumorigenesis of colorectal cancer. However, developing potential DUB inhibitors has been hindered because many DUBs harbor similar active site structures and show broad substrate specificities.. By performing in vitro deubiquitinating activity assays using a chemical library, we identified several potential DUB inhibitors. Among them, only neutral red (NR) showed selective inhibitory activity on USP4 in a cell-based assay system. In colon cancer cells, NR affected the protein stability of β-catenin, as shown by immunoblotting, and it affected the target gene expression of β-catenin, as shown by quantitative real-time PCR. NR's potential as an anticancer drug was further estimated by colony formation and cell migration assays and by using a mouse xenograft model.. We identified NR as an uncompetitive inhibitor of USP4 and validated its effects in colorectal cancer. NR-treated cells showed decreased β-catenin stability and reduced expression of β-catenin target genes. Additionally, treating colon cancer cells with NR significantly reduced colony formation and cell migration, and injecting NR into a mouse xenograft model reduced the tumor volume.. The current results suggest that NR could be developed as an anticancer drug targeting USP4, and they support the possibility of developing specific DUB inhibitors as therapeutic agents.

Topics: Animals; beta Catenin; Cell Line, Tumor; Cell Movement; Cell Proliferation; Colorectal Neoplasms; Cyclin D1; Disease Progression; Humans; Mice; Mice, Inbred BALB C; Mice, Nude; Neutral Red; Transplantation, Heterologous; Ubiquitin-Specific Proteases; Wnt Signaling Pathway

Proton radiation is a major component of the radiation field in outer space and is used clinically in radiation therapy of resistant cancers. Although epidemiologic studies in atom bomb survivors and radiologic workers have established radiation as a risk factor for colorectal cancer (CRC), we have yet to determine the risk of CRC posed by proton radiation owing to a lack of sufficient human or animal data. The purpose of the current study was to quantitatively and qualitatively characterize differential effects of acute and fractionated high-energy protons on colorectal carcinogenesis.. Significantly higher intestinal tumor number and grade, along with decreased differentiation, were observed after acute radiation relative to fractionated radiation. Acute protons induced upregulation of β-catenin and Akt pathways with increased proliferative marker phospho-histone H3. Increased DNA damage along with decreased DNA repair factors involved in mismatch repair and nonhomologous end joining were also observed after exposure to acute protons.. We show increased γH2AX, 53BP1, and 8-oxo-dG, suggesting that increased ongoing DNA damage along with decreased DNA repair factors and increased proliferative responses could be triggering a higher number of intestinal tumors after acute relative to fractionated proton exposures in Apc

Topics: 8-Hydroxy-2'-Deoxyguanosine; Animals; beta Catenin; Carcinogenesis; Cell Differentiation; Cell Proliferation; Colorectal Neoplasms; Cyclin D1; Disease Models, Animal; DNA Breaks, Double-Stranded; DNA End-Joining Repair; DNA Mismatch Repair; Dose Fractionation, Radiation; Female; Gene Expression; Genes, APC; Histones; Immunoblotting; Intestinal Neoplasms; Intestine, Small; Mice; Mice, Inbred C57BL; Neoplasms, Radiation-Induced; Proto-Oncogene Proteins c-akt; Protons; Radiation Dosage; Radiation Exposure; Space Flight; Tumor Suppressor p53-Binding Protein 1; Up-Regulation

The Wnt signaling pathway has been identified for its function in carcinogenesis and embryonic development. It is known to play a vital role in the initiation and development of colorectal cancer (CRC). Therefore, it is of great importance for CRC research to illuminate the mechanisms which regulate Wnt pathway activity. Here, we intended to examine the effect of hsa-miR-942 (miR-942) on the Wnt signaling activity, cell cycle progression, and its expression in CRC tissues. RT-qPCR results indicated that miR-942 is significantly upregulated in colorectal cancer. Then, overexpression of miR-942 promoted, whereas its inhibition decreased the Wnt signaling activity, detected by RT-qPCR and Top/Fop flash assay. Inhibition of Wnt signaling by using PNU-74654 or IWP-2 small molecules indicated that miR-942 applies its effect to the β-catenin degradation complex level. Then, RT-qPCR and dual luciferase assay showed that miR-942 upregulated Wnt signaling through direct targeting of APC, which is a tumor suppressor in Wnt signaling pathway. Furthermore, the western blotting analysis indicated that β.catenin, as a main member of Wnt signaling pathway is upregulated following the overexpression of miR-942. Finally, miR-942 overexpression resulted in cell cycle progression in SW480 cells. Taken together, our findings established an oncogenic role for miR-942 in CRC and indicated that this miRNA might be a crucial target for CRC therapy.

Topics: 3' Untranslated Regions; beta Catenin; Carcinogenesis; Cell Cycle; Cell Line, Tumor; Colorectal Neoplasms; Computational Biology; Cyclin D1; Disease Progression; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Luciferases; Male; MicroRNAs; Plasmids; Proto-Oncogene Proteins c-myc; RNA Interference; RNA, Long Noncoding; Signal Transduction; Up-Regulation; Wnt Signaling Pathway

Aberrant activation of Wnt/β‑catenin signaling is observed in >90% of colorectal cancer cases. microRNAs (miRNAs) regulate the expression of key genes in Wnt/β‑catenin signaling. As a result, abnormal expression of miRNAs regulates the activation of Wnt/β‑catenin signaling in several types of cancer. In the current study, it was demonstrated that miR‑501‑3p was overexpressed in colorectal tumor tissues compared to the adjacent normal tissues. Downregulation of miR‑501‑3p inhibited cell proliferation and sphere formation, while it induced cell cycle arrest at the G1 phase in colorectal cancer cells. Bioinformatics analysis results predicted that adenomatous polyposis coli (APC), a negative regulator of Wnt/β‑catenin signaling, was a potential target gene of miR‑501‑3p. Inhibition of miR‑501‑3p increased APC expression in colorectal cancer cells. Additionally, β‑catenin was destabilized following miR‑501‑3p inhibition; immunofluorescence analysis revealed that β‑catenin translocated from nucleus to cytoplasm. In addition, cyclin D1 and c‑Myc, two well‑characterized target genes of Wnt/β‑catenin signaling, were downregulated following miR‑501‑3p inhibition. Transfection of APC small interfering RNA re‑activated β‑catenin and stimulated the expression of cyclin D1 and c‑Myc. Furthermore, silencing of APC reversed the miR‑501‑3p inhibitor‑induced cell cycle disruption, and the inhibition of cell proliferation and sphere formation in colorectal cancer cells. In conclusion, the present study identified miR‑501‑3p as a novel regulator of Wnt/β‑catenin signaling in colorectal cancer cells via targeting APC, suggesting that miR‑501‑3p may act as a novel oncogenic miRNA in colorectal cancer.

Topics: 5' Untranslated Regions; Adenomatous Polyposis Coli Protein; Adult; Aged; Caco-2 Cells; Cell Line, Tumor; Cell Proliferation; Colorectal Neoplasms; Cyclin D1; Disease Progression; Female; Gene Expression Regulation, Neoplastic; HCT116 Cells; HT29 Cells; Humans; Male; MicroRNAs; Middle Aged; Proto-Oncogene Proteins c-myc; Up-Regulation; Wnt Signaling Pathway

Using microarray analysis, we previously showed that many lncRNAs are differentially expressed in colorectal cancer (CRC) tissues compared with normal tissues, suggesting that lncRNAs may be involved the initiation and progression of CRC. In this study, we investigated the expression and function of lncRNA-RP11-317J10.2 in human CRC tissues and cell lines.. LncRNA-RP11-317J10.2 expression level was analyzed in 52 colon cancer and cell lines. We used shRNA to knock-down the expression of RP11-317J10.2, and then proliferation assay, colony formation assay, Boyden chamber assay, FACS and Kaplan-Meier survival analysis were performed to explore the biological effect of RP11-317J10.2. Cyclin D1 protein level was detected by Western blot.. LncRNA-RP11-317J10.2 is downregulated in CRC and decreased expression is significantly associated with advanced tumor stage, larger tumor size and poor prognosis. RNA interference-mediated knockdown of lncRNA-RP11-317J10.2 in CRC cells promotes G1-to-S cell cycle transition, enhances invasiveness and facilitates cell growth in vitro and in mouse tumor xenograft models. Cyclin D1 was upregulated by lncRNA-RP11-317J10.2 knockdown, and co-expression of cyclin D1-targeting siRNA abrogates the pro-tumorigenic effects of lncRNA-RP11-317J10.2 knockdown.. This study reveals a crucial role for lncRNA-RP11-317J10.2 in CRC growth and invasion via upregulation of cyclin D1 expression and suggests that expression of this lncRNA may be a potential prognostic biomarker for CRC.

Topics: Animals; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Colorectal Neoplasms; Cyclin D1; Down-Regulation; Female; Gene Expression Regulation, Neoplastic; Humans; Kaplan-Meier Estimate; Male; Mice; Mice, Inbred BALB C; Middle Aged; Neoplasm Invasiveness; Prognosis; RNA, Long Noncoding; RNA, Small Interfering; Up-Regulation

Viticis Fructus (VF) as the dried fruit from Vitex rotundifolia L. used as a traditional medicine for treating inflammation, headache, migraine, chronic bronchitis, eye pain, and gastrointestinal infections has been reported to have antiproliferative effects against various cancer cells, including breast, lung and colorectal cancer cells. However, the molecular mechanisms by which VF mediates the inhibitory effect of the proliferation of cancer cells have not been elucidated in detail. In this study, we investigated the molecular mechanism of VF on the down-regulation of cyclin D1 and CDK4 level associated with cancer cell proliferation. VF suppressed the proliferation of human colorectal cancer cell lines such as HCT116 and SW480. VF induced decrease in cyclin D1 and CDK4 in both protein and mRNA levels. However, the protein levels of cyclin D1 and CDK4 were decreased by VF at an earlier time than the change of mRNA levels; rather it suppressed the expression of cyclin D1 and CDK4 via the proteasomal degradation. In cyclin D1 and CDK4 degradation, we found that Thr286 phosphorylation of cyclin D1 plays a pivotal role in VF-mediated cyclin D1 degradation. Subsequent experiments with several kinase inhibitors suggest that VF-mediated degradation of cyclin D1 may be dependent on GSK3[Formula: see text] and VF-mediated degradation of CDK4 is dependent on ERK1/2, p38 and GSK3[Formula: see text]. In the transcriptional regulation of cyclin D1 and CDK4, we found that VF inhibited Wnt activation associated with cyclin D1 transcriptional regulation through TCF4 down-regulation. In addition, VF treatment down-regulated c-myc expression associated CDK4 transcriptional regulation. Our results suggest that VF has potential to be a candidate for the development of chemoprevention or therapeutic agents for human colorectal cancer.

Topics: Cell Proliferation; Colorectal Neoplasms; Cyclin D1; Cyclin-Dependent Kinase 4; Down-Regulation; Fruit; Humans; Phytotherapy; Plant Extracts; Proteasome Endopeptidase Complex; RNA Stability; Transcription, Genetic; Tumor Cells, Cultured; Vitex

MicroRNAs (miRNAs) have an important role in the regulation of tumor development and metastasis. In this study, we investigated the clinical and prognostic value as well as biological function of miR-466 in colorectal cancer (CRC). Tumor and adjacent healthy tissues were obtained from 100 patients diagnosed with CRC. miR-466 expression was determined by quantitative reverse transcription polymerase chain reaction (qRT-PCR). mRNA and protein levels of cyclin D1, apoptosis regulator BAX (BAX), and matrix metalloproteinase-2 (MMP-2) were analyzed by qRT-PCR and Western blot, respectively, in SW-620 CRC cells transfected with miR-466 mimics or negative control miRNA. Effects of miR-466 on SW-620 cell proliferation, cell cycle and apoptosis, and invasion were investigated using CCK-8 assay, flow cytometry and Transwell assay, respectively. miR-466 expression was significantly downregulated in tumor tissues compared to matched adjacent non-tumor tissues. Low expression of miR-466 was significantly correlated with the tumor size, Tumor Node Metastasis stage, lymph node metastasis, and distant metastasis. The overall survival of CRC patients with low miR-466 expression was significantly shorter compared to high-miR-466 expression group (log-rank test: p = 0.0103). Multivariate analysis revealed that low miR-466 expression was associated with poor prognosis in CRC patients. The ectopic expression of miR-466 suppressed cell proliferation and migration/invasion, as well as induced G0/G1 arrest and apoptosis in SW-620 cells. Moreover, the ectopic expression of miR-466 decreased the expression of cyclin D1 and MMP-2, but increased BAX expression in SW-620 cells. In conclusion, our findings demonstrated that miR-466 functions as a suppressor miRNA in CRC and may be used as a prognostic factor in these patients.

Topics: Aged; Apoptosis; bcl-2-Associated X Protein; Biomarkers, Tumor; Cell Cycle; Cell Movement; Cell Proliferation; Colorectal Neoplasms; Cyclin D1; Female; Gene Expression Regulation, Neoplastic; Genes, Tumor Suppressor; Humans; Kaplan-Meier Estimate; Lymphatic Metastasis; Male; Matrix Metalloproteinase 2; MicroRNAs; Middle Aged; Neoplasm Invasiveness; Neoplasm Metastasis; Prognosis

Insulin receptor substrate 4 (IRS-4) is an adaptor protein for which new evidence suggests plays a role in tumour promotion.. We described nuclear IRS-4 in RKO colon cancer cell lines in biopsies of patients with colorectal cancer (CRC) (n = 20) and in matched adjacent normal colorectal (MANC) tissue (n = 20).. Treatment with physiological doses of IGF-1 promoted nuclear influx of IRS-4 from cellular cytosol in RKO cells. When exogenous IRS-4 was overexpressed in RKO cells, there was an increase in cyclin D1, cyclin E, E2F1, pRB Ser 809/811 and pRB Ser 705 levels compared with the empty vector-transfected cells. Some of these changes returned to control values after wortmannin treatment. Subcellular fractionation showed an overexpression of IRS-4 in the cytoplasm, membrane, and nuclei of tumour samples, whereas the levels of the protein were barely detectable in the three compartments of normal samples. Immunohistochemical studies showed positive nuclear IRS-4 staining in over 74% of the tumour cells. IRS-4 was strongly overexpressed in tumoural tissues from CRC patients compared to MANC tissues. The up-regulation of IRS-4 in CRC samples correlated significantly with the increase of several G1 checkpoint proteins including cyclin D1 (r = 0.6662), Rb (r = 0.7779), pRb Serine 809/811 (r = 0.6864), pRb serine 705 (r = 0.6261) and E2F1 (r = 0.8702).. Taken together, our findings suggest that IRS-4 promotes retinoblastoma-cyclin-dependent kinase activation and it may serve as a pharmacological target since its expression is very low in normal tissue, including colonic epithelium.

Topics: Adenocarcinoma; Adenoma; Aged; Aged, 80 and over; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Membrane; Cell Nucleus; Cell Proliferation; Colon; Colorectal Neoplasms; Cyclin D1; Cytoplasm; E2F1 Transcription Factor; Female; Humans; Insulin Receptor Substrate Proteins; Insulin-Like Growth Factor I; Liver Neoplasms; Male; Middle Aged; Neoplasm Grading; Neoplasm Staging; Proliferating Cell Nuclear Antigen; Protein Transport; Rectum; Retinoblastoma Protein; Signal Transduction; Up-Regulation

To investigate the expression features of long non-coding RNA (lncRNA) DLEU7-AS1 in colorectal cancer (CRC), so as to further study its role in the occurrence and development of CRC and its potential regulatory mechanism.. The expression levels of lncRNA DLEU7-AS1 in 82 pairs of CRC tissues and para-carcinoma normal tissues were detected via quantitative Real-time polymerase chain reaction (qRT-PCR), and the correlation of DLEU7-AS1 expression with pathological indexes of CRC and patients' prognosis was analyzed. Besides, the expression of DLEU7-AS1 in CRC cells was further detected via qRT-PCR. The DLEU7-AS1 knockdown expression model was established using small interfering RNA in CRC cell lines HT-29 and HCT-116, and the effect of DLEU7-AS1 on biological functions of CRC cells was analyzed via Cell Counting Kit-8 (CCK-8) and transwell invasion/migration assay. Finally, its potential mechanism was investigated via Western blotting.. The results of qRT-PCR showed that the expression level of DLEU7-AS1 in CRC was significantly higher than that in normal tissues, and the difference was statistically significant. Compared with those in patients with low DLEU7-AS1 expression, the tumor stage in patients with high DLEU7-AS1 expression was higher, the prevalence rates of lymph node metastasis and distant metastasis were higher, and the overall survival rate was lower. Compared with those in the negative control group, the cell proliferation, invasion, and migration capacities were decreased significantly in DLEU7-AS1 knockdown expression group. Moreover, the results of Western blotting revealed that the expressions of key proteins in Wnt/β-catenin pathway, including β-catenin, c-myc, and cyclinD1, were decreased in si-DLEU7-AS1.. The expression of DLEU7-AS1 is significantly increased in CRC, which is markedly associated with CRC staging, lymph node metastasis, distant metastasis and poor prognosis. DLEU7-AS1 may promote the proliferation, invasion and migration capacities of CRC through regulating the Wnt/β-catenin pathway.

Topics: beta Catenin; Cell Line, Tumor; Cell Movement; Cell Proliferation; Colorectal Neoplasms; Cyclin D1; Female; Humans; Lymphatic Metastasis; Male; Middle Aged; Neoplasm Staging; Prognosis; RNA Interference; RNA, Long Noncoding; RNA, Small Interfering; Survival Rate; Wnt Signaling Pathway

Thyroid hormone status has long been implicated in cancer development. Here we investigated the role of thyroxine (T

Topics: beta Catenin; Carcinogenesis; Cell Proliferation; Colorectal Neoplasms; Cyclin D1; Gene Expression Regulation, Neoplastic; Genes, APC; Genes, myc; HCT116 Cells; HT29 Cells; Humans; Proliferating Cell Nuclear Antigen; RNA, Small Interfering; Thyroxine; Transcriptional Activation

Because twigs of Cinnamomum cassia (TC) have been reported to exert anti-cancer activity, the mechanistic study for TC's anti-cancer activity is required. Thus, we elucidated the potential molecular mechanism of TC's anti-proliferative effect and the induction of apoptosis in human colorectal cancer cells.. How water extracts form TC (TC-HW) was used in this study. Anti-cell proliferative effect of TC-HW was evaluated by MTT assay. The change of protein or mRNA level by TC-HW was evaluated by Western blot and RT-RCR, respectively. The promoter construct for ATF3, NF-κB, TOP-FLASH or FOP-FLASH was used for the investigation of the transcriptional activity for ATF3, NF-κB or Wnt. siRNA for ATF3 or p65 was used for the knockdown of ATF3 and p65.. TC-HW reduced the cell viability in human colorectal cancer cells. TC-HW decreased cyclin D1 protein level through cyclin D1 degradation via GSK3β-dependent threonine-286 (T286) phosphorylation of cyclin D1, indicating that cyclin D1 degradation may contribute to TC-HW-mediated decrease of cyclin D1 protein level. TC-HW downregulated the expression of cyclin D1 mRNA level and inhibited Wnt activation through the downregulation of β-catenin and TCF4 expression, indicating that inhibition of cyclin D1 transcription may also result in TC-HW-mediated decrease of cyclin D1 protein level. In addition, TC-HW was observed to induce apoptosis through ROS-dependent DNA damage. TC-HW-induced ROS increased NF-κB and ATF3 activation, and inhibition of NF-κB and ATF3 activation attenuated TC-HW-mediated apoptosis.. Our results suggest that TC-HW may suppress cell proliferation through the downregulation of cyclin D1 via proteasomal degradation and transcriptional inhibition, and may induce apoptosis through ROS-dependent NF-κB and ATF3 activation. These effects of TC-HW may contribute to the reduction of cell viability in human colorectal cancer cells. From these findings, TC-HW has potential to be a candidate for the development of chemoprevention or therapeutic agents for human colorectal cancer.

Topics: Activating Transcription Factor 3; Apoptosis; beta Catenin; Cell Line, Tumor; Cell Proliferation; Cinnamomum aromaticum; Colorectal Neoplasms; Cyclin D1; Glycogen Synthase Kinase 3 beta; Growth Inhibitors; Humans; NF-kappa B; Phosphorylation; Plant Extracts; Plant Stems

Cell division cycle associated 3 (CDCA3) is required for mitotic entry, and mediates the degradation of the inhibitory kinase Wee1. New evidence suggests CDCA3 plays a role in tumor promotion. However, little is known about the relevance of CDCA3 in colorectal cancer(CRC), especially in the regulation of NF-κB activity. In this study, we found that colorectal tumors significantly expressed more CDCA3 than non-cancer tissues. In addition, CDCA3 promoted CRC cell proliferation in vitro. Furthermore, downregulation of CDCA3 not only induced cell cycle arrest but also facilitated apoptosis. Mechanistically, CDCA3 activates the NF-κB signaling pathway by interacting with TRAF2 in CRC. Together, these results define a tumor-supportive role for CDCA3, which may also provide a new promising strategy for treating CRC.

Topics: Cell Cycle Proteins; Cell Line, Tumor; Cell Proliferation; Colorectal Neoplasms; Cyclin D1; Disease Progression; Female; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Humans; Male; Middle Aged; NF-kappa B; Signal Transduction; TNF Receptor-Associated Factor 2; Up-Regulation

Astragaloside IV showed a pivotal anti-cancer efficacy in multiple types of cancers and reversed chemoresistance in colorectal cancer (CRC). However, it remained unknown whether and how Astragaloside IV suppressed the progression of CRC. In the present study, we found that Astragaloside IV treatment significantly and dose-dependently reduced cell proliferation of CRC cell lines (SW620 and HCT116), whereas it showed no significant influence on the cell proliferation of normal colonic cells (FHC). Flow cytometry (FCM) analysis indicated that there was a significant cell cycle arrest in G0/G1 phase of SW620 cells and HCT116 cells which were treated with Astragaloside IV. The mRNA levels and protein levels of several key cell cycle relative proteins (cyclin D1 and CDK4) were also dramatically decreased during the process of G0/G1 arrest after the administration of Astragaloside IV. In addition, we observed an obvious decrease of B7-H3 protein level upon Astragaloside IV treatment, which was a result of mRNA reduction that was verified by real-time quantitative polymerase chain reaction (RT-qPCR) and CHX chase. We further identified that Astragaloside IV suppressed B7-H3 expression by elevating the expression of miR-29c level. Inhibition of miR-29c could dramatically reverse Astragaloside IV-induced B7-H3 decrease and cell growth arrest. This study suggests that Astragaloside IV is a promising anti-cancer drug in CRC.

Topics: B7 Antigens; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Colorectal Neoplasms; Cyclin D1; Cyclin-Dependent Kinase 4; Down-Regulation; G1 Phase; Gene Expression Regulation, Neoplastic; Humans; MicroRNAs; Resting Phase, Cell Cycle; RNA, Messenger; Saponins; Triterpenes; Up-Regulation

To investigate the effects of miR-519d on the 5-fluorouracil resistance in colorectal cancer cells and to explore the mechanism.. Colorectal cancer cells HCT116 and SW480 were transfected with miR519d-mimic or siCCND1 by transient transfection. The sensitivity of cells to 5-Fu was assayed by MTT assay. Dual luciferase assay was used to examine the effect of CCND1 on the sensitivity of cells to 5-Fu mediated by miR-519d.. MiR-519d was overexpressed in colorectal cancer cells after transient transfection with miR-519d mimics. Overexpression of miR-519d increased the sensitivity of colorectal cancer cells to 5-Fu. MiR-519d negatively regulated the expression of CCND1 via directly bound to CCND1 3`UTR. si-CCND1 could downregulate the CCND1 expression in colorectal cancer HCT116 and SW480 cells. si-CCND1 increased the sensitivity of colorectal cancer cells to 5-Fu.. miR-519d inhibits the expression of CCND1 and then plays a role in alleviating 5-Fu resistance in colorectal cancer cells.

Topics: Antimetabolites, Antineoplastic; Cell Line, Tumor; Colorectal Neoplasms; Cyclin D1; Down-Regulation; Drug Resistance, Neoplasm; Fluorouracil; Gene Expression Regulation, Neoplastic; HCT116 Cells; Humans; MicroRNAs

Objective To investigate the relationship between serum adiponectin level and risk of colorectal cancer, and to explore the effect of recombinant adiponectin on the proliferation and apoptosis of colorectal cancer HCT116 cells. Methods Serum adiponectin levels in patients with colorectal cancer and healthy controls were dectected by ELISA. With the level of adiponectin as a risk factor, ROC curve was acquired using SPSS software. HCT116 cells were treated with recombinant adiponectin (2.5, 5, 10, 20 μg/mL). Then cell proliferation activity was detected by MTT assay. Cell cycle and apoptosis were detected by flow cytometry. The protein expressions of p21, NF-κBp65, phosphorylated NF-κBp65 (p-NF-κBp65), cyclin D1 and cleaved caspase 3 (c-caspase-3) were tested by Western blot analysis. Results The levels of serum adiponectin in the patients were significantly lower than those in the healthy controls. When the level of adiponectin was used as a risk factor, the area under ROC curve was 0.887 (95% CI: 0.807-0.966). Recombinant adiponectin inhibited the survival rate of HCT116 cells in a time- and dose-dependent manner. After the treatment with recombinant adiponectin, the percentage of HCT116 cells in G1/G0 phase increased and the expression of p21 was upregulated, while the expressions of p-NF-κBp65 and cyclin D1 were down-regulated. Meanwhile, the expression of c-caspase-3 increased and the percentage of apoptotic cells also increased. Conclusion The decreased level of serum adiponectin is closely related to the risk of colorectal cancer. Recombinant human adiponectin can inhibit the proliferation of colorectal cancer HCT116 cells, induce cell arrest at G1/G0 phase and promote apoptosis, which may be related to the down-regulation of NF-κB, cyclin D1, and activation of p21 and caspase-3.

Topics: Adiponectin; Adult; Aged; Apoptosis; Caspase 3; Cell Proliferation; Colorectal Neoplasms; Cyclin D1; Down-Regulation; Female; G1 Phase Cell Cycle Checkpoints; HCT116 Cells; Humans; Male; Middle Aged

Mutations in the Ras/Raf/MEK/ERK pathway are detected in 50% of colorectal cancer cases and play a crucial role in cancer development and progression. Cobimetinib is a MEK inhibitor approved for the treatment of advanced melanoma and inhibits the cell viability of other types of cancer cells.. HCT116 colorectal cancer cells were treated with cobimetinib, and MTT assay, colony formation assay, and flow cytometry were used to evaluate cell viability, cell cycle, and apoptosis, respectively. The expression of genes associated with the cell cycle and apoptosis were evaluated by quantitative real-time PCR and western blotting. To explore use of cobimetinib in colorectal cancer treatment and further understand its mechanisms, RNA-seq technology was used to identify differentially expressed genes (DEGs) between cobimetinib-treated and untreated HCT116 cells. Furthermore, we compared these DEGs with Gene Expression Omnibus data from colorectal cancer tissues and normal colonic epithelial tissues.. We found that cobimetinib not only inhibited cell proliferation but also induced G1 phase arrest and apoptosis in HCT116 colorectal cancer cells, suggesting that cobimetinib may useful in colorectal cancer therapy. After cobimetinib treatment, 3,495 DEGs were obtained, including 2,089 upregulated genes and 1,406 downregulated genes, and most of these DEGs were enriched in the cell cycle, DNA replication, and DNA damage repair pathways. Our results revealed that some genes with high expression in colorectal cancer tissues were downregulated by cobimetinib in HCT116 cells, including CCND1, E2F1, CDC25C, CCNE2, MYC, and PCNA. These genes have vital roles in DNA replication and the cell cycle. Furthermore, genes with low expression in colorectal cancer tissues were upregulated by cobimetinib, including PRKCA, PI3K, RTK, and PKC. Based on our results, the PKC and PI3K pathways were activated after cobimetinib treatment, and inhibition of these two pathways can increase the cytotoxicity of cobimetinib in HCT116 cells. Notably, cobimetinib appeared to enhance the efficacy of 5-fluorouracil (5-FU) by decreasing TYMS expression, high expression of which is responsible for 5-FU resistance in colorectal cancer.. Our results suggest the potential use of cobimetinib in colorectal cancer therapy.

Topics: Apoptosis; Azetidines; Cell Line, Tumor; Colorectal Neoplasms; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Down-Regulation; Drug Resistance, Neoplasm; Fluorouracil; G1 Phase Cell Cycle Checkpoints; HCT116 Cells; Humans; MAP Kinase Kinase Kinases; Phosphatidylinositol 3-Kinases; Piperidines; Protein Kinase Inhibitors; Thymidylate Synthase; Tumor Suppressor Protein p53; Up-Regulation

Tumor necrosis factor ligand-related molecule 1 A (TLlA) is closely related to the occurrence and development of inflammatory bowel disease.. We aimed to explore whether TLlA was involved in the occurrence of colitis-associated colorectal cancer (CAC).. Firstly, azoxymethane (AOM) and dextran sulfate sodium (DSS) were used to construct the CAC mice model in wild-type (WT) and TL1A transgenic (Tg) mice with TL1A high expression. The histopathological analysis was used for the evaluation of inflammation level, and the immunohistochemistry staining analysis was used to test the expression and location of proliferating cell nuclear antigen (PCNA) and β-catenin. Secondly, the HCT116 and HT29 cell lines were used for knockdown of TL1A gene for further assay including cell viability, cell clone, cell apoptosis and matrigel invasion. Western blot were used for quantitative protein expression of β-catenin and downstream oncogenes including c-myc and Cyclin D1 after knockdown of TL1A gene.. The evaluation of inflammation level showed that the disease activity index score and tumor formation rate were significantly higher in AOM + DSS/Tg group than that in AOM + DSS/WT group. The expression of PCNA, β-catenin, c-myc, and Cyclin D1 in AOM + DSS/Tg group was significantly higher than that in AOM + DSS/WT group. The cell experiment showed that TL1A knockdown inhibited the cell proliferation, invasion, and migration. Moreover, the expression of c-myc and Cyclin D1 was significantly decreased after TL1A knockdown.. TL1A can induce tumor cell proliferation and promote the occurrence of CAC by activating Wnt/β-catenin pathway.

Topics: Animals; Azoxymethane; beta Catenin; Cell Movement; Cell Proliferation; Colitis; Colorectal Neoplasms; Cyclin D1; Dextran Sulfate; Disease Models, Animal; Gene Expression Regulation, Neoplastic; HCT116 Cells; HT29 Cells; Humans; Mice, Inbred C57BL; Mice, Transgenic; Neoplasm Invasiveness; Proliferating Cell Nuclear Antigen; Proto-Oncogene Proteins c-myc; Time Factors; Tumor Necrosis Factor Ligand Superfamily Member 15; Wnt Signaling Pathway

Cancer stem cells (CSCs) possess a self‑renewal ability and display tumorigenic potential in immunodeficient mice. Colorectal CSCs are thought to be a uniform population and no functionally distinct subpopulations have been identified. Because E‑cadherin is an essential molecule for self‑renewal of embryonic stem cells, we examined E‑cadherin expression, which may play a role in maintaining the properties of CSCs, in EpCAMhigh/CD44+ colorectal CSCs from human primary colorectal cancers. We obtained 18 surgical specimens of human primary colorectal cancer. CD44, EpCAM, and E‑cadherin expression were analyzed by fluorescence‑activated cell sorting. Sorted EpCAMhigh/CD44+ colorectal CSCs were injected into immunodeficient mice to estimate the tumorigenic potential. Genetic profiles were analyzed by cDNA microarray. Notably, colorectal CSCs could be divided into two populations based on the E‑cadherin expression status, and they exhibited different pathological characteristics. Compared to E‑cadherin‑negative colorectal CSCs, E‑cadherin‑positive (EC+) colorectal CSCs demonstrated higher tumor growth potential in vivo. EC+ colorectal CSCs revealed a higher expression of the pluripotency factor NANOG, which contributed to the higher tumor growth potential of EC+ colorectal CSCs through control of cyclin D1 expression. These findings are the first demonstration of functionally distinct subpopulations of colorectal CSCs in human clinical samples.

Topics: Adult; Aged; Animals; Cadherins; Cell Proliferation; Colorectal Neoplasms; Cyclin D1; Epithelial Cell Adhesion Molecule; Female; Flow Cytometry; Humans; Hyaluronan Receptors; Male; Mice; Middle Aged; Nanog Homeobox Protein; Neoplastic Stem Cells

Ultraconserved regions (UCRs) are 481 segments that have been strictly conserved among many mammalian species for hundreds of years. Their transcribed products belong to long non‑coding RNAs and are closely involved in the progression of various tumors. uc.338 has been reported to promote cell proliferation in hepatocellular and lung cancers. However, the role of uc.338 in colorectal cancer (CRC) proliferation remains unclear. In the present study, we first detected the expression of uc.338 in human tissues and analyzed the relationship between uc.338 expression and clinical features of CRC. We then investigated the biological function of uc.338 in CRC proliferation in vitro and in vivo. Finally, we explored the potential mechanism by which uc.338 promotes the proliferation of CRC cells. Our results indicated that uc.338 was upregulated in CRC tissues and higher uc.338 expression was associated with a larger tumor size, deeper invasion, increased lymph node metastasis and poorer prognosis. Further investigations in vivo and in vitro revealed that uc.338 could promote proliferation and cell cycle G1/S transition, and might target p21 downregulation and cyclin D1 upregulation via the PI3K/AKT pathway in CRC cells. Thus, our findings suggested that uc.338 acts as an oncogene by promoting proliferation in CRC cells, and could become a novel target for CRC detection and therapy.

Topics: Animals; Cell Line, Tumor; Cell Proliferation; Colorectal Neoplasms; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Down-Regulation; G1 Phase Cell Cycle Checkpoints; Gene Expression Regulation, Neoplastic; HCT116 Cells; Humans; Lymphatic Metastasis; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Oncogenes; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; RNA, Long Noncoding; Up-Regulation

Although PTHrP is implicated in several cancers, its role in chemoresistance is not fully elucidated. We found that in CRC cells, PTHrP exerts proliferative and protective effects and induces cell migration. The aim of this work was to further study the effects of PTHrP in CRC cells. Herein we evidenced, for the first time, that PTHrP induces resistance to CPT-11 in Caco-2 and HCT116 cells; although both cell lines responded to the drug through different molecular mechanisms, the chemoresistance by PTHrP in these models is mediated through ERK, which in turn is activated by PCK, Src and Akt. Moreover, continue administration of PTHrP in nude mice xenografts increased the protein levels of this MAPK and of other markers related to tumorigenic events. The understanding of the molecular mechanisms leading to ERK 1/2 activation and the study of ERK targets may facilitate the development of new therapeutic strategies for CRC treatment.

Topics: Animals; Apoptosis; Caco-2 Cells; Camptothecin; Carcinogenesis; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Colorectal Neoplasms; Cyclin D1; Drug Resistance, Neoplasm; HCT116 Cells; Humans; MAP Kinase Signaling System; Mice, Nude; Models, Biological; Molecular Targeted Therapy; Parathyroid Hormone-Related Protein; Phosphorylation; Protein Kinase C-alpha; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-myc; Signal Transduction; src-Family Kinases; Xenograft Model Antitumor Assays

Colorectal cancer (CRC) is a commonly diagnosed digestive malignancy worldwide. Ras-related protein 1A (RAP1A) is a member of the Ras superfamily of small GTPases and has been recently identified as a novel oncoprotein in several human malignancies. However, its specific role in CRC remains unclear.. In this study, we firstly analyzed its expression and clinical significance in a retrospective cohort of 144 CRC patients. Then, cellular assays in vitro and in vivo were performed to clarify its biological role in CRC cells. Finally, microarray analysis was utilized to investigate the molecular mechanisms regulated by RAP1A in CRC progression.. Firstly, RAP1A expression was abnormally higher in CRC tissues as compared with adjacent normal tissues, and significantly correlated tumor invasion. High RAP1A expression was an independent unfavourable prognostic factor for CRC patients. Combining RAP1A expression and preoperative CEA level contributed to a more accurate prognostic stratification in CRC patients. Secondly, knockdown of RAP1A dramatically inhibited the growth of CRC cells, while it was opposite for RAP1A overexpression. Finally, the microarray analysis revealed RAP1A promoted CRC growth partly through phosphatase and tensin homolog (PTEN)/forkhead box O3(FOXO3)/cyclin D1(CCND1) signaling pathway. FOXO3 overexpression could partly mimic the inhibitory effect of RAP1A knockdown in CRC growth. Moreover, FOXO3 overexpression inhibited CCND1 expression, but had no impact on RAP1A and PTEN expression.. RAP1A promotes CRC development partly through PTEN/FOXO3 /CCND1 signaling pathway. It has a great potential to be an effective clinical biomarker and therapeutic target for CRC patients.

Topics: Cohort Studies; Colorectal Neoplasms; Cyclin D1; Female; Forkhead Box Protein O3; Humans; Male; Middle Aged; Phenotype; Prognosis; PTEN Phosphohydrolase; rap1 GTP-Binding Proteins; Retrospective Studies; Signal Transduction

Tyrosine phosphorylation of Fas (TNFRSF6/CD95) in its death domain turns off Fas-mediated apoptosis, turns on the pro-survival signal, and has implications in different cancers types. We show here that Fas in its pro-survival state, phosphorylated at Y291 (pY291-Fas), functionally interacts with the epidermal growth factor receptor (EGFR), a key cancer-driving protein and major therapeutic target. Using an evolution-guided pY291-Fas proxy, RNA interference, and site-specific phospho-protein detection, we show that pY291-Fas significantly intensifies EGFR signaling in anti-EGFR-resistant colorectal cancer cells via the Yes-1/STAT3-mediated pathway. The pY291-Fas is essential for the EGF-induced formation of the Fas-mediated nuclear EGFR/STAT3 signaling complex consisting of Fas, EGFR, Yes-1, Src, and STAT3. The pY291-Fas accumulates in the nucleus upon EGF treatment and promotes the nuclear localization of phospho-EGFR and phospho-STAT3, the expression of cyclin D1, the activation of STAT3-mediated Akt and MAPK pathways, and cell proliferation and migration. This novel cancer-promoting function of phosphorylated Fas in the nuclear EGFR signaling constitutes the foundation for developing pro-survival-Fas targeted anti-cancer therapies to overcome disease recurrence in patients with anti-EGFR resistant cancer.

Topics: Apoptosis; Cell Line, Tumor; Cell Movement; Cell Nucleus; Cell Proliferation; Colorectal Neoplasms; Cyclin D1; Epidermal Growth Factor; ErbB Receptors; fas Receptor; HCT116 Cells; Humans; Neoplasm Recurrence, Local; Phosphorylation; Signal Transduction; STAT3 Transcription Factor; Tyrosine

Zinc is a transition metal and catalytic cofactor involved in many biological processes including proliferation, development, differentiation, and metabolism. Zinc transporters (ZnTs) play a fundamental role in cellular zinc homeostasis. ZnTs are responsible of zinc efflux and are encoded by 10 genes belonging to solute carrier family 30A (SLC30A1-10), while zinc-regulated transporter (ZRT)/iron-regulated transporter (IRT)-like protein (ZIP) transporters are responsible for the influx of zinc into the cytoplasm and are encoded by 14 genes belonging to solute carrier family 39A (SLC39A1-14). In this study, we analyzed, by transcriptome analysis, the microRNA levels of ZnT-encoding and ZIP-encoding genes in colorectal cancer (CRC) samples matched to normal colon tissues and in CRC cell lines. Results revealed an upregulation of specific ZnT and ZIP transcripts in CRC. Upregulation of SLC30A5, SLC30A6, SLC30A7 transcripts, encoding zinc efflux transporters ZnT5, ZnT6, ZnT7, localized on endoplasmic reticulum membranes, might be part of a coordinated transcriptional program associated to the increased activity of the early secretory pathway, while transcriptional upregulation of several specific ZIP transporters (SLC39A6, SLC39A7, SLC39A9, SLC39A10, and SLC39A11) could contribute in meeting the increased demand of zinc in cancer cells. Moreover, exon-level analysis of SLC30A9, a nuclear receptor coactivator involved in the transcriptional regulation of Wnt-responsive genes, revealed the differential expression of alternative transcripts in CRC and normal colonic mucosa.

Topics: Aged; Breast Neoplasms; Cation Transport Proteins; Cell Cycle Proteins; Colorectal Neoplasms; Cyclin D1; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Male; Middle Aged; Nuclear Proteins; Transcription Factors; Wnt Signaling Pathway; Zinc

Topics: Adenomatous Polyposis Coli Protein; Animals; Antineoplastic Agents; Bacteria; Berberine; beta Catenin; Colorectal Neoplasms; Cyclin D1; Diet, High-Fat; DNA, Bacterial; DNA, Ribosomal; Gastrointestinal Microbiome; Gene Expression Regulation, Neoplastic; High-Throughput Nucleotide Sequencing; Male; Mice; Neoplasms, Experimental; RNA, Ribosomal, 16S; Sequence Analysis, DNA; Xenograft Model Antitumor Assays

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

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

The present study was aimed at examining Ezrin expression in human colorectal cancer (CRC) tissues and elucidating the influence of baicalein on the proliferation of HCT116 cells.. The expression of Ezrin was determined by qRT-PCR and immunohistochemistry. HCT116 cells were divided into four groups－ baicalein groups with various concentrations, pcDNA3.1-Ezrin group, si-Ezrin group and dual inhibitory group (baicalein + si-Ezrin). CCK-8 assay and flow cytometry (FCM) were employed to assess cell proliferation and to detect the distribution of cell cycle respectively. The expression levels of Ezrin protein and cell cycle-associated proteins were detected by using western blot. The proliferation ability of CRC cells was also evaluated in vivo.. Ezrin expression in CRC tissues was observably higher than that in adjacent colorectal tissues. With drug concentration and action time of baicalein increasing, the cell propagation capacity of HCT116 cells was decreased and the cell cycle progression was arrested. Ezrin expression was inhibited by the administration of baicalein in a dose-dependent way. The levels of CyclinD1 and CDK4 were also significantly decreased, but the expression of P53 pathway proteins P53 and P21 was markedly upregulated.. Baicalein repressed proliferation of human colorectal cancer cells HCT116 and blocked cell cycle through downregulating Ezrin and upregulating P53 pathway-related proteins.

Topics: Animals; Cell Cycle Checkpoints; Cell Proliferation; Colorectal Neoplasms; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase Inhibitor p21; Cytoskeletal Proteins; Down-Regulation; Female; Flavanones; HCT116 Cells; Humans; Male; Mice; Mice, Nude; Middle Aged; RNA Interference; RNA, Small Interfering; Tumor Suppressor Protein p53

Objective To investigate the effect of miR-107 on the proliferation of colorectal cancer cells. Methods The expression of miR-107 was detected in colorectal cancer tissues and colorectal cancer cell lines by real-time quantitative PCR (qRT-PCR). miR-107 expression was up-regulated and down-regulated by the transfection of miR-107 mimic and miR-107 inhibitor in HT29 cells, respectively. The effect of miR-107 on the proliferation of HT29 cells was evaluated by MTT assay. The tumorigenic ability of HT29 cells was detected by soft-agar colony formation assay and nude mouse tumorigenic assay. The expression of cyclin D1 in HT29 cells was tested by Western blot analysis. Results The expression of miR-107 was significantly up-regulated in both colorectal cancer tissues and cells. Overexpression of miR-107 promoted the proliferation and tumorigenicity of HT29 cells, while the decrease of miR-107 expression inhibited the proliferation and tumorigenicity of HT29 cells. Overexpression of miR-107 up-regulated the expression of cyclin D1 in HT29 cells. Conclusion Overexpression of miR-107 in both colon cancer tissues and cells promotes the proliferation and tumor formation potential of HT29 cells via up-regulating cyclin D1 expression.

Topics: Animals; Cell Proliferation; Colorectal Neoplasms; Cyclin D1; Gene Expression Regulation, Neoplastic; HT29 Cells; Humans; Mice; Mice, Nude; MicroRNAs

The comprehensive screening of intracellular and extracellular microRNAs was performed to identify novel tumor suppressors. We found that miR-8073 was present in exosome and predominantly exported from colorectal cancer cells. Treatment with a synthetic miR-8073 mimic resulted in a dramatic decrease in the proliferation of various types of cancer cells, which was not observed in similarly treated normal cells. As little is known about the biological functions of miR-8073, its target mRNAs were analyzed by both mRNA expression and in silico sequence analyses, leading to five probable target candidates (FOXM1, MBD3, CCND1, KLK10, and CASP2) that enhance survival during the regulation of the cell cycle, cell proliferation, and apoptosis. We experimentally confirmed that miR-8073 binds the 3'-UTR of each of these mRNA target candidates and that the introduction of a synthetic miR-8073 mimic into cancer cells reduced levels of protein expression. Finally, the antiproliferative effects of miR-8073 were validated in vivo: the subcutaneous injection of a synthetic miR-8073 mimic suppressed colorectal tumor volume to 43% in tumor-bearing xenografted mice. These results suggest that because miR-8073 binds, and thus reduces the levels of, these oncogenic targets, cancer cells must actively downregulate miR-8073 as a survival mechanism. The introduction of miR-8073 into tumors could thus inhibit tumor growth, indicating its great potential for cancer therapeutics.

Topics: 3' Untranslated Regions; A549 Cells; Animals; Apoptosis; Caspase 2; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Colorectal Neoplasms; Cyclin D1; Cysteine Endopeptidases; DNA-Binding Proteins; Female; Forkhead Box Protein M1; Gene Expression Regulation, Neoplastic; HCT116 Cells; HEK293 Cells; HT29 Cells; Humans; Kallikreins; MCF-7 Cells; Mice; Mice, Nude; MicroRNAs; RNA, Messenger

Long non-coding RNAs (lncRNAs) function in the development and progression of cancer, but only a small portion of lncRNAs have been characterized to date. A novel lncRNA transcript, 2.53 kb in length, was identified by transcriptome sequencing analysis, and was named p53-inducible cancer-associated RNA transcript 1 (PICART1). PICART1 was found to be upregulated by p53 through a p53-binding site at -1808 to -1783 bp. In breast and colorectal cancer cells and tissues, PICART1 expression was found to be decreased. Ectopic expression of PICART1 suppressed the growth, proliferation, migration, and invasion of MCF7, MDA-MB-231 and HCT116 cells whereas silencing of PICART1 stimulated cell growth and migration. In these cells, the expression of PICART1 suppressed levels of p-AKT (Thr308 and Ser473) and p-GSK3β (Ser9), and accordingly, β-catenin, cyclin D1 and c-Myc expression were decreased, while p21Waf/cip1 expression was increased. Together these data suggest that PICART1 is a novel p53-inducible tumor-suppressor lncRNA, functioning through the AKT/GSK3β/β-catenin signaling cascade.

Topics: beta Catenin; Breast Neoplasms; Cell Proliferation; Colorectal Neoplasms; Cyclin D1; Female; Gene Expression Regulation, Neoplastic; Glycogen Synthase Kinase 3 beta; HCT116 Cells; Humans; MCF-7 Cells; Oncogene Protein v-akt; RNA, Long Noncoding; Tumor Suppressor Protein p53

One long-term complication of chronic intestinal inflammation is the development of colorectal cancer. However, the mechanisms linking inflammation to the colorectal tumorigenesis are poorly defined. Previously, we have demonstrated that galectin-4 is predominantly expressed in the luminal epithelia of the gastrointestinal tract, and its loss of expression plays a key role in the colorectal tumorigenesis. However, the mechanism by which galectin-4 regulates inflammation-induced tumorigenesis is unclear. Here, we show that galectin-4 secreted by the colorectal cancer cell lines was bound to the cell surface. Neutralization of surface-bound galectin-4 with anti-galectin-4 antibody resulted in increased cell proliferation with concomitant secretion of several chemokines into the extracellular medium. Neutralization of the surface-bound galectin-4 also resulted in the up-regulation of transcription of 29 genes, several of which are components of multiple inflammation signaling pathways. In an alternate experiment, binding of recombinant galectin-4 protein to cell surface of the galectin-4-negative colorectal cancer cells resulted in increased p27, and decreased cyclin D1 and c-Myc levels, leading to cell cycle arrest and apoptosis. Together, these data demonstrated that surface-bound galectin-4 is a dual function protein-down-regulating cell proliferation and chemokine secretion in galectin-4-expressing colorectal cancer cells on one hand and inducing apoptosis in galectin-4-negative colorectal cancer cells on the other hand.

Topics: Apoptosis; Carcinogenesis; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Chemokines; Colorectal Neoplasms; Cyclin D1; Galectin 4; Gene Expression Regulation, Neoplastic; HCT116 Cells; HT29 Cells; Humans; Inflammation; Intestinal Mucosa; Proliferating Cell Nuclear Antigen; Proto-Oncogene Proteins c-myc

MTA3 overexpression has been implicated in carcinogenesis. The aim of the present study was to explore the clinical significance and biological roles of MTA3 in human colorectal cancer and colorectal cancer cells. A total of 80 cases of colorectal cancer tissues were examined by immunohistochemistry for MTA3 protein expression. We analyzed the relationship between MTA3 and clinical factors and the results showed that MTA3 was overexpressed in 51.25% (41/80) cancer cases. There was significant associations between MTA3 overexpression and advanced TNM stage (p = 0.0086) and Ki67 index (p = 0.001). We overexpressed MTA3 in LoVo cells and depleted its expression in HCT15 cells. The results showed that MTA3 promoted cancer cell proliferation, invasion, migration, and cell cycle progression, and inhibited 5-fluorouracil-induced apoptosis in LoVo cell line. MTA3 depletion in HCT15 cell line showed the opposite effects. In addition, we found that MTA3 positively regulated cell cycle proteins including cyclin D1 and cyclin E. It also upregulated Bcl2 and downregulated Bax expression. Furthermore, we found that MTA3 could activate Wnt signaling pathway by upregulating Wnt target proteins. Our results demonstrated that MTA3 overexpression contributes to colorectal cancer carcinogenesis, progression, and chemoresistance. MTA3 could serve as a potential therapeutic target in colorectal cancer.

Topics: Adult; Aged; Apoptosis; Carcinogenesis; Cell Proliferation; Colorectal Neoplasms; Cyclin D1; Cyclin E; Disease Progression; Female; Fluorouracil; Gene Expression Regulation, Neoplastic; Humans; Male; Middle Aged; Neoplasm Proteins; Neoplasm Staging; Oncogene Proteins; Wnt Signaling Pathway

The infrequent manifestation of SIRT1 and Aurora B kinase has shown to play a pivotal role in colorectal cancer (CRC) progression by regulating Wnt signaling pathway. The present study investigates the signaling events that regulate the modulation of SIRT1 and Aurora B kinase expression and it's mediated cell proliferation in SW480 human primary adenocarcinoma CRC cells using Butea monosperma floral compounds (BMFC). In this, cell viability, mitochondrial mediated apoptosis, cell cycle arrest and inhibition of Wnt pathway were examined. Interestingly, the active novel compound, sodium salt of butrin, from BMFC significantly enhances the apoptosis activity, where SIRT1 and Aurora B kinase were ectopically overexpressed in CRC cells. Moreover, mRNA and protein expressions analysis indicates that the expression of GSK-3β, β-catenin, cyclin D1, pAKT, TGF-3β, SIRT1 and Aurora B kinase were down regulated in BMFC treated cells. These findings provide valuable information that the active BMFC having great impact on SIRT1 and Aurora B kinase mediated Wnt signaling down regulation in SW480 CRC cells.

Topics: Apoptosis; Aurora Kinase B; beta Catenin; Butea; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Cell Survival; Colorectal Neoplasms; Cyclin D1; Flavonoids; Flowers; Gene Expression Regulation, Neoplastic; Glycogen Synthase Kinase 3 beta; Humans; Mitochondria; RNA, Messenger; Sirtuin 1; Sodium; Wnt Signaling Pathway

The canonical Wnt signaling pathway activates the transcriptional activity of β-catenin. This pathway is often activated in colorectal cancer cells, but strategies to block it in tumors have not been effective. The SAM pointed domain containing ETS transcription factor (SPDEF) suppresses formation of colon tumors by unclear mechanisms. We investigated these mechanisms and the effects of SPDEF on β-catenin activity in mouse models of colorectal cancer (CRC), CRC cell lines, and mouse and human normal and cancer colonoids.. We performed studies of Lgr5. Expression of SPDEF was sufficient to inhibit intestinal tumorigenesis by activated β-catenin, block tumor cell proliferation, and restrict growth of established tumors. In tumor cells with activated β -catenin, expression of SPDEF induced a quiescent state, which was reversed when SPDEF expression was stopped. In mouse and human normal and tumor-derived enteroids/colonoids, those that expressed SPDEF for 3 days were significantly smaller. SPDEF inhibited the transcriptional activity of β-catenin via a protein-protein interaction, independent of SPDEF DNA binding capacity. SPDEF disrupted β-catenin binding to TCF1 and TCF3, displacing β-catenin from enhancer regions of genes that regulate the cell cycle but not genes that regulate stem cell activities.. In studies of mice and human CRC, we found that SPDEF induces a quiescent state in CRC cells by disrupting binding of β-catenin to TCF1 and TCF3 and regulation of genes that control the cell cycle. In this model, β-catenin activity determines the proliferation or quiescence of CRC cells based on the absence or presence of SPDEF.

Topics: Adenoma; Animals; Axin Protein; beta Catenin; Carcinogenesis; Cell Cycle; Cell Proliferation; Chromatin; Colon; Colorectal Neoplasms; Cyclin D1; Gene Expression Regulation, Neoplastic; HCT116 Cells; HEK293 Cells; Humans; Hyaluronan Receptors; Intestinal Mucosa; Lymphoid Enhancer-Binding Factor 1; Male; Mice; Organoids; Promoter Regions, Genetic; Proto-Oncogene Proteins c-ets; Proto-Oncogene Proteins c-myc; Receptor, EphB2; Stem Cells; TCF Transcription Factors; Transcription, Genetic; Transfection; Wnt Signaling Pathway

Topics: Animals; beta Catenin; Carcinogenesis; Cell Line, Tumor; Chemoprevention; Colorectal Neoplasms; Cyclin D1; Diet; DNA, Bacterial; Enterococcus faecalis; Face; Female; Functional Food; HCT116 Cells; Hot Temperature; Humans; Intestinal Polyps; Male; Mice; Mice, Inbred C57BL; Promoter Regions, Genetic; Proto-Oncogene Proteins c-myc; RNA, Messenger; Signal Transduction; TCF Transcription Factors; Transcriptional Activation

We examined a set of 805 cases that underwent DNA sequencing using the FoundationOne Heme (F1H) targeted sequencing panel and gene expression profiling. Known and likely variant calls from the mutational data were analyzed for significant associations with gene expression defined translocation cyclin D (TC) molecular subgroups. The spectrum of KRAS, NRAS, and BRAF codon mutations varied across subgroups with NRAS mutations at Q61 codon being common in hyperdiploid (HRD) and t(11;14) myeloma while being rare in MMSET and MAF. In addition, the presence of RAS-RAF mutations was inversely associated with NFκB pathway activation in all subgroups excluding MAF. In the MMSET subgroup, cases with low FGFR3 expression frequently had RAS-RAF mutations. Conditional inference tree analysis determined that mutation and homozygous deletion of TP53, CDKN2C, and RB1 were key prognostic factors associated with adverse outcome in a non-relapse clinical setting. In conclusion, this study highlights the heterogeneity in the distribution and clinical outcomes of RAS codon and other mutations in multiple myeloma dependent upon primary molecular subgroup.

Topics: Codon; Colorectal Neoplasms; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p18; DNA; DNA Mutational Analysis; Gene Expression Profiling; GTP Phosphohydrolases; Humans; Membrane Proteins; Multiple Myeloma; Mutation; NF-kappa B; Prognosis; Proto-Oncogene Proteins B-raf; Proto-Oncogene Proteins p21(ras); Receptor, Fibroblast Growth Factor, Type 3; Retinoblastoma Binding Proteins; Sequence Analysis, DNA; Signal Transduction; Translocation, Genetic; Tumor Suppressor Protein p53; Ubiquitin-Protein Ligases

As a major in vivo condensation product of indole-3-carbinol, which is mostly present in cruciferous vegetables, 3,3'-diindolylmethane (DIM) has been previously reported with anti-proliferative action in different types of cancer by our group and others. To further elucidate these underlying mechanisms, we examined the effect of DIM on cyclin D1, which was aberrantly overexpressed in various cancer cells and tumors. Herein, we found that DIM downregulated cyclin D1 expression in colorectal cancer cells (CRC), which was independent of PPARγ expression and protease activity. Furthermore, DIM did not affect cyclin D1 mRNA expression, suggesting DIM modulated cyclin D1 expression at the translational level. Subsequently, blocking eIF2α phosphorylation resulted from endoplasmic reticulum (ER) stress restored cyclin D1 in the presence of DIM. Thus, the present study demonstrates that DIM downregulates cyclin D1 through triggering ER stress in human colorectal cancer cells.

Topics: Animals; Anticarcinogenic Agents; Cell Line, Tumor; Colorectal Neoplasms; Cyclin D1; Down-Regulation; Endoplasmic Reticulum Stress; Eukaryotic Initiation Factor-2; Humans; Indoles; Phosphorylation; PPAR gamma; RNA, Messenger

Protocadherin18 (PCDH18) was found to be preferentially methylated and inactivated in colorectal cancer (CRC) using bioinformatics tools. However, its biologic role in tumorgenesis remains unclear. Herein, we aimed to elucidate its epigenetic regulation and biological functions in CRC. The methylation status of PCDH18 was significant higher in CRC tissues than in adjacent non-tumor tissues (median, 15.17% vs. median, 0.4438%). Expression level of PCDH18 was significantly lower in primary CRCs than in nonmalignant tissues. Importantly, methylation status of PCDH18 in cell-free DNA of CRC patients was also significantly higher than in healthy subjects. PCDH18 was readily expressed in NCM460 cells, but downregulated in 100% (4/4) of CRC cell lines by promoter methylation, despite its expression could be restored through demethylation treatment. Overexpression of PCDH18 suppressed CRC cell viability, colony formation and migration. Meanwhile, the depletion of PCDH18 by siRNA in NCM460 cells enhanced the colonogenicity and migration ability and promoted β-catenin nuclear accumulation, whereas it inhibited cell cycle arrest. These effects were associated with upregulation of phospho-GSK-3β and cyclin D1, and downregulation of caspase3 and p21. Our results suggested that PCDH18 was a putative tumor suppressor with epigenetic silencing in CRC and a potential biomarker for CRC diagnosis.

Topics: Aged; beta Catenin; Cadherins; Caspase 3; Cell Line, Tumor; Cell Movement; Cell-Free Nucleic Acids; Colorectal Neoplasms; Cyclin D1; DNA Methylation; Epigenesis, Genetic; Female; Gene Expression Regulation, Neoplastic; Glycogen Synthase Kinase 3 beta; Humans; Male; Middle Aged; Promoter Regions, Genetic; Protocadherins; RNA, Small Interfering

The aim of the present study was to clarify the clinical implication and functional role of tripartite motif-59 (TRIM59) in colorectal carcinoma (CRC) and explore the underlying mechanism of aberrant high expression of TRIM59 in cancer. We validated that TRIM59 was upregulated in CRC samples, and also demonstrated that its upregulation was associated with advanced tumor stage of CRC patients; and its high expression indicated shorter overall survival and faster recurrence. Knockdown of TRIM59 significantly inhibited cell proliferation, migration and invasion. Cell cycle analysis showed that TRIM59-depleted cells accumulated in S-phase. In addition, the cell cycle regulators CDC25C, cyclin B1 and cyclin D1 were decreased by TRIM59 siRNA mediated knockdown. Furthermore, the depletion of TRIM59 promoted apoptosis in cell culture as indicated by the cleavage of caspase-3 and PARP when TRIM59 was depleted. These results suggested that TRIM59 is upregulated in human colorectal tumors compared with non-tumor tissues. The level of TRIM59 is correlated with malignant features of CRC and may serve as potential therapeutic and preventive strategies for CRC.

Topics: Aged; Caspase 3; cdc25 Phosphatases; Cell Line, Tumor; Cell Movement; Cell Proliferation; Colorectal Neoplasms; Cyclin B1; Cyclin D1; Female; Gene Expression Regulation, Neoplastic; Humans; Intracellular Signaling Peptides and Proteins; Male; Membrane Proteins; Metalloproteins; Middle Aged; Neoplasm Invasiveness; Neoplasm Recurrence, Local; Poly(ADP-ribose) Polymerases; Tripartite Motif Proteins

Although it has been reported to contain high polyphenols, the pharmacological studies of the calyx of Diospyros kaki Thunb (DKC) have not been elucidated in detail. In this study, we elucidated anti-cancer activity and potential molecular mechanism of DKC against human colorectal cancer cells.. Anti-cell proliferative effect of 70% ethanol extracts from the calyx of Diospyros kaki (DKC-E70) was evaluated by MTT assay. The effect of DKC-E70 on the expression of cyclin D1 in the protein and mRNA level was evaluated by Western blot and RT-PCR, respectively.. DKC-E70 suppressed the proliferation of human colorectal cancer cell lines such as HCT116, SW480, LoVo and HT-29. Although DKC-E70 decreased cyclin D1 expression in protein and mRNA level, decreased level of cyclin D1 protein by DKC-E70 occurred at the earlier time than that of cyclin D1 mRNA, which indicates that DKC-E70-mediated downregulation of cyclin D1 protein may be a consequence of the induction of degradation and transcriptional inhibition of cyclin D1. In cyclin D1 degradation, we found that cyclin D1 downregulation by DKC-E70 was attenuated in presence of MG132. In addition, DKC-E70 phosphorylated threonine-286 (T286) of cyclin D1 and T286A abolished cyclin D1 downregulation by DKC-E70. We also observed that DKC-E70-mediated T286 phosphorylation and subsequent cyclin D1 degradation was blocked in presence of the inhibitors of ERK1/2, p38 or GSK3β. In cyclin D1 transcriptional inhibition, DKC-E70 inhibited the expression of β-catenin and TCF4, and β-catenin/TCF-dependent luciferase activity.. Our results suggest that DKC-E70 may downregulate cyclin D1 as one of the potential anti-cancer targets through cyclin D1 degradation by T286 phosphorylation dependent on ERK1/2, p38 or GSK3β, and cyclin D1 transcriptional inhibition through Wnt signaling. From these findings, DKC-E70 has potential to be a candidate for the development of chemoprevention or therapeutic agents for human colorectal cancer.

Topics: Antineoplastic Agents; beta Catenin; Cell Proliferation; Colorectal Neoplasms; Cyclin D1; Diospyros; HCT116 Cells; HT29 Cells; Humans; Phosphorylation; Plant Extracts; Proteasome Endopeptidase Complex; Proteolysis

Colorectal cancer (CRC) is the third most common cancer in the world.

Topics: Animals; beta Catenin; Cell Line, Tumor; Colorectal Neoplasms; Cyclin D1; Gene Expression Regulation, Neoplastic; HCT116 Cells; Humans; Male; Mice; Mice, Inbred C57BL; Mice, Mutant Strains; Oplopanax; Plant Extracts; Polyynes; Wnt Proteins

To determine the expression level of long non-coding RNA (lncRNA) imprinted maternallyexpressed transcript (. Real-time quantitative PCR (qRT-PCR) was applied to detect the expression of. The expression levels of

Topics: Cadherins; Cell Line, Tumor; Cell Proliferation; Colorectal Neoplasms; Cyclin D1; Humans; RNA, Long Noncoding; RNA, Small Interfering; Transfection

Microorganisms have been regarded as important sources of novel bioactive natural products. In this study, we evaluated the anti-cancer activity and the potential mechanism of Bacillus amyloliquefaciens AK-0 newly isolated from the rhizosphere soil of Korean ginseng. The ethyl acetate fraction from the culture medium of B. amyloliquefaciens AK-0 (EA-AK0) inhibited markedly the proliferation of human colorectal cancer cells such as HCT116, SW480, LoVo and HT-29. EA-AK0 effectively decreased cyclin D1 protein level in human colorectal cancer cells, while cyclin D1 mRNA level was not changed by EA-AK0 treatment. Inhibition of proteasomal degradation by MG132 blocked EA-AK0-mediated cyclin D1 downregulation and the half-life of cyclin D1 was decreased in the cells treated with MRB. In addition, EA-AK0 increased threonine-286 (T286) phosphorylation of cyclin D1, and a point mutation of T286 to alanine attenuated cyclin D1 degradation by EA-AK0. Inhibition of GSK3β by LiCl suppressed cyclin D1 phosphorylation and downregulation by EA-AKO. From these results, EA-AK0 may suppress the proliferation of human colorectal cancer cells by inducing cyclin D1 proteasomal degradation through GSK3β-dependent T286 phosphorylation. These results indicate that EA-AK0 could be used for treating colorectal cancer and serve as a potential candidate for anticancer drug development. In addition, these findings will be helpful for expanding the knowledge on the molecular anti-cancer mechanisms of EA-AK0.

Topics: Antineoplastic Agents; Bacillus amyloliquefaciens; Cell Line, Tumor; Cell Proliferation; Colorectal Neoplasms; Cyclin D1; Down-Regulation; Gene Expression Regulation, Neoplastic; Glycogen Synthase Kinase 3 beta; Humans; Phosphorylation; Rhizosphere; Soil Microbiology; Threonine

Intervention strategies in familial adenomatous polyposis (FAP) patients and other high-risk colorectal cancer (CRC) populations have highlighted a critical need for endoscopy combined with safe and effective preventive agents. We performed transcriptome profiling of colorectal adenomas from FAP patients and the polyposis in rat colon (Pirc) preclinical model, and prioritized molecular targets for prevention studies in vivo. At clinically relevant doses in the Pirc model, the drug Clotam (tolfenamic acid, TA) was highly effective at suppressing tumorigenesis both in the colon and in the small intestine, when administered alone or in combination with Sulindac. Cell proliferation in the colonic crypts was reduced significantly by TA, coincident with increased cleaved caspase-3 and decreased Survivin, β-catenin, cyclin D1 and matrix metalloproteinase 7. From the list of differentially expressed genes prioritized by transcriptome profiling, Mmp7, S100a9, Nppb and Aldh1a3 were defined as key oncogene candidates downregulated in colon tumors after TA treatment. Monthly colonoscopies revealed the rapid onset of tumor suppression by TA in the Pirc model, and the temporal changes in Mmp7, S100a9, Nppb and Aldh1a3, highlighting their value as potential early biomarkers for prevention in the clinical setting. We conclude that TA, an "old drug" repurposed from migraine, offers an exciting new therapeutic avenue in FAP and other high-risk CRC patient populations.

Topics: Adenoma; Adenomatous Polyposis Coli; Aldehyde Oxidoreductases; Animals; beta Catenin; Biomarkers, Tumor; Calgranulin B; Carcinogenesis; Caspase 3; Cell Proliferation; Colorectal Neoplasms; Cyclin D1; Down-Regulation; Gene Expression Profiling; Humans; Male; Matrix Metalloproteinase 7; Oncogenes; ortho-Aminobenzoates; Rats

Colorectal cancer (CRC) is a major cause of cancer morbidity and mortality worldwide. Although response rates and overall survival have been improved in recent years, resistance to multiple drug combinations is inevitable. Therefore, the development of more efficient drugs, with fewer side effects is urgently needed. To this end, we have investigated in the present report the effect of PAC, a novel cucumin analogue, on CRC cells both in vitro and in vivo. We have shown that PAC induces apoptosis, mainly via the internal mitochondrial route, and inhibits cell proliferation through delaying the cell cycle at G2/M phase. Interestingly, the pro-apoptotic effect was mediated through STAT3-dependent down-regulation of cyclin D1 and its downstream target survivin. Indeed, change in the expression level of cyclin D1 modulated the expression of survivin and the response of CRC cells to PAC. Furthermore, using the ChIP assay, we have shown PAC-dependent reduction in the binding of STAT3 to the cyclin D1 promoter in vivo. Additionally, PAC suppressed the epithelial-to-mesenchymal process through down-regulating the mesenchymal markers (N-cadherin, vimentin and Twist1) and inhibiting the invasion/migration abilities of the CRC cells via repressing the pro-migration/invasion protein kinases AKT and ERK1/2. In addition, PAC inhibited tumor growth and repressed the JAK2/STAT3, AKT/mTOR and MEK/ERK pathways as well as their common downstream effectors cyclin D1 and survivin in humanized CRC xenografts. Collectively, these results indicate that PAC has potent anti-CRC effects, and therefore could constitute an effective alternative chemotherapeutic agent, which may consolidate the adjuvant treatment of colon cancer.

Topics: Animals; Antineoplastic Agents; Apoptosis; Cell Proliferation; Colon; Colorectal Neoplasms; Curcumin; Cyclin D1; Epithelial-Mesenchymal Transition; G2 Phase Cell Cycle Checkpoints; HCT116 Cells; Humans; M Phase Cell Cycle Checkpoints; Mice, Nude; Rectum; Signal Transduction; STAT3 Transcription Factor

Keratinocyte growth factor (KGF) stimulates normal growth, development and intestinal epithelial cell proliferation. Cyclin D1 promotes the cell cycle by inhibiting retinoblastoma protein (RB1). The activated aryl hydrocarbon receptor (AhR) has an important influence on the development of tumors through its interactions with the cell cycle.. The aim of the present study was to explore a new role for AhR in KGF-induced colon cancer cell growth.. Real-time PCR, western blot or immunofluorescence analysis were used to detect the expression of KGF, AhR, cyclin D1 and CYP1A1. Immunohistochemistry was used to observe the localization of AhR. MTT assay and flow cytometric analyses were performed to measure cell viability and the cell cycle.. Real-time PCR analysis revealed that KGF, AhR, and CYP1A1 mRNAs were overexpressed in colorectal cancer tissues. Meanwhile, overexpression of AhR was primarily observed in epithelial cells. In in vitro assay, KGF promoted colon cancer cell growth, as well as up-regulated and activated AhR. At the same time, AhR-knockdown colon cancer cells were less responsive to KGF. Western blot analysis, real-time PCR, or immunofluorescence data indicated that cyclin D1 expression was up-regulated by KGF but this up-regulation was compromised when AhR was silenced, and the cell cycle was arrested in the G0/G1 stage in these cells.. Our study suggests that KGF, AhR, and CYP1A1 are overexpressed in colorectal cancer tissues. Moreover, we reveal a new mechanism by which KGF promotes cell proliferation through the AhR-cyclin D1 pathway in colon cancer cells.

Topics: Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Colorectal Neoplasms; Cyclin D1; Cytochrome P-450 CYP1A1; Fibroblast Growth Factor 7; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Humans; Receptors, Aryl Hydrocarbon; RNA, Messenger

Vitamin A is required for normal body function, including vision, epithelial integrity, growth, and differentiation. All trans-retinoic acid (ATRA), a family member of vitamin A, has been explored in treating acute promyelocytic leukemia and other types of cancer. Dysregulated Wnt/β-catenin signaling and disrupted cadherin-catenin complex often contribute to colorectal malignancy. MED28, a mammalian Mediator subunit, is found highly expressed in breast and colorectal cancers. Our laboratory has also reported that MED28 regulates cell growth, migration, and invasion in human breast cancer cells. In the current study we investigated the effect of ATRA on MED28 and Wnt/β-catenin signaling in colorectal cancer. HCT116, HT29, SW480, and SW620, four human colorectal cancer cell lines representing different stages of carcinogenesis and harboring critical genetic changes, were employed. Our data indicated that regardless of genetic variations among these cells, suppression of MED28 reduced the expression of cyclin D1, c-Myc, and nuclear β-catenin, but increased the expression of E-cadherin and HMG box-containing protein 1 (HBP1) where HBP1 has been described as a negative regulator of the Wnt/β-catenin signaling. The reporter activity of an HBP1 promoter increased upon MED28 knockdown, but decreased upon MED28 overexpression. ATRA reduced the expression of MED28 and mimicked the effect of MED28 suppression in down-regulating Wnt/β-catenin signaling. Taken together, ATRA can reverse the suppressive effect of MED28 on HBP1 and E-cadherin and inactivate the Wnt/β-catenin pathway in colorectal cancer, suggesting a protective effect of ATRA against colorectal cancer. J. Cell. Physiol. 231: 1796-1803, 2016. © 2015 Wiley Periodicals, Inc.

Topics: Antigens, CD; Antineoplastic Agents; beta Catenin; Cadherins; Cell Survival; Colorectal Neoplasms; Cyclin D1; Dose-Response Relationship, Drug; Gene Expression Regulation, Neoplastic; HCT116 Cells; High Mobility Group Proteins; HT29 Cells; Humans; Mediator Complex; Promoter Regions, Genetic; Proto-Oncogene Proteins c-myc; Repressor Proteins; RNA Interference; Signal Transduction; Transfection; Tretinoin

The Groucho transcriptional co-repressor TLE4 protein has been shown to be a tumor suppressor in a subset of acute myeloid leukemia. However, little is known about its role in development and progression of solid tumor. In this study, we found that the expression of TLE4 in colorectal cancer (CRC) tissues was significantly higher than that in their matched adjacent intestine epithelial tissues. In addition, high expression of TLE4 was significantly correlated with advanced Dukes stage, lymph node metastasis and poor prognosis of CRC. Moreover, enforced expression of TLE4 in CRC cell lines significantly enhanced proliferation, invasion and tumor growth. On the contrary, knock down of TLE4 repressed cell proliferation, invasion and tumor growth. Furthermore, our study exhibited that the TLE4 promoted cell proliferation and invasion partially via activation of JNK-c-Jun pathway and subsequently increased cyclinD1 and decreased P27Kip1 expression. In conclusion, these results suggested that TLE4, a potential prognostic biomarker for CRC, plays an important role in the development and progression of human CRC.

Topics: Animals; Biomarkers, Tumor; Caco-2 Cells; Cell Line, Tumor; Cell Proliferation; Colorectal Neoplasms; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p27; Disease Progression; Enzyme Activation; HCT116 Cells; HT29 Cells; Humans; JNK Mitogen-Activated Protein Kinases; Lymphatic Metastasis; MAP Kinase Signaling System; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Invasiveness; Neoplasm Transplantation; Nuclear Proteins; Prognosis; Repressor Proteins; RNA Interference; RNA, Small Interfering; Transplantation, Heterologous; Tumor Suppressor Proteins

The purpose of the present study was to explore the role and mechanism of extracellular ecto-5'-nucleotidase (CD73) in human colorectal cancer growth. Firstly, CD73 expression was detected in colorectal cancer cell lines both at the mRNA and protein levels. Secondly, recombinant CD73 interference and overexpression lentiviruses were used, respectively. Colony formation assay, CCK-8 assay and flow cytometry were used to investigate the impact of CD73 on colorectal cancer cell proliferation and cell cycle distribution. Then, adenosine and CD73 enzyme activity inhibitor (APCP) were used to study the effect of CD73 on Epidermal growth factor receptor (EGFR) and β-catenin/cyclin D1 signaling pathways. Finally, a human colorectal cancer transplantation nude mouse model was used to observe the effect of CD73 on tumor growth in vivo. As the results showed, CD73 was highly expressed in the colorectal cancer cell lines. CD73 promoted colorectal cancer cell proliferation both in vivo and in vitro. CD73 activated EGFR and the β-catenin/cyclin D1 signaling pathways through its enzyme and non-enzyme activities. All of the results confirmed that CD73 promotes the growth of human colorectal cancer cells through EGFR and the β-catenin/cyclin D1 signaling pathway. CD73 may be used as a valuable biomarker of colorectal cancer.

Topics: 5'-Nucleotidase; Animals; Apoptosis; beta Catenin; Biomarkers, Tumor; Cell Proliferation; Colorectal Neoplasms; Cyclin D1; Enzyme Inhibitors; ErbB Receptors; Gene Expression Regulation, Neoplastic; GPI-Linked Proteins; Humans; Mice; RNA, Small Interfering; Signal Transduction

To assess the risk of colorectal cancer in humans with inactivation of NRF2, Nrf2-proficient (Nrf2(+/+) ) and -deficient (Nrf2(-/-) ) mice were exposed to potassium bromate (KBrO3 ) at concentrations of 750 or 1500 ppm for 52 weeks. Neoplastic proliferative lesions were observed in the small intestine and exhibited accumulations of β-catenin and cyclin D1. The lesions had characteristics similar to those in experimental models of human hereditary colorectal cancer. An additional 13-week study was performed to examine the role of Nrf2 in the effects of oxidative stress. Significant increase in combined incidences of preneoplastic and neoplastic lesions in Nrf2(-/-) mice administered high-dose KBrO3 . In the short-term study, although 8-hydroxydeoxyguanosine (8-OHdG) levels in the epithelial DNA of Nrf2(-/-) mice at the high dose were significantly lower than those of the corresponding Nrf2(+/+) mice, the difference was very small. mRNA levels of Nrf2-regulated genes were increased in Nrf2(+/+) mice. Overexpression of cyclooxygenase 2 (COX2) and increased numbers of proliferating cell nuclear antigen (PCNA)-positive cells in the jejunal crypts were observed in Nrf2(-/-) mice administered high-dose KBrO3 . Overall, these data suggested that individuals having single-nucleotide polymorphisms in NRF2 may have a risk of colorectal cancer to some extent.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Animals; beta Catenin; Cell Transformation, Neoplastic; Colorectal Neoplasms; Cyclin D1; Cyclooxygenase 2; Cytokines; Deoxyguanosine; Disease Models, Animal; Female; Gene Expression; Gene Silencing; Humans; Intestinal Mucosa; Intestine, Small; Mice; Mice, Knockout; NF-E2-Related Factor 2; Oxidative Stress; Proliferating Cell Nuclear Antigen

Thyroid hormone induces cancer cell proliferation through its cell surface receptor integrin αvβ3. Acting via integrin αvβ3, the deaminated T4 analog tetraiodothyroacetic acid (tetrac), and its nanoparticle formulation nano-diamino-tetrac (NDAT) could inhibit cell proliferation and xenograft growth. In this study, we investigated the T4 effects on proliferation in colorectal cancer cell lines based on the proliferation marker expressions at both mRNA and protein levels. The effects of tetrac/NDAT, the monoclonal anti-EGFR antibody cetuximab, and their combinations on colorectal cancer cell proliferation were examined according to the relevant gene expression profiles and cell count analysis. The results showed that T4 significantly enhanced PCNA, Cyclin D1 and c-Myc levels in both K-ras wild type HT-29 and mutant HCT 116 cells. In HCT 116 cells, the combination of NDAT and cetuximab significantly suppressed the mRNA expressions of proliferative genes PCNA, Cyclin D1, c-Myc and RRM2 raised by T4 compared to cetuximab alone. In addition, T4-suppressed mRNA expressions of pro-apoptotic genes p53 and RRM2B could be significantly elevated by the combination of NDAT and cetuximab compared to cetuximab alone. In the K-ras mutant HCT 116 cells, but not in the K-ras wild type COLO 205 cells, the combinations of tetrac/NDAT and cetuximab significantly reduced cell proliferation compared to cetuximab alone. In conclusion, T4 promoted colorectal cancer cell proliferation which could be repressed by tetrac and NDAT. The combinations of tetrac/NDAT and cetuximab potentiated cetuximab actions in K-ras mutant colorectal cancer cells.

Topics: Antineoplastic Agents; Blotting, Western; Cell Line, Tumor; Cell Proliferation; Cetuximab; Colorectal Neoplasms; Cyclin D1; Gene Expression Regulation, Neoplastic; Genes, myc; HCT116 Cells; HT29 Cells; Humans; Proliferating Cell Nuclear Antigen; Reverse Transcriptase Polymerase Chain Reaction; RNA, Ribosomal, 18S; Thyroid Hormones; Thyroxine

Recent studies suggest that tumor-associated macrophage-produced IL-6 is an important mediator within the tumor microenvironment that promotes tumor growth. The activation of IL-6/STAT3 axis has been associated with chemoresistance and poor prognosis of a variety of cancers including colorectal carcinoma and thus serves as a potential immunotherapeutic target for cancer treatment. However, it is not fully understood whether anticytokine therapy could reverse chemosensitivity and enhance the suppressive effect of chemotherapy on tumor growth. In this study, we aimed to investigate the effect of IL-6 inhibition therapy on the antitumor effect of carboplatin. Enhanced expression of IL-6 and activation of STAT3 were observed in human colorectal carcinoma samples compared to normal colorectal tissue, with higher levels of IL-6/STAT3 in low grade carcinomas. Treatment of carboplatin (CBP) dose-dependently increased IL-6 production and STAT3 activation in human colorectal LoVo cells. Blockade of IL-6 with neutralizing antibody enhanced chemosensitivity of LoVo cells to carboplatin as evidenced by increased cell apoptosis. IL-6 blockade abolished carboplatin-induced STAT3 activation. IL-6 blockade and carboplatin synergistically reduced cyclin D1 expression and enhanced caspase-3 activity in LoVo cells. Our results suggest that inhibition of IL-6 may enhance chemosensitivity of colon cancers with overactive STAT3 to platinum agents.

Topics: Apoptosis; Carboplatin; Cell Line, Tumor; Colorectal Neoplasms; Cyclin D1; Enzyme-Linked Immunosorbent Assay; Humans; Immunohistochemistry; Interleukin-6; Signal Transduction; STAT3 Transcription Factor

The incidence of and mortality from colorectal cancers (CRC) can be reduced by early detection. Currently there is a lack of established markers to detect early neoplastic changes. We aimed to identify the copy number variations (CNVs) and the associated genes which could be potential markers for the detection of neoplasia in both ulcerative colitis-associated neoplasia (UC-CRN) and sporadic colorectal neoplasia (S-CRN).. We employed array comparative genome hybridization (aCGH) to identify CNVs in tissue samples of UC nonprogressor, progressor and sporadic CRC. Select genes within these CNV regions as a panel of markers were validated using quantitative real time PCR (qRT-PCR) method along with the microsatellite instability (MSI) in an independent cohort of samples. Immunohistochemistry (IHC) analysis was also performed.. Integrated analysis showed 10 overlapping CNV regions between UC-Progressor and S-CRN, with the 8q and 12p regions showing greater overlap. The qRT-PCR based panel of MYC, MYCN, CCND1, CCND2, EGFR and FNDC3A was successful in detecting neoplasia with an overall accuracy of 54% in S-CRN compared to that of 29% in UC neoplastic samples. IHC study showed that p53 and CCND1 were significantly overexpressed with an increasing frequency from pre-neoplastic to neoplastic stages. EGFR and AMACR were expressed only in the neoplastic conditions.. CNVs that are common and unique to both UC-associated and sporadic colorectal neoplasm could be the key players driving carcinogenesis. Comparative analysis of CNVs provides testable driver aberrations but needs further evaluation in larger cohorts of samples. These markers may help in developing more effective neoplasia-detection strategies during screening and surveillance programs.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Child; Colitis, Ulcerative; Colorectal Neoplasms; Comparative Genomic Hybridization; Cyclin D1; Cyclin D2; DNA Copy Number Variations; ErbB Receptors; Female; Fibronectins; Humans; Male; Microsatellite Instability; Middle Aged; N-Myc Proto-Oncogene Protein; Neoplasm Proteins; Proto-Oncogene Proteins c-myc

Novel, effective and safe agents are needed for the chemoprevention of colorectal cancer (CRC). This study investigated the effects of chitosan oligosaccharides (COS) on CRC progression and their underlying mechanisms and safety profiles in mice. Using a mouse model of colitis-associated CRC, we found that oral administration of COS (500mg/kg/day) resulted in a ∼60% reduction of tumor size and tumor numbers/sectioning. In addition, COS treatment increased AMPK activity, suppressed the NF-κB-mediated inflammatory response and reduced the expressions of cyclin D1, phosphorylated ribosomal protein S6, and MMP-9 in the colon tissues of these mice. Importantly, administration of COS (500mg/kg/day; 50 days) had no adverse effects on renal or liver functions. Our results indicate that COS suppressed CRC progression via AMPK activation and the suppression of NF-κB and mTOR signaling. COS may be of potential utility in the chemoprevention of CRC.

Topics: AMP-Activated Protein Kinases; Animals; Anticarcinogenic Agents; Chitosan; Colitis; Colon; Colorectal Neoplasms; Cyclin D1; Disease Models, Animal; Male; Matrix Metalloproteinase 9; Mice, Inbred C57BL; NF-kappa B; Oligosaccharides; TOR Serine-Threonine Kinases; Tumor Burden

Long intergenic noncoding RNAs (lincRNAs) have important roles in biological functions, molecular mechanisms and prognostic values in colorectal cancer (CRC). In this context, the roles of linc-UFC1 remain to be elucidated. In this study, linc-UFC1 was overexpressed in CRC patient tissues and positively correlated with tumor grade, N stage and M stage. Inhibition of linc-UFC1 resulted in cell proliferation inhibition and G1 cell cycle arrest, which was mediated by cyclin D1, CDK4, Rb and phosphorylated Rb. In addition, inhibition of linc-UFC1 induced cell apoptosis through the intrinsic apoptosis signaling pathway, as evidenced by the activation of caspase-9 and caspase-3. An investigation of the signaling pathway revealed that the effects on proliferation and apoptosis following linc-UFC1 knockdown were mediated by suppression of β-catenin and activation of phosphorylated P38. Furthermore, the P38 inhibitor SB203580 could attenuate the apoptotic effect achieved by linc-UFC1 knockdown, confirming the involvement of P38 signaling in the induced apoptosis. Taken together, linc-UFC1 might have a critical role in pro-proliferation and anti-apoptosis in CRC by regulating the cell cycle, intrinsic apoptosis, and β-catenin and P38 signaling. Thus, linc-UFC1 could be a potential therapeutic target and novel molecular biomarker for CRC.

Topics: Aged; Apoptosis; beta Catenin; Caspase 3; Caspase 9; Cell Line, Tumor; Cell Proliferation; Colorectal Neoplasms; Cyclin D1; Cyclin-Dependent Kinase 4; G1 Phase Cell Cycle Checkpoints; Gene Expression Regulation, Neoplastic; Humans; Imidazoles; Middle Aged; Neoplasm Grading; Neoplasm Staging; Oligoribonucleotides, Antisense; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Pyridines; Retinoblastoma Protein; RNA, Long Noncoding; Signal Transduction

Kahweol as a coffee-specific diterpene has been reported to exert anti-cancer properties. However, the mechanism responsible for the anti-cancer effects of kahweol is not fully understood. The main aim of this investigation was to determine the effect of kahweol on cell proliferation and the possible mechanisms in human colorectal cancer cells. Kahweol inhibited markedly the proliferation of human colorectal cancer cell lines such as HCT116, SW480. Kahweol decreased cyclin D1 protein level in HCT116 and SW480 cells. Contrast to protein levels, cyclin D1 mRNA level and promoter activity did not be changed by kahweol treatment. MG132 treatment attenuated kahweol-mediated cyclin D1 downregulation and the half-life of cyclin D1 was decreased in kahweol-treated cells. Kahweol increased phosphorylation of cyclin D1 at threonine-286 and a point mutation of threonine-286 to alanine attenuated cyclin D1 degradation by kahweol. Inhibition of ERK1/2 by PD98059, JNK by SP600125 or GSK3β by LiCl suppressed cyclin D1 phosphorylation and downregulation by kahweol. Furthermore, the inhibition of nuclear export by LMB attenuated cyclin D1 degradation by kahweol. In conclusion, kahweol-mediated cyclin D1 degradation may contribute to the inhibition of the proliferation in human colorectal cancer cells.

Topics: Antineoplastic Agents; Blotting, Western; Cell Proliferation; Coffee; Colorectal Neoplasms; Cyclin D1; Diterpenes; Glycogen Synthase Kinase 3 beta; Humans; MAP Kinase Kinase 4; MAP Kinase Signaling System; Phosphorylation; Proteasome Endopeptidase Complex; Proteolysis; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Threonine; Tumor Cells, Cultured

This study aimed to investigate the anticancer potential of indigocarpan (1), a pterocarpan isolated from Indigofera aspalathoides, a plant found in India which has been used in Ayurveda for centuries for the treatment of oedematous tumours.. The antiproliferative activity in a panel of four human cancer cell lines was studied. The mechanism of its antiproliferative activity in human colorectal adenocarcinoma LS174T cells was investigated in detail.. Indigocarpan (1) showed antiproliferative activity in a panel of four human cancer cell lines with IC50 s ranging from 180 to 250 μm. Indigocarpan induces p53-dependent p21 upregulation and apoptosis in LS174T cells, upregulates p53 and p21(WAF1) protein levels, enhances cleavage of caspase-3 and downregulates cyclin D1, cyclin B1 and PCNA protein levels, indicating its role in modulating cell cycle progression. Indigocarpan also exhibited a strong antioxidative effect in LS174T cells.. Along with the antiproliferative capacity, the strong antioxidative property of the compound makes it a promising candidate for further development as an anticancer and chemopreventive compound.

Topics: Adenocarcinoma; Antioxidants; Apoptosis; Cell Cycle; Cell Cycle Proteins; Cell Line, Tumor; Cell Proliferation; Colorectal Neoplasms; Cyclin B1; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Down-Regulation; Humans; India; Indigofera; Plant Extracts; Proliferating Cell Nuclear Antigen; Pterocarpans; Tumor Suppressor Protein p53; Up-Regulation

Increasing evidences have shown that B-cell translocation gene 3 (BTG3) inhibits metastasis of multiple cancer cells. However, the role of BTG3 in colorectal cancer (CRC) and its possible mechanism have not yet been reported. In our study, we evaluated BTG3 expression in several CRC cell lines. Then, pcDNA3.1-BTG3 was transfected into SW480 cells. We found that BTG3 was upregulated in SW480 cells after overexpression plasmid transfection. BTG3 overexpression significantly inhibited cell growth and decreased PCNA (proliferating cell nuclear antigen) and Ki67 levels. BTG3 overexpression markedly downregulated Cyclin D1 and Cyclin E1 levels, whereas elevated p27. Overexpression of BTG3 arrested the cell cycle at G1 phase, which was abrogated by p27 silencing. Furthermore, migration, invasion and EMT of SW480 cells were significantly suppressed by BTG3 overexpression. Further investigations showed the inhibition of Wnt/β-catenin signaling pathway. We then used GSK3β specific inhibitor SB-216763 to activate the Wnt/β-catenin signaling pathway. We found that Wnt/β-catenin signaling pathway activation reversed the effect of BTG3 overexpression on cell proliferation, cell cycle progression, invasion and EMT. In conclusion, BTG3 overexpression inhibited cell growth, induced cell cycle arrest and suppressed the metastasis of SW480 cells via the Wnt/β-catenin signaling pathway. BTG3 may be considered as a therapeutic target in CRC treatment.

Topics: Caco-2 Cells; Cell Cycle Proteins; Cell Line, Tumor; Cell Movement; Cell Proliferation; Colorectal Neoplasms; Cyclin D1; Cyclin E; Epithelial-Mesenchymal Transition; G1 Phase Cell Cycle Checkpoints; Glycogen Synthase Kinase 3 beta; Humans; Indoles; Ki-67 Antigen; Maleimides; Neoplasm Invasiveness; Oncogene Proteins; Proliferating Cell Nuclear Antigen; Proteins; RNA Interference; RNA, Small Interfering; Wnt Signaling Pathway

NHERF1/EBP50, an adaptor molecule that interacts with β-catenin, YAP, and PTEN, has been recently implicated in the progression of various human malignancies, including colorectal cancer. We report here that NHERF1 acts as a tumor suppressor in vivo for intestinal adenoma development. NHERF1 is highly expressed at the apical membrane of mucosa intestinal epithelial cells (IECs) and serosa mesothelial cells. NHERF1-deficient mice show overall longer small intestine and colon that most likely could be attributed to a combination of defects, including altered apical brush border of absorbtive IECs and increased number of secretory IECs. NHERF1 deficiency in Apc(Min/+) mice resulted in significantly shorter animal survival due to markedly increased tumor burden. This resulted from a moderate increase of the overall tumor density, more pronounced in females than males, and a massive increase in the number of large adenomas in both genders. The analysis of possible pathways controlling tumor size showed upregulation of Wnt-β-catenin pathway, higher expression of unphosphorylated YAP, and prominent nuclear expression of cyclin D1 in NHERF1-deficient tumors. Similar YAP changes, with relative decrease of phosphorylated YAP and increase of nuclear YAP expression, were observed as early as the adenoma stages in the progression of human colorectal cancer. This study discusses a complex role of NHERF1 for intestinal morphology and presents indisputable evidence for its in vivo tumor suppressor function upstream of Wnt-β-catenin and Hippo-YAP pathways.

Topics: Adaptor Proteins, Signal Transducing; Adenoma; Animals; beta Catenin; Carcinoma; Cell Cycle Proteins; Cell Transformation, Neoplastic; Colorectal Neoplasms; Cyclin D1; Genes, APC; Humans; Intestinal Mucosa; Mice; Mice, Knockout; Mutation; Phosphoproteins; Phosphorylation; Sodium-Hydrogen Exchangers; Tumor Burden; Wnt Proteins; Wnt Signaling Pathway; YAP-Signaling Proteins

We previously identified TSC22D2 (transforming growth factor β-stimulated clone 22 domain family, member 2) as a novel cancer-associated gene in a rare multi-cancer family. However, its role in tumor development remains completely unknown. In this study, we found that TSC22D2 was significantly downregulated in colorectal cancer (CRC) and that TSC22D2 overexpression inhibited cell growth. Using a co-immunoprecipitation (co-IP) assay combined with mass spectrometry analysis to identify TSC22D2-interacting proteins, we demonstrated that TSC22D2 interacts with pyruvate kinase isoform M2 (PKM2). These findings were confirmed by the results of immunoprecipitation and immunofluorescence assays. Moreover, overexpression of TSC22D2 reduced the level of nuclear PKM2 and suppressed cyclin D1 expression. Collectively, our study reveals a growth suppressor function of TSC22D2 that is at least partially dependent on the TSC22D2-PKM2-cyclinD1 regulatory axis. In addition, our data provide important clues that might contribute to future studies evaluating the role of TSC22D2.

Topics: Carrier Proteins; Cell Cycle; Cell Line, Tumor; Cell Nucleus; Cell Proliferation; Colorectal Neoplasms; Cyclin D1; DNA-Binding Proteins; Down-Regulation; Gene Expression Regulation, Neoplastic; Humans; Membrane Proteins; Thyroid Hormone-Binding Proteins; Thyroid Hormones; Transcription Factors

Tumor heterogeneity implies the possibility of significantly different expression of key pathways between primary and metastatic clones. Colon adenocarcinoma is one of the few tumors where current practice includes resection of primary and isolated organ metastases simultaneously without neoadjuvant therapy. We performed a pilot study on 28 cases of colon adenocarcinoma resected simultaneously with metastases in patients with no history of neoadjuvant therapy. We assayed matched primary and metastatic tumors from each patient with common diagnostic antibodies to Bcl-2, Cyclin D1, AMACR, and ALDH-1 by immunohistochemistry with semi-quantitative interpretation on archived formalin fixed, paraffin embedded samples. We were powered for large, consistent differences between primary and metastatic expression, and found 21 of 28 had a significant difference in expression of at least one of the four proteins, accounting for multiplicity of testing. Cyclin D1 had significantly more cases with differential metastatic:primary expression than would be expected by chance alone (p-value 0.0043), favoring higher expression in the metastatic sample. Bcl-2 and ALDH-1 had trends in this direction (p-value 0.078 each). Proportionately more cases with significant differences were identified when a liver metastasis was tested. We conclude differences in expression between metastatic and primary colon adenocarcinoma within the same patient exist, and may have therapeutic and biomarker testing consequences.

Topics: Adenocarcinoma; Aldehyde Dehydrogenase 1 Family; Biomarkers, Tumor; Colorectal Neoplasms; Cyclin D1; Humans; Immunohistochemistry; Isoenzymes; Neoplasm Metastasis; Pilot Projects; Proto-Oncogene Proteins c-bcl-2; Racemases and Epimerases; Retinal Dehydrogenase; Retrospective Studies

Deubiquitinases are important components of the protein degradation regulatory network. We report the discovery of ML364, a small molecule inhibitor of the deubiquitinase USP2 and its use to interrogate the biology of USP2 and its putative substrate cyclin D1. ML364 has an IC

Topics: Cell Cycle Checkpoints; Cell Line, Tumor; Colorectal Neoplasms; Cyclin D1; DNA Damage; DNA Repair; Endopeptidases; Humans; Lymphoma, Mantle-Cell; Neoplasm Proteins; Protease Inhibitors; Proteolysis; Ubiquitin Thiolesterase

Accumulative evidence suggests that alterations due to mutations or genetic polymorphisms in the TCF7L2 and CCND1 genes, which are components of the Wnt signaling pathway, contributes to carcinogenesis. The present study was designated to clarify whether common single nucleotide polymorphisms (SNPs) of the transcription factor 7- like 2 (TCF7L2) and cyclin D1 (CCND1) genes are associated with colorectal cancer risk in Mexican patients. A case-control study including 197 colorectal cancer patients and 100 healthy subjects was conducted in a Mexican population. Identification of polymorphisms was made by the polymerase chain reaction-restriction fragment length polymorphism methodology. The association was calculated by the odds ratio (OR) test. The results demonstrate that patients with the T/T genotype for the rs12255372 polymorphism of the TCF7L2 gene present an increased colorectal cancer risk (OR=2.64, P=0.0236). Also, the risk analysis for Tumor-Nodule-Metastasis (TNM) stage and tumor location showed association with this polymorphism under the over-dominant model of inheritance (OR=1.75, P=0.0440). A similar relation was observed for the genotype T/T of the rs7903146 polymorphism and the rectal location of cancer (OR=7.57, P=0.0403). For the rs603965 polymorphism of the CCND1 gene, we observed a protection effect for the colon cancer location under the dominant model (OR=0.49, P=0.0477). These results reveal a significant role of the analyzed polymorphisms in the TCF7L2 and CCND1 genes on the susceptibility or protection for developing colorectal cancer in the Mexican population.

Topics: Adult; Alleles; Case-Control Studies; Colorectal Neoplasms; Cyclin D1; Demography; Female; Gene Frequency; Genetic Predisposition to Disease; Genotype; Humans; Logistic Models; Male; Mexico; Middle Aged; Neoplasm Staging; Odds Ratio; Polymorphism, Single Nucleotide; Transcription Factor 7-Like 2 Protein

Cortactin is a scaffolding protein that regulates Arp2/3-mediated actin polymerization. We showed in a previous study that cortactin was highly expressed in human stage II-III colorectal cancer (CRC) tissues. In the present study, using colony formation and CCK-8 assays, we showed that overexpression of cortactin accelerated the proliferation of CRC cells. Flow cytometric assays revealed that cortactin promoted G1/S phase cell cycle transition. Later, we constructed the phosphorylation mutation of cortactin at the Tyr421 residue. Colony formation and CCK-8 assays showed that cortactin/Tyr421A lost its ability to promote cell proliferation. Western blot analysis indicated that cortactin activated cyclin D1, but not cortactin/Tyr421A. Further study in nude mice revealed that there was a greater decrease in both tumor volume and tumor weight in animals injected with SW480/cortactin/Tyr421A cells than in those injected with SW480/cortactin/WT cells. Thus, the present study demonstrates that the cortactin Tyr421 residue is required to promote cell proliferation both in vitro and in vivo.

Topics: Animals; Carcinogenesis; Cell Line, Tumor; Cell Proliferation; Colorectal Neoplasms; Cortactin; Cyclin D1; G1 Phase Cell Cycle Checkpoints; Gene Expression Regulation, Neoplastic; Humans; Mice, Inbred BALB C; Mice, Nude; Mutation, Missense; Transcriptional Activation

Topics: Adenocarcinoma; Adenoma; beta Catenin; Biomarkers, Tumor; Cancer-Associated Fibroblasts; Carcinogenesis; Colorectal Neoplasms; Cyclin D1; Disease Progression; Humans; Immunohistochemistry; Ki-67 Antigen; Neoplasm Grading; Neoplasm Recurrence, Local; Neprilysin; Predictive Value of Tests; Tumor Suppressor Protein p53

Colorectal cancer is the third most common cancer worldwide. Aberrant overexpression of antiapoptotic BCL-2 (B-cell lymphoma 2) family proteins is closely linked to tumorigenesis and poor prognosis in colorectal cancer. Obatoclax is an inhibitor targeting all antiapoptotic BCL-2 proteins. A previous study has described the antiproliferative action of obatoclax in one human colorectal cancer cell line without elucidating the underlying mechanisms. We herein reported that, in a panel of human colorectal cancer cell lines, obatoclax inhibits cell proliferation, suppresses clonogenicity, and induces G₁-phase cell cycle arrest, along with cyclin D1 downregulation. Notably, ectopic cyclin D1 overexpression abrogated clonogenicity suppression but also G₁-phase arrest elicited by obatoclax. Mechanistically, pre-treatment with the proteasome inhibitor MG-132 restored cyclin D1 levels in all obatoclax-treated cell lines. Cycloheximide chase analyses further revealed an evident reduction in the half-life of cyclin D1 protein by obatoclax, confirming that obatoclax downregulates cyclin D1 through induction of cyclin D1 proteasomal degradation. Lastly, threonine 286 phosphorylation of cyclin D1, which is essential for initiating cyclin D1 proteasomal degradation, was induced by obatoclax in one cell line but not others. Collectively, we reveal a novel anticancer mechanism of obatoclax by validating that obatoclax targets cyclin D1 for proteasomal degradation to downregulate cyclin D1 for inducing antiproliferation.

Topics: Carcinoma; Cell Proliferation; Colorectal Neoplasms; Cyclin D1; Down-Regulation; G1 Phase Cell Cycle Checkpoints; HCT116 Cells; HT29 Cells; Humans; Indoles; Proteolysis; Proto-Oncogene Proteins c-bcl-2; Pyrroles

Colorectal cancer, a leading cause of cancer death, has been linked to inflammation and obesity. Berberine, an isoquinoline alkaloid, possesses anti-inflammatory, anti-diabetes and anti-tumor properties. In the azoxymethane initiated and dextran sulfate sodium (AOM/DSS) promoted colorectal carcinogenesis mouse model, berberine treated mice showed a 60% reduction in tumor number (P = 0.009), a 48% reduction in tumors <2 mm, (P = 0.05); 94% reduction in tumors 2-4 mm, (P = 0.001), and 100% reduction in tumors >4 mm (P = 0.02) compared to vehicle treated mice. Berberine also decreased AOM/DSS induced Ki-67 and COX-2 expression. In vitro analysis showed that in addition to its anti-proliferation activity, berberine also induced apoptosis in colorectal cancer cell lines. Berberine activated AMP-activated protein kinase (AMPK), a major regulator of metabolic pathways, and inhibited mammalian target of rapamycin (mTOR), a downstream target of AMPK. Furthermore, 4E-binding protein-1 and p70 ribosomal S6 kinases, downstream targets of mTOR, were down regulated by berberine treatment. Berberine did not affect Liver kinase B1 (LKB1) activity or the mitogen-activated protein kinase pathway. Berberine inhibited Nuclear Factor kappa-B (NF-κB) activity, reduced the expression of cyclin D1 and survivin, induced phosphorylation of p53 and increased caspase-3 cleavage in vitro. Berberine inhibition of mTOR activity and p53 phosphorylation was found to be AMPK dependent, while inhibition NF-κB was AMPK independent. In vivo, berberine also activated AMPK, inhibited mTOR and p65 phosphorylation and activated caspase-3 cleavage. Our data suggests that berberine suppresses colon epithelial proliferation and tumorigenesis via AMPK dependent inhibition of mTOR activity and AMPK independent inhibition of NF-κB.

Topics: AMP-Activated Protein Kinases; Animals; Apoptosis; Azoxymethane; Berberine; Carcinogenesis; Caspase 3; Cell Line, Tumor; Colon; Colorectal Neoplasms; Cyclin D1; Cyclooxygenase 2; eIF-2 Kinase; Female; HCT116 Cells; Humans; Mice; NF-kappa B; Phosphorylation; Protein Serine-Threonine Kinases; Signal Transduction; TOR Serine-Threonine Kinases; Tumor Suppressor Protein p53

β1 integrin (ITGB1) is the major expressed integrin protein of normal cells and tumor-associated cells. It is often up-regulated in human malignancies and is involved in many developmental processes, such as tumor progression and metastasis. However, little is known about the function of ITGB1 in colorectal cancer. We constructed lentiviral vectors expressing ITGB1 or ITGB1-specific RNA interference (RNAi) and an unrelated control vector. After infecting HT29 cells in vitro, proliferation and migration were evaluated by Cell Counting Kit 8 (CCK-8) assays, transwell invasion assays, and Western blots. The influence of lentivirus infection on the tumor development capacity of HT29 cells in vivo was examined by xenografting the tumor cells. The expression of ITGB1 in the xenografted tumor cells was analyzed by immunohistochemistry. The up-regulation of ITGB1 significantly increased the proliferation in HT29 cells in vitro. Moreover, we found that the overexpression of ITGB1 up-regulated sonic hedgehog (Shh) while down-regulating Gli1 and SuFu in HT29-ITGB1 cells compared to controls. Moreover, the levels of c-myc and cyclin D1 proteins were up-regulated. Transwell assays showed that the number of migrating HT29-RNAi cells was lower than that in the other cell groups, indicating that ITGB1 significantly enhances the invasive ability of HT29 cells. In addition to these in vitro results, ITGB1 was found to be a significantly effective growth factor in a xenografted tumor mouse model. These results suggest that ITGB1 induces growth and invasion in a human colorectal cancer cell line through the hedgehog (Hh) signaling pathway in vitro and in vivo.

Topics: Animals; Cell Line, Tumor; Cell Movement; Cell Proliferation; Colorectal Neoplasms; Cyclin D1; Down-Regulation; Gene Expression Regulation, Neoplastic; HCT116 Cells; Hedgehog Proteins; HT29 Cells; Humans; Integrin beta Chains; Male; Mice; Mice, Inbred BALB C; Mice, Nude; RNA Interference; Signal Transduction; Up-Regulation

Silymarin from milk thistle (Silybum marianum) plant has been reported to show anti-cancer, anti-inflammatory, antioxidant and hepatoprotective effects. For anti-cancer activity, silymarin is known to regulate cell cycle progression through cyclin D1 downregulation. However, the mechanism of silymarin-mediated cyclin D1 downregulation still remains unanswered. The current study was performed to elucidate the molecular mechanism of cyclin D1 downregulation by silymarin in human colorectal cancer cells. The treatment of silymarin suppressed the cell proliferation in HCT116 and SW480 cells and decreased cellular accumulation of exogenously-induced cyclin D1 protein. However, silymarin did not change the level of cyclin D1 mRNA. Inhibition of proteasomal degradation by MG132 attenuated silymarin-mediated cyclin D1 downregulation and the half-life of cyclin D1 was decreased in the cells treated with silymarin. In addition, silymarin increased phosphorylation of cyclin D1 at threonine-286 and a point mutation of threonine-286 to alanine attenuated silymarin-mediated cyclin D1 downregulation. Inhibition of NF-κB by a selective inhibitor, BAY 11-7082 suppressed cyclin D1 phosphorylation and downregulation by silymarin. From these results, we suggest that silymarin-mediated cyclin D1 downregulation may result from proteasomal degradation through its threonine-286 phosphorylation via NF-κB activation. The current study provides new mechanistic link between silymarin, cyclin D1 downregulation and cell growth in human colorectal cancer cells.

Topics: Antineoplastic Agents; Cell Cycle; Cell Proliferation; Colorectal Neoplasms; Cyclin D1; HCT116 Cells; Humans; Leupeptins; NF-kappa B; Nitriles; Phosphorylation; Point Mutation; Proteasome Endopeptidase Complex; Proteolysis; Silybum marianum; Silymarin; Sulfones; Threonine

Patrinia scabiosaefolia (PS) has long been used as an important component in traditional Chinese medicine formulas to treat gastrointestinal malignancies including colorectal cancer (CRC). We recently reported that PS can inhibit CRC growth through induction of apoptosis and inhibition of tumor angiogenesis. To further elucidate the mode of action of PS, in the present study, we used a CRC mouse xenograft model and a human CRC cell line HT-29 to evaluate the effect of the ethanol extract of PS (EEPS) on cancer cell proliferation and investigated the underlying molecular mechanisms. We found that EEPS inhibited CRC growth both in vivo and in vitro, which was associated with the inhibitory effects of EEPS on cancer cell proliferation. In addition, EEPS treatment significantly blocked G1 to S phase cell cycle progression in HT-29 cells. Moreover, EEPS treatment decreased the expression of pro-proliferative CyclinD1 and CDK4, at both the mRNA and protein levels. Thus, inhibition of cell proliferation via G1/S cell cycle arrest might be a potential mechanism whereby PS effectively treats cancers.

Topics: Animals; Apoptosis; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cell Survival; Colorectal Neoplasms; Cyclin D1; Cyclin-Dependent Kinase 4; Disease Progression; Ethanol; G1 Phase Cell Cycle Checkpoints; Humans; Male; Mice; Mice, Inbred BALB C; Neoplasm Transplantation; Neovascularization, Pathologic; Patrinia; Plant Extracts; RNA, Messenger; S Phase

Catechol-O-methyltransferase (COMT) has been reported as an important molecule in various types of cancers. The biological function of COMT in colorectal cancer (CRC) has not yet been fully investigated.. We constructed a transient transfection of a CRC cell lines to up- and downregulate COMT expression level and tested the proliferative, invasion ability in vitro. We also constructed a stable transduced CRC cell line and conducted tumor-forming capacity experiment in mouse xenograft model in vivo.. In vitro experiment showed that COMT inhibited the cell proliferation by regulating p-Akt, PTEN and inhibited G1 to S phase transition by regulating p53, p27, and cyclinD1. COMT inhibited invasion by regulating E-cadherin. In vivo experiment showed decreased tumor growth in COMT overexpressing cell line.. COMT has tumor-suppressive functions for CRC cell lines in vitro and in vivo experiments.

Topics: Animals; Cadherins; Catechol O-Methyltransferase; Cell Line, Tumor; Cell Proliferation; Colorectal Neoplasms; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p27; Down-Regulation; Female; G1 Phase Cell Cycle Checkpoints; HCT116 Cells; Humans; Mice; Mice, Inbred BALB C; Neoplasm Invasiveness; Proto-Oncogene Proteins c-akt; PTEN Phosphohydrolase; Transfection; Tumor Suppressor Protein p53

Tanshinone I (TAN I) as one of the naturally occurring diterpenes from Salvia miltiorrhizae Bunge (Danshen) has been reported to exhibit an anti-cancer activity. However, the underlying mechanisms are still poorly understood. Thus, we performed in vitro study to elucidate the biological mechanism by which TAN I may induce the inhibition of cell growth in human colorectal cancer cells. The treatment of TAN I suppressed the cell proliferation in HCT116 and SW480 cells and decreased the level of cyclin D1 protein. However, the mRNA level of cyclin D1 did not changed by TAN I treatment. Inhibition of proteasomal degradation by MG132 blocked TAN I-mediated cyclin D1 downregulation and the half-life of cyclin D1 was decreased in the cells treated with TAN I. In addition, phosphorylation of cyclin D1 at threonine-286 was increased by TAN I and a point mutation of threonine-286 to alanine attenuated TAN I-mediated cyclin D1 downregulation. Inhibition of ERK1/2 suppressed cyclin D1 phosphorylation and subsequent downregulation by TAN I. From these results, we suggest that TAN I-mediated cyclin D1 downregulation may result from proteasomal degradation through its ERK1/2-mediated phosphorylation of threonine-286. In conclusion, the current study provides new mechanistic link between TAN I, cyclin D1 downregulation and cell growth in human colorectal cancer cells.

Topics: Abietanes; Antineoplastic Agents, Phytogenic; Cell Line, Tumor; Cell Proliferation; Colorectal Neoplasms; Cyclin D1; Down-Regulation; Humans; MAP Kinase Signaling System; Phosphorylation; Proteasome Endopeptidase Complex

Chemotherapeutic resistance indicated the poor prognosis of colorectal cancer.. Our study aimed to investigate the role of STAT3/cyclinD1 pathway in the chemotherapeutic resistance of colorectal cancer.. We firstly measured the expression of cyclinD1 in the colorectal cancer tissues using immunohistochemistry in tissue microarray. Then cell viability and apoptosis were investigated in the HT-29 cell lines dealing with recombinant lentivirus and shRNA to increase or decrease cyclinD1 expression. Furthermore, luciferase and ChIP assays were applied to investigate whether STAT3 regulated cyclinD1 expression by binding to its promoter. Finally, we determined whether inhibition of STAT3 could decrease cyclinD1 and increase the chemotherapy sensitivity.. CyclinD1 expression was significantly increased in the cancer cells and high level of cyclinD1 indicated the poor prognosis. Inhibition of cyclinD1 decreased the cell viability assessed by MTT and increased rate of apoptosis when exposed to 5-FU treatment while overexpression of cyclinD1 showed the reverse effect. ChIP assay showed that STAT3 directly bind to cyclinD1 promoter. Subclone of full promoter of cyclinD1 into pGL4 increased the luciferase activity while delete or mutation of any of STAT3 binding sites resulted in reductions of luciferase activity. Inhibition of STAT3 decreased cyclinD1 expression to decrease the cell viability and increase rate of apoptosis when exposed to 5-FU treatment.. Inhibition of STAT3/cyclinD1 pathway increased the sensitivity of colorectal cancer cell to chemotherapy.

Topics: Aged; Antimetabolites, Antineoplastic; Colon; Colorectal Neoplasms; Cyclin D1; Drug Resistance, Neoplasm; Female; Fluorouracil; Gene Expression Regulation, Neoplastic; HT29 Cells; Humans; Male; Prognosis; Promoter Regions, Genetic; Rectum; RNA Interference; Signal Transduction; STAT3 Transcription Factor; Up-Regulation

Abnormal activation of β-catenin has been reported in 90% in the sporadic and hereditary colorectal cancer. The suppression of abnormally activated β-catenin is one of the good strategies for chemoprevention and treatment of colorectal cancer. In this study, we have isolated two main compounds from root of Saussurea lappa, dehydrocostus lactone (DCL) and costunolide (CL), and investigated their anti-colorectal cancer activities. DCL and CL suppressed cyclin D1 and survivin through inhibiting nuclear translocation of β-catenin. They also suppressed the nuclear translocation of galectin-3 that is one of the coactivators of β-catenin in SW-480 colon cancer cells. Furthermore, DCL and CL suppressed proliferation and survival of SW-480 colon cancer cells through the induction of cell cycle arrest and cell death. Taken together, DCL and CL from root of S. lappa have anti-colorectal cancer activities through inhibiting Wnt/β-catenin pathway.

Topics: beta Catenin; Blood Proteins; Cell Cycle Checkpoints; Cell Line, Tumor; Colorectal Neoplasms; Cyclin D1; Galectin 3; Galectins; Humans; Inhibitor of Apoptosis Proteins; Lactones; Plant Roots; Saussurea; Sesquiterpenes; Survivin; Wnt Signaling Pathway

Colorectal cancer, one million cases of diagnosis worldwide annually, is one of the most common malignant tumors and 20 % incidence caused by low penetrance susceptibility genes. Cyclin D1 (CCND1) regulating cell cycle transition may determine the susceptible individuals to genomic instability and carcinogenesis. The study aimed at examining the contribution of CCND1 genotypes to colorectal cancer risk in Taiwan. The genotypes of CCND1 A870G (rs9344) and G1722C (rs678653) were determined among 362 colorectal cancer patients and 362 age- and gender-matched cancer-free controls. Significant differences were observed between colorectal cancer and control groups in the distributions of genotypic (P = 9.71 × 10(-4)) and allelic (P = 0.0017) frequencies at CCND1 A870G. Additionally, individuals carried AG or GG genotype had 0.56- or 0.51-fold higher of odds ratios for developing colorectal cancer than the AA genotype (95 % confidence intervals = 0.40-0.78 and 0.32-0.81, respectively). Furthermore, G allele of CCND1 A870G performed a protective effects for nonsmokers and nonalcohol drinkers (P = 0.0012 and 0.0007, respectively) on colorectal cancer risk. These findings support the concept that the cell cycle regulation may play a role in colorectal cancer initiation and development and CCND1 A870G genotyping maybe a feasible technology for colorectal cancer early detection.

Topics: Aged; Biomarkers, Tumor; Colorectal Neoplasms; Cyclin D1; Female; Genetic Association Studies; Genetic Predisposition to Disease; Genotype; Humans; Middle Aged; Polymorphism, Single Nucleotide; Prognosis; Risk Factors; Smoking; Taiwan

Epithelial cells affected by somatic mutations undergo transition from a tumour-suppressive to a carcinogenic Smad pathway during sporadic colorectal carcinogenesis, and the specific linker threonine phosphorylation of Smad2/3 in colon epithelial cells indicates stem-like cells. This study extends previous observations to a model of colitis-associated colorectal cancer.. After Crl:CD-1 mice received an administration of azoxymethane [AOM], the mice were given dextran sodium sulfate [DSS] for 7 days. AOM/DSS-treated mice [AOM/DSS mice] were killed at 10 or 20 weeks. After macroscopic observations, a histopathological analysis was conducted. Immunohistochemical staining was performed using the avidin-biotin immunoperoxidase method [pSmad3C-Ser, pSmad3L-Ser, c-Myc] and immunofluorescent methods [Ki67, β-catenin, CDK4, cyclin D1, Sox9, pSmad2/3L-Thr].. The colons from AOM/DSS mice were shorter than those from control mice. The number of colon tumours at Week 20 was higher than at Week 10. The inflammation scores for AOM/DSS mice were greater than those for control mice. Immunostaining-positive cells (staining by Ki67, β-catenin [nuclear and cytoplasmic], cyclin D1, and Sox9) were diffusely distributed in colon tumours. The percentage of pSmad3L-Ser-positive cells in colon tumours was higher than in sites of pre-neoplastic colitis, and that in sites of pre-neoplastic colitis was higher than in control mice. pSmad2/3L-Thr-positive cells were sparsely detected around crypt bases in non-neoplastic colon epithelia and at the tops of tumours, and immunohistochemical co-localisation of pSmad2/3L-Thr with Ki67 was not observed. Immunohistochemical co-localisation of pSmad2/3L-Thr with β-catenin and CDK4 was observed.. pSmad3L-Ser signalling is an early event in colitis-associated colorectal cancer, and pSmad2/3L-Thr immunostaining-positive cells might be cancer stem cells.

Topics: Animals; Azoxymethane; beta Catenin; Biomarkers, Tumor; Carcinogenesis; Colitis; Colorectal Neoplasms; Cyclin D1; Dextran Sulfate; Disease Models, Animal; Ki-67 Antigen; Male; Mice; Neoplastic Stem Cells; Phosphorylation; Proto-Oncogene Proteins c-myc; Serine; Signal Transduction; Smad2 Protein; Smad3 Protein; SOX9 Transcription Factor

Cryptotanshinone (CPT) is a natural compound extracted from herbal medicine that has been previously shown to possess antitumor properties in various types of human cancer cells. In the present study, we examined the potential role of CPT in the treatment of colorectal cancer. Using SW480, HCT116, and LOVO colorectal cancer cell lines, the effects of CPT on cell viability, apoptosis, and tumorigenicity were evaluated. The results showed that CPT significantly inhibited the growth and viability of SW480, HCT116, and LOVO cell lines by inducing apoptosis and prevented anchorage dependent growth on agar. In addition, CPT inhibited the activation of Signal transducer and activator of transcription 3 (Stat3) pathways in colorectal cancer cells. Stat3 is a transcription factor that mediates the expression of various genes associated with many cellular processes, such as inflammation and cell growth, and has been shown to promote several cancer types, including colorectal cancer. These findings indicate that CPT may be a potential candidate for the treatment and prevention of colorectal cancer in part by inhibiting the activation of Stat3.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Cell Proliferation; Cell Survival; Colorectal Neoplasms; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase Inhibitor p27; Drug Screening Assays, Antitumor; HCT116 Cells; Humans; Inhibitor of Apoptosis Proteins; Phenanthrenes; STAT3 Transcription Factor; Survivin

Inhibitors of apoptosis proteins (IAPs) have been well investigated in human cancers, where they are frequently overexpressed and associated with poor prognosis. Here we explored the role of baculoviral IAP repeat containing 6 (BIRC6), a member of IAPs, in human colorectal cancer (CRC).. We used Western blotting and immunohistochemistry to examine BIRC6 expression in 7 CRC cell lines and 126 CRC clinical samples. We determined the biological significance of BIRC6 in CRC cell lines by a lentivirus-mediated silencing method.. We reported that BIRC6 was overexpressed in CRC cell lines and clinical CRC tissues. BIRC6 overexpression was correlated with tumor size and invasion depth of CRC. BIRC6 overexpression is associated with worse overall survival (OS) (P = 0.001) and shorter disease-free survival (DFS) (P = 0.010). BIRC6 knockdown inhibited cell proliferation, arrested cell cycle at S phase, downregulated cyclin A2, B1, D1 and E1 levels, and sensitized CRC cells to chemotherapy in vitro and in vivo.. Taken together, these data suggests that BIRC6 overexpression is a predictor of poor prognosis in colorectal cancer and BIRC6 could be a potential target of CRC therapy.

Topics: Adenocarcinoma; Antineoplastic Agents; Cell Line, Tumor; Cell Proliferation; Colorectal Neoplasms; Cyclin A2; Cyclin B1; Cyclin D1; Cyclin E; Drug Resistance, Neoplasm; Female; Gene Expression Regulation, Neoplastic; Genetic Vectors; Humans; Inhibitor of Apoptosis Proteins; Lentivirus; Male; Middle Aged; Neoplasm Invasiveness; Oncogene Proteins; Prognosis; RNA, Small Interfering; S Phase Cell Cycle Checkpoints; Signal Transduction; Survival Analysis; Tumor Burden

3-Phosphoinositide-dependent protein kinase 1 (PDK1) is centrally involved in cancer progression, including proliferation, apoptosis and invasion. However, its expression pattern and possible cellular functions in human colorectal cancer remain unclear. In the present study, we show that PDK1 expression is up-regulated at both mRNA and protein levels in colorectal cancer clinical specimens and cell lines. Transient knockdown of PDK1 suppresses cellular growth, induces cellular apoptosis and causes abnormal cell cycle distribution. Meanwhile, decreased PDK1 level is closely associated with reduced Akt/cyclin D1 activity. Activating AKT activity and reintroducing cyclin D1 expression significantly compromised the oncogenic activity induced by PDK1. Together, our findings elucidate a key role for PDK1 in colorectal cellular functions trigged by the Akt/cyclin D1 pathway, thus providing a novel insight of PDK1 in colorectal carcinogenesis.

Topics: Apoptosis; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Colorectal Neoplasms; Cyclin D1; Gene Expression; HCT116 Cells; Humans; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins c-akt; Pyruvate Dehydrogenase Acetyl-Transferring Kinase; RNA, Messenger; Up-Regulation

Lysophosphatidic acid (LPA) and its analogs are well-known mitogens for various cell types. Many reports have confirmed that several types of cancer cell produce LPA to promote survival, growth and tumorigenesis. This indicates that the interface between the LPA signaling pathway and the cell cycle signaling system is critical to the control of cancer cell proliferation. However, our previous study indicated that cyclic phosphatidic acid (cPA), which is structurally similar to LPA, inhibits the proliferation and migration of colon cancer cells. It has been reported that cPA shows several biological activities not shown by LPA. However, understanding of the detailed molecular and cellular mechanism underlying the regulation of the cell cycle by cPA is still in its infancy. In this study, we investigated the effect of cPA treatment on human DLD-1 colon cancer cells by analyzing cell cycle dynamics, gene expression, and AKT phosphorylation. Our findings indicate that cPA inhibits cell cycle progression in DLD-1 colon cancer cells via the downregulation of cyclin D1 and the inhibition of AKT phosphorylation.

Topics: Antineoplastic Agents; Cell Line, Tumor; Colorectal Neoplasms; Cyclin D1; Down-Regulation; G1 Phase Cell Cycle Checkpoints; Humans; Phosphatidic Acids; Phosphorylation; Proto-Oncogene Proteins c-akt

It was reported that the receptor tyrosine kinase (RTK) family often highly expressed in several mucinous carcinomas. In the present study, we established a murine model of colorectal mucinous adenocardinoma (CRMAC) by treating C57 mice [both wild type (WT) and loss-of-function c-kit mutant type (Wads-/-)] with AOM+DSS for 37 weeks and found that c-kit, a member of RTK family, clearly enhanced the tumor cell proliferation by decreasing p53 and increasing cyclin D1 through AKT pathway. Significantly, c-kit strongly promoted tumor cell invasiveness by increasing ETV4, which induced MMP7 expression and epithelial-mesenchymal transition (EMT) via ERK pathway. In vitro up- or down-regulating c-kit activation in human colorectal cancer HCT-116 cells further consolidated these results. In conclusion, our data suggested that the c-kit signaling obviously promoted proliferation and invasion of CRMAC. Therefore, targeting the c-kit signaling and its downstream molecules might provide the potential strategies for treatment of patients suffering from CRMAC in the future.

Topics: Adenocarcinoma; Adenocarcinoma, Mucinous; Animals; Cell Line, Tumor; Cell Proliferation; Colorectal Neoplasms; Cyclin D1; Disease Models, Animal; Epithelial-Mesenchymal Transition; Extracellular Signal-Regulated MAP Kinases; Gene Expression Regulation, Neoplastic; Genotype; HCT116 Cells; Humans; Lentivirus; Matrix Metalloproteinase 7; Mice; Mice, Inbred C57BL; Mice, Transgenic; Neoplasm Invasiveness; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-kit; Signal Transduction; Tumor Suppressor Protein p53

Human colorectal cancers are known to possess multiple mutations, though how these mutations interact in tumor development and progression has not been fully investigated. We have previously described the FCPIK3ca* murine colon cancer model, which expresses a constitutively activated phosphoinositide-3 kinase (PI3K) in the intestinal epithelium. The expression of this dominantly active form of PI3K results in hyperplasia and invasive mucinous adenocarcinomas. These cancers form via a non-canonical mechanism of tumor initiation that is mediated through activation of PI3K and not through aberrations in WNT signaling. Since the Adenomatous Polyposis Coli (APC) gene is mutated in the majority of human colon cancers and often occurs simultaneously with PIK3CA mutations, we sought to better understand the interaction between APC and PIK3CA mutations in the mammalian intestine. In this study, we have generated mice in which the expression of a constitutively active PI3K and the loss of APC occur simultaneously in the distal small intestine and colon. Here, we demonstrate that expression of a dominant active PI3K synergizes with loss of APC activity resulting in a dramatic change in tumor multiplicity, size, morphology and invasiveness. Activation of the PI3K pathway is not able to directly activate WNT signaling through the nuclear localization of CTNNB1 (β-catenin) in the absence of aberrant WNT signaling. Alterations at the transcriptional level, including increased CCND1, may be the etiology of synergy between these activated pathways.

Topics: Adenocarcinoma; Adenomatous Polyposis Coli Protein; Animals; beta Catenin; Cell Nucleus; Class I Phosphatidylinositol 3-Kinases; Colorectal Neoplasms; Cyclin D1; Disease Models, Animal; Epistasis, Genetic; Female; Gene Expression; Humans; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Microsatellite Instability; Phosphatidylinositol 3-Kinases; Tumor Burden; Wnt Signaling Pathway

Ku70, a known nonhomologous end-joining (NHEJ) factor, also functions in tumor suppression, although this molecular mechanism remains uncharacterized. Previously, we showed that mice deficient for DNA ligase IV (Lig4), another key NHEJ factor, succumbed to aggressive lymphoma in the absence of tumor suppressor p53. However, the tumor phenotype is abrogated by the introduction of a hypomorphic mutant p53(R172P), which impaired p53-mediated apoptosis but not cell-cycle arrest. However, Lig4(-/-)p53(R172P) mice succumbed to severe diabetes. To further elucidate the role of NHEJ and p53-mediated apoptosis in vivo, we bred Ku70(-/-) p53(R172P) mice. Unexpectedly, these mice were free of diabetes, although 80% of the mutant mice had abnormally enlarged colons with pronounced inflammation. Remarkably, most of these mutant mice progressed to dysplasia, adenoma and adenocarcinoma; this is in contrast to the Lig4(-/-)p53(R172P) phenotype, strongly suggesting an NHEJ-independent function of Ku70. Significantly, our analyses of Ku70(-/-)p53(R172P) colonic epithelial cells show nuclear stabilization of β-catenin accompanied by higher expression of cyclin D1 and c-Myc in affected colon sections than in control samples. This is not due to the p53 mutation, as Ku70(-/-) mice share this phenotype. Our results not only unravel a novel function of Ku70 essential for colon homeostasis, but also establish an excellent in vivo model in which to study how chronic inflammation and abnormal cellular proliferation underlie tumorigenesis and tumor progression in the colon.

Topics: Adenocarcinoma; Animals; Antigens, Nuclear; Carcinogenesis; Cell Proliferation; Cells, Cultured; Cellular Senescence; Colon; Colorectal Neoplasms; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; DNA Damage; DNA End-Joining Repair; DNA-Binding Proteins; Epithelial Cells; Homeostasis; Humans; Interleukin-6; Ku Autoantigen; Mice; Mice, Knockout; Proto-Oncogene Proteins c-myc; T-Lymphocytes; Tumor Necrosis Factor-alpha; Tumor Suppressor Protein p53; Wnt Signaling Pathway

Krüppel-like factor 5 (KLF5) is a pro-proliferative transcriptional regulator primarily expressed in the intestinal crypt epithelial cells. Constitutive intestine-specific deletion of Klf5 is neonatal lethal suggesting a crucial role for KLF5 in intestinal development and homeostasis. We have previously shown Klf5 to play an active role regulating intestinal tumorigenesis. Here we examine the effect of inducible intestine-specific deletion of Klf5 in adult mice. Klf5 is lost from the intestine beginning at day 3 after the start of a 5-day treatment with the inducer tamoxifen. Although the mice have no significant weight loss or lethality, the colonic tissue shows signs of epithelial distress starting at day 3 following induction. Accompanying the morphological changes is a significant loss of proliferative crypt epithelial cells as revealed by BrdU or Ki67 staining at days 3 and 5 after start of tamoxifen. We also observed a loss of goblet cells from the colon and Paneth cells from the small intestine upon induced deletion of Klf5. In addition, loss of Klf5 from the colonic epithelium is accompanied by a regenerative response that coincides with an expansion in the zone of Sox9 expression along the crypt axis. At day 11, both proliferation and Sox9 expression return to baseline levels. Microarray and quantitative PCR analyses reveal an up-regulation of several regeneration-associated genes (Reg1A, Reg3G and Reg3B) and down-regulation of many Klf5 targets (Ki-67, cyclin B, Cdc2 and cyclin D1). Sox9 and Reg1A protein levels are also increased upon Klf5 loss. Lentiviral-mediated knockdown of KLF5 and exogenous expression of KLF5 in colorectal cancer cell lines confirm that Sox9 expression is negatively regulated by KLF5. Furthermore, ChIP assays reveal a direct association of KLF5 with both the Sox9 and Reg1A promoters. We have shown that disruption of epithelial homeostasis due to Klf5 loss from the adult colon is followed by a regenerative response led by Sox9 and the Reg family of proteins. Our study demonstrates that adult mouse colonic tissue undergoes acute physiological changes to accommodate the loss of Klf5 withstanding epithelial damage further signifying importance of Klf5 in colonic homeostasis.

Topics: Animals; Antineoplastic Agents, Hormonal; CDC2 Protein Kinase; Cell Line, Tumor; Cell Proliferation; Colon; Colorectal Neoplasms; Cyclin B; Cyclin D1; Down-Regulation; Goblet Cells; HCT116 Cells; HEK293 Cells; Humans; Ki-67 Antigen; Kruppel-Like Transcription Factors; Lithostathine; Mice; Mice, Inbred C57BL; Mice, Knockout; Pancreatitis-Associated Proteins; Paneth Cells; Promoter Regions, Genetic; Proteins; Regeneration; RNA Interference; RNA, Small Interfering; Sequence Deletion; Signal Transduction; SOX9 Transcription Factor; Tamoxifen; Up-Regulation

Temsirolimus (TEM) is a novel, water-soluble mammalian target of rapamycin (mTOR) inhibitor that has shown activity against a wide range of cancers in preclinical models, but its efficacy against colorectal cancer (CRC) has not been fully explored.. We evaluated the antitumor effect of TEM in CRC cell lines (CaR-1, HT-29, Colon26) in vitro and in vivo. In vitro, cell growth inhibition was assessed using a MTS assay. Apoptosis induction and cell cycle effects were measured using flow cytometry. Modulation of mTOR signaling was measured using immunoblotting. Antitumor activity as a single agent was evaluated in a mouse subcutaneous tumor model of CRC. The effects of adding chloroquine, an autophagy inhibitor, to TEM were evaluated in vitro and in vivo.. In vitro, TEM was effective in inhibiting the growth of two CRC cell lines with highly activated AKT, possibly through the induction of G1 cell cycle arrest via a reduction in cyclin D1 expression, whereas TEM reduced HIF-1α and VEGF in all three cell lines. In a mouse subcutaneous tumor model, TEM inhibited the growth of tumors in all cell lines, not only through direct growth inhibition but also via an anti-angiogenic effect. We also explored the effects of adding chloroquine, an autophagy inhibitor, to TEM. Chloroquine significantly potentiated the antitumor activity of TEM in vitro and in vivo. Moreover, the combination therapy triggered enhanced apoptosis, which corresponded to an increased Bax/Bcl-2 ratio.. Based on these data, we propose TEM with or without chloroquine as a new treatment option for CRC.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Cell Line, Tumor; Chloroquine; Colorectal Neoplasms; Cyclin D1; Gene Expression Regulation, Neoplastic; HT29 Cells; Humans; Mice; Sirolimus; TOR Serine-Threonine Kinases; Xenograft Model Antitumor Assays

The key role of the Wnt/β-catenin signaling in colorectal cancer (CRC) insurgence and progression is now recognized and several therapeutic strategies targeting this pathway are currently in developing. Wnt/β-catenin signaling not only dominates the early stages of sporadic colorectal cancer (SCC), but could also represent the connection between inflammatory bowel diseases (IBD) and increased risk of developing SCC. The knowledge on the sequential molecular events of Wnt-signaling cascade in IBD and during colorectal carcinogenesis, might provide new diagnostic/prognostic markers and could be helpful for optimizing the treatment protocols, thus improving the efficacy of Wnt-targeting therapies. We performed a comparative evaluation of the expression of some crucial molecules participating to Wnt signaling in an animal model of chemically-induced CRC and in human tissues obtained from patients suffering from IBD or at sequential stages of SCC. Specifically, we analyzed upstream events of Wnt signaling including β-catenin nuclear translocation and loss of E-cadherin and APC functions, and downstream events including c-Myc and Cyclin-D1 expression. We demonstrated that these crucial components of the Wnt/β-catenin pathway, when evaluated by immunohistochemistry using a multiparametric approach that includes the analyses of both expression and localization, could be potent markers for diagnosis, prevention and therapy in IBD and SCC, also possessing a predictive value for responsiveness to Wnt-targeting therapies. Furthermore, we showed that the animal model of chemically-induced CRC mimics the molecular events of Wnt signaling during IBD and SCC development in humans and may therefore be suitable for testing chemopreventive or therapeutic drugs targeting this pathway.

Topics: Adenomatous Polyposis Coli Protein; Animals; beta Catenin; Cadherins; Carcinogenesis; Colorectal Neoplasms; Cyclin D1; Disease Models, Animal; Disease Progression; Humans; Inflammatory Bowel Diseases; Intestinal Mucosa; Male; Paraffin Embedding; Proto-Oncogene Proteins c-myc; Rats; Signal Transduction; Wnt Signaling Pathway

Postmenopausal hormone therapy (HRT) and oral contraceptive (OC) use have in several studies been reported to be associated with a decreased colorectal cancer (CRC) risk. However, data on the association between HRT and OC and risk of different clinicopathological and molecular subsets of CRC are lacking. The aim of this molecular pathological epidemiology study was therefore to evaluate the associations between HRT and OC use and risk of specific CRC subgroups, overall and by tumour site.. In the population-based prospective cohort study Mamö Diet and Cancer, including 17035 women, 304 cases of CRC were diagnosed up until 31 December 2008. Immunohistochemical expression of beta-catenin, cyclin D1, p53 and MSI-screening status had previously been assessed in tissue microarrays with tumours from 280 cases. HRT was assessed as current use of combined HRT (CHRT) or unopposed oestrogen (ERT), and analysed among 12583 peri-and postmenopausal women. OC use was assessed as ever vs never use among all women in the cohort. A multivariate Cox regression model was applied to determine hazard ratios for risk of CRC, overall and according to molecular subgroups, in relation to HRT and OC use.. There was no significantly reduced risk of CRC by CHRT or ERT use, however a reduced risk of T-stage 1-2 tumours was seen among CHRT users (HR: 0.24; 95% CI: 0.09-0.77).Analysis stratified by tumour location revealed a reduced overall risk of rectal, but not colon, cancer among CHRT and ERT users, including T stage 1-2, lymph node negative, distant metastasis-free, cyclin D1 - and p53 negative tumours.In unadjusted analysis, OC use was significantly associated with a reduced overall risk of CRC (HR: 0.56; 95% CI: 0.44-0.71), but this significance was not retained in adjusted analysis (HR: 1.05: 95% CI: 0.80-1.37). A similar risk reduction was seen for the majority of clinicopathological and molecular subgroups.. Our findings provide information on the relationship between use of HRT and OC and risk of clinicopathological and molecular subsets of CRC.

Topics: beta Catenin; Biomarkers, Tumor; Colorectal Neoplasms; Contraceptives, Oral, Hormonal; Cyclin D1; Estrogen Replacement Therapy; Female; Genetic Predisposition to Disease; Humans; Immunohistochemistry; Microsatellite Instability; Middle Aged; Multivariate Analysis; Neoplasm Staging; Proportional Hazards Models; Prospective Studies; Risk Assessment; Risk Factors; Sweden; Time Factors; Tissue Array Analysis; Tumor Suppressor Protein p53

Root bark of mulberry (Morus alba L.) has been used in herbal medicine as anti-phlogistic, liver protective, kidney protective, hypotensive, diuretic, anti-cough and analgesic agent. However, the anti-cancer activity and the potential anti-cancer mechanisms of mulberry root bark have not been elucidated. We performed in vitro study to investigate whether mulberry root bark extract (MRBE) shows anti-inflammatory and anti-cancer activity.. In anti-inflammatory activity, NO was measured using the griess method. iNOS and proteins regulating NF-κB and ERK1/2 signaling were analyzed by Western blot. In anti-cancer activity, cell growth was measured by MTT assay. Cleaved PARP, ATF3 and cyclin D1 were analyzed by Western blot.. In anti-inflammatory effect, MRBE blocked NO production via suppressing iNOS over-expression in LPS-stimulated RAW264.7 cells. In addition, MRBE inhibited NF-κB activation through p65 nuclear translocation via blocking IκB-α degradation and ERK1/2 activation via its hyper-phosphorylation. In anti-cancer activity, MRBE deos-dependently induced cell growth arrest and apoptosis in human colorectal cancer cells, SW480. MRBE treatment to SW480 cells activated ATF3 expression and down-regulated cyclin D1 level. We also observed that MRBE-induced ATF3 expression was dependent on ROS and GSK3β. Moreover, MRBE-induced cyclin D1 down-regulation was mediated from cyclin D1 proteasomal degradation, which was dependent on ROS.. These findings suggest that mulberry root bark exerts anti-inflammatory and anti-cancer activity.

Topics: Activating Transcription Factor 3; Animals; Anti-Inflammatory Agents; Antineoplastic Agents; Cell Line, Tumor; Cell Proliferation; Colorectal Neoplasms; Cyclin D1; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Humans; Lipopolysaccharides; Macrophages; Mice; Morus; NF-kappa B; Nitric Oxide; Nitric Oxide Synthase Type II; Plant Extracts; Reactive Oxygen Species; RNA, Messenger; Signal Transduction

Autophagy is reported to suppress tumor proliferation, whereas deficiency of autophagy is associated with tumorigenesis. ATG4B is a deubiquitin-like protease that plays dual roles in the core machinery of autophagy; however, little is known about the role of ATG4B on autophagy and proliferation in tumor cells. In this study, we found that ATG4B knockdown induced autophagic flux and reduced CCND1 expression to inhibit G 1/S phase transition of cell cycle in colorectal cancer cell lines, indicating functional dominance of ATG4B on autophagy inhibition and tumor proliferation in cancer cells. Interestingly, based on the genetic and pharmacological ablation of autophagy, the growth arrest induced by silencing ATG4B was independent of autophagic flux. Moreover, dephosphorylation of MTOR was involved in reduced CCND1 expression and G 1/S phase transition in both cells and xenograft tumors with depletion of ATG4B. Furthermore, ATG4B expression was significantly increased in tumor cells of colorectal cancer patients compared with adjacent normal cells. The elevated expression of ATG4B was highly correlated with CCND1 expression, consistently supporting the notion that ATG4B might contribute to MTOR-CCND1 signaling for G 1/S phase transition in colorectal cancer cells. Thus, we report that ATG4B independently plays a role as a positive regulator on tumor proliferation and a negative regulator on autophagy in colorectal cancer cells. These results suggest that ATG4B is a potential biomarker and drug target for cancer therapy.

Topics: Animals; Autophagy; Autophagy-Related Proteins; Cell Line, Tumor; Cell Proliferation; Colorectal Neoplasms; Cyclin D1; Cysteine Endopeptidases; G1 Phase Cell Cycle Checkpoints; Gene Knockdown Techniques; Gene Silencing; Humans; Mice, Nude; Phosphorylation; S Phase; TOR Serine-Threonine Kinases; Xenograft Model Antitumor Assays

The anti cancer properties and underlying cell death mechanisms induced by an extract of the roots of Cnidium officinale Makino (COM) were investigated. An ethanolic extract of COM inhibited proliferation of human colon cancer cells (HT-29) with both dose- and time-dependence. Analysis of the cell cycle after treatment of HT-29 cells with various concentrations of COM showed reduction of cellular proliferation via G1 phase arrest. Apoptotic effects of COM and Western blotting both revealed that COM extract dose-dependently increased the expression of p53, p21,Bax and caspase-3. Anti-apoptotic factor Bcl-2 expression was down regulated as well as those of cyclin D1 and CDK4. These data suggest that COM has anti cancer properties by inducing apoptosis and cell cycle arrest in HT-29 cells and could have possible therapeutic potential against human colon adenocarcinoma.

Topics: Antineoplastic Agents; Apoptosis; bcl-2-Associated X Protein; Caspase 3; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Cnidium; Colorectal Neoplasms; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Down-Regulation; G1 Phase; HT29 Cells; Humans; Plant Extracts; Proto-Oncogene Proteins c-bcl-2; Tumor Suppressor Protein p53

Abnormal expression of FOXC2 has been found in several human cancers. However, the role of FOXC2 in the progression of colorectal cancer (CRC) has not been well characterized. In analysis of 206 CRC specimens, we revealed that both high expression and nuclear localization of FOXC2 were correlated to aggressive characteristics and poor survival of patients with CRC. FOXC2 promoted cell proliferation through activation of MAPK and AKT pathways, subsequently down-regulating p27, up-regulating cyclin D1 and p-FOXO3a. Furthermore, FOXC2 nuclear localization was required for its promotion of cell proliferation. These findings suggest that FOXC2 plays an essential role in CRC progression and may serve as a valuable clinical prognostic marker of this disease.

Topics: Adenocarcinoma; Adolescent; Adult; Aged; Biomarkers, Tumor; Caco-2 Cells; Cell Proliferation; Colorectal Neoplasms; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p27; Disease-Free Survival; Enzyme Activation; Female; Forkhead Box Protein O3; Forkhead Transcription Factors; Gene Expression Regulation, Neoplastic; HCT116 Cells; HT29 Cells; Humans; Male; Middle Aged; Mitogen-Activated Protein Kinases; Proto-Oncogene Proteins c-akt; RNA Interference; Signal Transduction; Time Factors; Transfection; Up-Regulation; Young Adult

The Wnt and Notch1 signaling pathways play major roles in intestinal development and tumorigenesis. Sub-cellular localization of β-catenin has been implicated in colorectal carcinogenesis. However, the β-catenin and Notch intracellular domain (NICD) interaction has to be addressed. Immunohistochemistries of β-catenin, NICD, and dual immunofluorescence of β-catenin and NICD were analyzed in colorectal tissues and HT29 cell line. Moreover, real-time PCR analysis of CyclinD1, Hes1 and MUC2 was done in HT29 cells upon N-[N-(3, 5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT) treatment. Dual staining emphasized the strong interaction of β-catenin and NICD in adenoma and adenocarcinoma than in normal tissues. Hes1 transcript levels were decreased 1.5- and 7.1-fold in 12.5 and 25 µM DAPT-treated HT29 cells. CyclinD1 transcript levels decreased 1.2- and 1.6-fold, and MUC2 transcript level increased 4.3- and 7.5-fold in 12.5 and 25 µM DAPT-treated HT29 cells. The results of this study showed that the sub-cellular localization of β-catenin converges with NICD inducing proliferation through the activation of CyclinD1 and Hes1. Moreover, the inhibition of Notch1 signaling by DAPT leads to the arrest of cell proliferation and induces apoptosis leading to the upregulation of MUC2, a secretory cell lineage marker.

Topics: Adenocarcinoma; Adenoma; Basic Helix-Loop-Helix Transcription Factors; beta Catenin; Cell Proliferation; Colonic Neoplasms; Colorectal Neoplasms; Cyclin D1; Dipeptides; Dose-Response Relationship, Drug; Gene Expression Regulation, Neoplastic; Homeodomain Proteins; HT29 Cells; Humans; Mucin-2; Protein Structure, Tertiary; Receptor, Notch1; Reference Values; Signal Transduction; Transcription Factor HES-1

Colorectal cancer (CRC) is one of the major causes of cancer death worldwide. The development of novel anti-CRC agents able to overcome drug resistance and/or off-target toxicity is of pivotal importance. The mammalian target of rapamycin (mTOR) plays a critical role in CRC, regulating protein translation and controlling cell growth, proliferation, metabolism and survival. The aim of this study was to explore the effect of a combination of three natural compounds, eicosapentaenoic acid-free fatty acid (EPA-FFA), epigallocatechin-3-gallate (EGCG) and proanthocyanidins (grape seed [GS] extract) at low cytotoxic concentrations on CRC cells and test their activity on mTOR and translational regulation. The CRC cell lines HCT116 and SW480 were treated for 24h with combinations of EPA-FFA (0-150 µM), EGCG (0-175 µM) and GS extract (0-15 µM) to evaluate the effect on cell viability. The low cytotoxic combination of EPA-FFA 150 µM, EGCG 175 µM and GS extract 15 µM completely inhibited the mTOR signaling in HCT116 and SW480 cells, reaching an effect stronger than or comparable to that of the mTOR inhibitor Rapamycin in HCT116 or SW480 cells, respectively. Moreover, the treatment led to changes of protein translation of ribosomal proteins, c-Myc and cyclin D1. In addition, we found a reduction of clonal capability in both cell lines, with block of cell cycle in G0G1 and induction of apoptosis. Our data suggest that the low cytotoxic combination of EPA-FFA, EGCG and GS extract, tested for the first time here, inhibits mTOR signaling and thus could be considered for CRC treatment.

Topics: Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Catechin; Cell Line, Tumor; Cell Proliferation; Colorectal Neoplasms; Cyclin D1; Eicosapentaenoic Acid; Grape Seed Extract; Humans; Proanthocyanidins; Proto-Oncogene Proteins c-myc; Signal Transduction; TOR Serine-Threonine Kinases

Cyclin D1 (CCND1) and E-cadherin (CDH1) are two important genes of the β-catenin/LEF pathway that is involved in tumorigenesis of various cancers including colorectal cancer (CRC). However, studies of the association between genetic variants of these two genes and CRC have shown conflicting results. We conducted a genetic association study in South Indian population (cases, 103; controls, 107) to assess the association of CCND1 870G/A and CDH1 -160C/A single nucleotide polymorphisms (SNPs) with CRC risk. Genotyping of SNPs was performed by PCR sequencing analysis. Haplotype frequencies for multiple loci and the standardized disequilibrium coefficient (D') for pair-wise linkage disequilibrium (LD) were assessed by Haploview Software. In addition, to better understand the role of CCND1 and CDH1 in the pathophysiology of CRC, the expression pattern was evaluated in analogous tumor and adjacent normal tissues from 23 CRC patients by Western blot analysis. The frequencies of CCND1 870A/A (P = 0.045) genotype, CDH1 -160A allele (P = 0.042), and 870A/-160A haplotype (P = 0.002) were significantly higher in patients as compared with controls. Strong LD was observed between 870G/A and -160C/A SNPs in cases (D' = 0.76) as compared to controls (D' = 0.32). Furthermore, elevated CCND1 and diminished CDH1 expression was observed in tumor tissue as compared with analogous normal tissue of CRC patients. Interestingly, advanced-stage tumors showed wider expression alterations than in early-stage tumors. In conclusion, CCND1 870G/A and CDH1 -160C/A SNPs may modify the risk of CRC susceptibility in South Indian population. In addition, elevated CCND1 and diminished CDH1 expression appears to be useful prognostic markers for CRC.

Topics: Adult; Aged; Alleles; Cadherins; Case-Control Studies; Colorectal Neoplasms; Cyclin D1; Female; Genetic Predisposition to Disease; Genetic Variation; Genotype; Haplotypes; Humans; India; Linkage Disequilibrium; Male; Middle Aged; Neoplasm Invasiveness; Neoplasm Staging; Polymorphism, Single Nucleotide; Risk Factors; Sequence Analysis, DNA

Despite the fact that new treatment regimes have improved overall survival of patients challenged by colorectal cancer (CRC), prognosis in the metastatic situation is still restricted. The Bcl-2 family of proteins has been identified as promising anti cancer drug target. Even though small molecules targeting Bcl-2 proteins are in clinical trials, little is known regarding their effects on CRC. The aim of this study was to preclinically investigate the value of ABT-737 and Obatoclax as anticancer drugs for CRC treatment. The effects of the BH3-mimetics ABT-737 and Obatoclax on CRC cells were assessed using viability and apoptosis assays. Wound healing migration and boyden chamber invasion assays were applied. 3-dimensional cell cultures were used for long term assessment of invasion and proliferation. Clinically relevant concentrations of pan-Bcl-2 inhibitor Obatoclax did not induce cell death. In contrast, the BH3-mimetic ABT-737 induced apoptosis in a dose dependent manner. Obatoclax caused a cell line specific slowdown of CRC cell growth. Furthermore, Obatoclax, but not ABT-737, recovered E-Cadherin expression and led to impaired migration and invasion of CRC cells. The proliferative capacity and invasiveness of CRC cells was strikingly inhibited by low dose Obatoclax in long term 3-dimensional cell cultures. Obatoclax, but not ABT-737, caused a G1-phase arrest accompanied by a downregulation of Cyclin D1 and upregulation of p27 and p21. Overexpression of Mcl-1, Bcl-xL or Bcl-2 reversed the inhibitory effect of Obatoclax on migration but failed to restore the proliferative capacity of Obatoclax-treated CRC cells. The data presented indicate broad and multifaceted antitumor effects of the pan-Bcl-2 inhibitor Obatoclax on CRC cells. In contrast to ABT-737, Obatoclax inhibited migration, invasion and proliferation in sublethal doses. In summary, this study recommends pan-Bcl-2 inhibition as a promising approach for clinical trials in CRC.

Topics: Antineoplastic Agents; Apoptosis; Biphenyl Compounds; Cadherins; Cell Cycle; Cell Line, Tumor; Cell Movement; Cell Proliferation; Colorectal Neoplasms; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase Inhibitor p27; G1 Phase Cell Cycle Checkpoints; Gene Expression; Humans; Indoles; Nitrophenols; Piperazines; Proto-Oncogene Proteins c-bcl-2; Pyrroles; Sulfonamides

Colorectal cancer (CRC) is the second leading cause of death from cancer in the United States. Colorectal cancers have a prolonged latency following initiation that may span decades providing ample time for implementing a chemoprevention strategy that could block or reverse the progression to CRC. Cdk4 pathway alterations have been linked to a number of cancers including CRC. In these experiments we focused on the Cdk4 pathway and its role in intestinal tumorigenesis as a possible target in chemoprevention strategies.. We evaluated the effect of Cdk4 blockade on the prevention of intestinal tumor formation by crossing Cdk4(-/-) mice to Apc(-/+) mice. In addition, we tested the effect of the dietary compound silibinin on the Cdk4 pathway in Apc(-/+) mice and HT-29 colon cancer cells in culture.. Cdk4(-/-) mice backcrossed to Apc(-/+) mice reduced intestinal adenoma formation compared to Apc(-/+) controls. Silibinin effectively targeted the Cdk4 pathway causing hypophosphorylation of the retinoblastoma protein, inhibited cell growth, and induced apoptosis. As a result silibinin blocked the development of intestinal adenomas by 52% in this genetic model (Apc(-/+) mice) of early events in colorectal cancer formation. No toxic abnormalities were detected in mice which received silibinin.. Modification of the Cdk4 pathway using a natural plant-derived compound such as silibinin may be a useful chemopreventive strategy for colorectal carcinomas.

Topics: Adenoma; Animals; Apoptosis; Cell Line, Tumor; Cell Proliferation; Cell Survival; Chemoprevention; Colorectal Neoplasms; Cyclin D1; Cyclin-Dependent Kinase 4; Disease Models, Animal; Female; Genes, APC; Humans; Ki-67 Antigen; Mice; Mice, Knockout; Retinoblastoma Protein; Signal Transduction; Silybin; Silymarin

Conventional classifications of gastroenteropancreatic neuroendocrine tumours (GEP- NETs) are rather unsatisfactory because of the variation in survival within each subgroup. Molecular markers are being found able to predict patient outcome in more and more tumours. The aim of this study was to characterize the expression of the proteins cyclin D1, cyclin E and P53 in GEP- NETs and assess any prognostic impact. Tumor specimens from 68 patients with a complete follow-up were studied immunohistochemically for cyclin D1, cyclin E and P53 expression. High cyclin D1 and cyclin E immunostaining (≥ 5% positive nuclei) was found in 48 (71%) and 24 (35%) cases, and high P53 staining (≥ 10% positive nuclei) in 33 (49%) . High expression of P53 was more common in gastric neuroendocrine tumors and related to malignant behavior, being associate with a worse prognosis on univariate analysis (RR=1.9, 95%CI=1.1-3.2). High expression of cyclin E was significantly associated with shorter survival in the univariate analysis (RR=2.0, 95%CI=1.2-3.6) and multivariate analysis (RR=2.1, 95%CI=1.1-4.0). We found no significant correlation between the expression of cyclin D1 and any clinicopathological variables. Our study indicated a prognostic relevance for cyclin E and P53 immunoreactivity. Cyclin E may be an independent prognostic factor from the 2010 WHO Classification which should be evaluated in further studies.

Topics: Aged; Colorectal Neoplasms; Cyclin D1; Cyclin E; Female; Humans; Kaplan-Meier Estimate; Male; Middle Aged; Multivariate Analysis; Neuroendocrine Tumors; Pancreatic Neoplasms; Proportional Hazards Models; Retrospective Studies; Stomach Neoplasms; Tumor Suppressor Protein p53

The Hippo signaling pathway is a critical regulator of organ size control during development, and its deregulation is associated with cancers. Acting downstream of this pathway, Yes-associated protein (YAP) was implicated in tumorigenesis. The present study aimed to explore the expression patterns and clinical significance of YAP in human colorectal cancer (CRC). In addition, we investigated the relationship between YAP expression and Wnt/β-catenin pathway activation in CRC. A total of 139 cases of CRC tissues were investigated by immunohistochemistry for the expression of YAP, cyclin D1, and β-catenin. The association between YAP expression and clinicopathologic features was analyzed. Our results showed that YAP was overexpressed in 52.5 % (73/139) cases of CRC and predominantly presented in the nucleus. There was an excellent correlation between YAP expression and pTNM stage (p = 0.0024). YAP expression in CRC was significantly correlated with nodal status (p = 0.0034), tumor status (p = 0.0382), and cyclin D1 overexpression (p < 0.0001). Importantly, YAP expression was associated with short overall survival (p < 0.001). Furthermore, patients with YAP-positive and nuclear β-catenin-positive profiles had worse overall survival. Univariate and multivariate analyses revealed that YAP expression was an independent prognostic indicator of CRC (p = 0.0207). Our results indicated that YAP overexpression contributed to the tumorigenesis and played a pivotal role in the progression in CRC, and the interaction of YAP and Wnt/β-catenin pathways needs further exploration.

Topics: Adaptor Proteins, Signal Transducing; beta Catenin; Cell Transformation, Neoplastic; Colorectal Neoplasms; Cyclin D1; Female; Humans; Male; Middle Aged; Phosphoproteins; Prognosis; Survival; Transcription Factors; Wnt Proteins; Wnt Signaling Pathway; YAP-Signaling Proteins

Hepatic metastasis is responsible for the majority of colorectal cancer (CRC)-related mortalities. Although Gankyrin (PSMD10) has been implicated in cancer metastasis, its exact role and underlying mechanisms of CRC hepatic metastasis remain largely unknown. Herein, we showed that the expression of Gankyrin was higher in primary CRC with hepatic metastasis compared with CRC without metastasis. RNAi-mediated silencing of Gankyrin expression in highly metastatic human CRC cells impaired their migratory and metastatic capacity in vivo. Genome-wide transcriptome profiling revealed activation of the interleukin (IL)-8 signaling pathway by Gankyrin. Protein levels of IL-8 and cyclin D1 (CCND1), the two important molecules in the IL-8 pathway, were positively correlated with Gankyrin expression in human CRC specimens. Furthermore, genetic deletion of cyclin D1 showed its requirement in Gankyrin-mediated cell migration. Finally, administration of recombinant IL-8 rescued the migratory defect of CRC cells where Gankyrin expression was silenced. Together, our findings highlight the importance of Gankyrin in hepatic metastasis of CRC and point out its candidature as a potential prognostic marker and therapeutic target to improve the clinical management of metastatic CRC.

Topics: 3T3 Cells; Animals; Cell Line; Cell Movement; Colorectal Neoplasms; Cyclin D1; HEK293 Cells; Humans; Interleukin-8; Liver Neoplasms; Male; Mice; Mice, Inbred C57BL; Neoplasm Invasiveness; Neoplasm Metastasis; Proteasome Endopeptidase Complex; Proto-Oncogene Proteins; Signal Transduction

Sex determining region Y-box 2 (SOX2), is a high mobility group box transcription factor involved in the maintenance of pluripotency and the self-renewal of embryonic and neuronal stem cells, which also plays differential roles in the cell proliferation of several tumors. However, its role in colorectal adenocarcinoma cell proliferation and the underlying mechanisms remain unclear. The mammalian target of rapamycin (mTOR) signaling pathway has recently emerged as an important regulator of cell proliferation in many types of cancer. In this study, we examined the effect of SOX2 on the proliferation of colorectal adenocarcinoma cells and evaluated the role of the mTOR pathway in this process. Our results indicated that the overexpression of SOX2 significantly inhibited the proliferation of colorectal adenocarcinoma cells. Of note, mechanistic investigations revealed that SOX2 inhibited the activation of the mTOR pathway in HT-29 cells. We then examined the effect of SOX2 on the cell cycle, and the results revealed that SOX2 downregulated cyclin D1 expression and induced G0/G1 arrest in the HT-29 cells. Moreover, we also analyzed the correlation between SOX2 expression and patient clinicopathological characteristics in colorectal adenocarcinoma tissues, as well as the level of phospho-S6 (S235/236), phospho-Akt (S473) and cyclin D1. The results revealed a significant negative correlation between SOX2 expression and tumor size and the levels of phospho-S6 (S235/236), phospho-Akt (S473) and cyclin D1. Taken together, to our knowledge, our findings suggest for the first time that SOX2 suppresses colorectal adenocarcinoma cell proliferation through the inhibition of the mTOR pathway.

Topics: Adenocarcinoma; Adult; Aged; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Colorectal Neoplasms; Cyclin D1; Female; Gene Expression Regulation, Neoplastic; HT29 Cells; Humans; Male; Middle Aged; Neoplasm Grading; Proto-Oncogene Proteins c-akt; Ribosomal Protein S6 Kinases; Signal Transduction; SOXB1 Transcription Factors; TOR Serine-Threonine Kinases; Tumor Burden

Patchouli alcohol (PA) is one of the important compounds isolated from the essential oil of Pogostemon cablin (patchouli). PA has neuroprotective, anti-influenza and anti-inflammatory activities. However, anti-cancer activity of PA has not been studied so far. We performed in vitro study to investigate whether PA affects proliferation and apoptosis of human colorectal cancer cells, and to define potential molecular mechanisms. PA suppressed cell growth and induced apoptosis in a dose-dependent manner in human colorectal cancer cells (HCT116, SW480). In addition, PA decreased cell growth in MCF7, BxPC3, PC3, and HUVEC cells. Exposure of PA to HCT116 and SW480 cells activated p21 expression and suppressed the expressions of cyclin D1 and cyclin-dependent kinase 4 (CDK4) in a dose-dependent manner. In addition, PA attenuated the expressions of HDAC2 (histone deacetylase 2) and c-myc, and HDAC enzyme activity. We also observed that PA induced the transcriptional activity of NF-κB through an increase of nuclear translocation of p65. These findings suggest that PA exerts an anti-cancer activity by decreasing cell growth and increasing apoptosis in human colorectal cancer cells. The proposed mechanisms include the inhibition of HDAC2 expression and HDAC enzyme activity, and subsequent downregulation of c-myc and activation of NF-κB pathway.

Topics: Antineoplastic Agents; Apoptosis; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cells, Cultured; Colorectal Neoplasms; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase Inhibitor p21; Histone Deacetylase 2; Histone Deacetylase Inhibitors; Histone Deacetylases; Human Umbilical Vein Endothelial Cells; Humans; Lamiaceae; NF-kappa B; Proto-Oncogene Proteins c-myc; Sesquiterpenes

To evaluate the expression of special AT-rich sequence-binding protein 1 (SATB1) gene in colorectal cancer and its role in colorectal cancer cell proliferation and invasion.. Immunohistochemistry was used to detect the protein expression of SATB1 in 30 colorectal cancer (CRC) tissue samples and pair-matched adjacent non-tumor samples. Cell growth was investigated after enhancing expression of SATB1. Wound-healing assay and Transwell assay were used to investigate the impact of SATB1 on migratory and invasive abilities of SW480 cells in vitro. Nude mice that received subcutaneous implantation or lateral tail vein were used to study the effects of SATB1 on tumor growth or metastasis in vivo.. SATB1 was over-expressed in CRC tissues and CRC cell lines. SATB1 promotes cell proliferation and cell cycle progression in CRC SW480 cells. SATB1 overexpression could promote cell growth in vivo. In addition, SATB1 could significantly raise the ability of cell migration and invasion in vitro and promote the ability of tumor metastasis in vivo. SATB1 could up-regulate matrix metalloproteases 2, 9, cyclin D1 and vimentin, meanwhile SATB1 could down-regulate E-cadherin in CRC.. SATB1 acts as a potential growth and metastasis promoter in CRC. SATB1 may be useful as a therapeutic target for CRC.

Topics: Animals; Antigens, CD; Cadherins; Cell Line, Tumor; Cell Movement; Cell Proliferation; Colorectal Neoplasms; Cyclin D1; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Matrix Attachment Region Binding Proteins; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Mice; Mice, Nude; Neoplasm Invasiveness; Neoplasm Metastasis; Up-Regulation; Vimentin; Wound Healing

Colorectal cancer is one of the leading causes of cancer-related mortality worldwide. Cancer stem cells are cell populations with stem cell nature presenting in tumor tissues and are the root of tumor formation and metastasis. CCND1 and E2F3 play important roles in cell cycle regulation. The 3'UTRs of CCND1 and E2F3 contain miR-449 binding sites. By transfecting pre-miR-449b and inhibiting miR-449b, we found that cell cycle, cell proliferation ability and cell cycle regulatory protein expression levels of colon cancer stem cells were altered. The correlation between CCND1, E2F3 and miR-449b showed that miR-449b could downregulate CCND1 and E2F3 expression. This, in turn, reduced the proliferative ability of colon cancer stem cells. These data suggest that miR-449b plays a tumor-suppressive role in colon cancer stem cells.

Topics: Cell Cycle; Cell Cycle Proteins; Cell Line, Tumor; Cell Proliferation; Colorectal Neoplasms; Cyclin D1; Down-Regulation; E2F3 Transcription Factor; Gene Expression Regulation, Neoplastic; Humans; MicroRNAs; Neoplastic Stem Cells; Signal Transduction

G-protein-coupled receptor 48 (GPR48) is an orphan receptor belonging to the G-protein-coupled receptors family, which plays an important role in the development of various organs and cancer development and progression such as gastric cancer and colorectal cancer (CRC). However, the prognostic value of GPR48 expression in patients with CRC has not been reported. In this study, we observed that GPR48 was overexpressed in primary CRC and metastatic lymph nodes and closely correlated with tumor invasion and metastasis. Multivariate analysis indicated that high GPR48 expression was a poor prognostic factor for overall survival in CRC patients. In vitro and in vivo assays demonstrated that enforced expression of GPR48 contributed to enhance migration and invasion of cancer cells and tumor metastasis. In addition, we found that GPR48 increased nuclear β-catenin accumulation, T-cell factor 4 (TCF4) transcription activity, and expression of its target genes including Cyclin D1 and c-Myc in CRC cells. Correlation analysis showed that GPR48 expression in CRC tissues was positively associated with β-catenin expression. Upregulation of GPR48 resulted in increased phosphorylation of glycogen synthase kinase 3β, Akt and extracellular signal-regulated kinase 1/2 (ERK1/2) in CRC cells, while inhibition of PI3K/Akt and mitogen-activated protein kinase /ERK1/2 pathways was sufficient to abolish the effect of GPR48 on β-catenin/TCF signaling. Taken together, GPR48 could serve as both a prognostic biomarker and a therapeutic target for resectable CRC patients.

Topics: Aged; Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; beta Catenin; Cell Line; Cell Line, Tumor; Cell Movement; Colorectal Neoplasms; Cyclin D1; Female; HCT116 Cells; HEK293 Cells; Humans; Lymph Nodes; Lymphatic Metastasis; Male; MAP Kinase Signaling System; Mitogen-Activated Protein Kinases; Neoplasm Invasiveness; Phosphatidylinositol 3-Kinases; Phosphorylation; Prognosis; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-myb; Receptors, G-Protein-Coupled; Signal Transduction; Transcription Factor 4; Transcription Factors; Transcription, Genetic; Up-Regulation

To investigate the effects of a proliferation-inducing ligand (APRlL) on colorectal cancer (CRC) cell growth and migration, and to observe the role of APRIL in CRC biological behavior.. The siRNA plasmid vector targeting APRIL gene (APRIL-siRNA) was transfected into human colorectal cancer SW480 cells and recombinant human APRIL (rhAPRIL) was used to stimulate human colorectal cancer HCT-116 cells. Cell proliferation activity was analyzed using cell counting kit-8 (CCK-8), cell cycle was detected by flow cytometry, and the protein expression of cyclin D1, p21 and Bcl-2 was detected by Western blot analysis. Tumor cell migration and invasion were measured by Transwell chambers. RT-PCR was applied to examine the mRNA expression level of MMP-2 and MMP-9. APRIL-siRNA was used to transfect directly SW480 cells, which were injected subcutaneously into nude mice, then the tumor growth and metastasis were observed.. Cell proliferation ability of APRIL-siRNA-transfected SW480 cells was drastically repressed, and the percentage of G0/G1 phase cells was significantly increased (t = 4.12, P < 0.05), accompanied with depressed cyclin D1, Bcl-2 expression and elevated p21 expression. Cell proliferation ability of rhAPRIL-stimulated HCT-116 cells was promoted with a decreased G0/G1 phase ratio (t = 3.31, P < 0.05). cyclin D1 and Bcl-2 protein expression was up-regulated while p21 was down-regulated by rhAPRIL stimulation. Metastatic and invasive capacities of APRIL-siRNA-transfected SW480 cells were significantly inhibited compared with their respective controls (both P < 0.05), accompanied with the deregulated MMP-2 and MMP-9 mRNA expression. Metastatic and invasive capacities of rhAPRIL-stimulated HCT-116 cells were promoted with up-regulated MMP-2 and MMP-9 mRNA expression(both P < 0.05). Tumor growth in the group transfected with APRIL-siRNA appeared to be slower than that in the control groups and the expression of MMP-2, MMP-9 in tumor tissues was depressed in the APRIL-siRNA group.. APRIL facilitates tumor growth and metastasis, and is associated with carcinogenesis and prognosis. Our findings suggest that APRIL might be used as a novel target for the intervention and therapy of colorectal cancer.

Topics: Animals; Cell Cycle; Cell Line, Tumor; Cell Movement; Cell Proliferation; Colorectal Neoplasms; Cyclin D1; Female; Genetic Vectors; HCT116 Cells; HT29 Cells; Humans; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Mice; Mice, Nude; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasm Transplantation; Plasmids; Proto-Oncogene Proteins c-bcl-2; Proto-Oncogene Proteins p21(ras); RNA, Messenger; RNA, Small Interfering; Transfection; Tumor Burden; Tumor Necrosis Factor Ligand Superfamily Member 13

Colorectal cancer (CRC) is one of the most common cancers worldwide and a leading cause of cancer related death. Although the mortality rate of CRC is decreasing, finding novel targets for its therapy remains urgent. Carboxypeptidase E (CPE), a member of the pro-protein convertases, which are involved in the maturation of protein precursors, has recently been reported as elevated in many types of cancer. However, its role and mechanisms in tumor progression are poorly understood.. In the present study, we investigated expression of CPE in CRC cell lines and tumor tissues using Western blot and real-time qRT-PCR. Plasmids for overexpression and depletion of CPE were constructed and analyzed by Western blot, MTT and colony formation assays and bromodeoxyuridine incorporation assays. The relative expression of p21, p27, and cyclin D1 were analyzed by Real-time qRT-PCR in the indicated cells.. Our study showed that CPE was significantly upregulated in CRC cell lines and tumor tissues. MTT and colony formation assays indicated that overexpression of CPE enhanced cell growth rates. BrdU incorporation and flow-cytometry assays showed that ectopic expression of CPE increased the S-phase fraction cells. Soft agar assay proved enhanced tumorigenicity activity in CPE over-expressing CRC cells. Further studies of the molecular mechanisms of CPE indicated that is promoted cell proliferation and tumorigenicity through downregulation of p21 and p27, and upregulation of cyclin D1.. Taken together, these data suggest that CPE plays an important role in cell cycle regulation and tumorigenicity, and may serve as a potential target for CRC therapeutics.

Topics: Carboxypeptidase H; Cell Line, Tumor; Cell Proliferation; Cell Transformation, Neoplastic; Colorectal Neoplasms; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase Inhibitor p27; Gene Expression; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Humans; S Phase; Up-Regulation

We recently reported that the tumor suppressor Runt-related transcription factor 3 (RUNX3) is silenced in colorectal cancer cells via oxidative stress-induced hypermethylation of its promoter. The resulting downregulation of RUNX3 expression influences cell proliferation. Activation of the Akt signaling pathway is also associated with cell survival and proliferation; however, the effects of oxidative stress on the relationship between RUNX3 and Akt signaling are largely unknown. Therefore, this study investigated the mechanisms involved in cell proliferation caused by oxidative stress-induced silencing of RUNX3. The levels of RUNX3 mRNA and protein were downregulated in response to treatment of the human colorectal cancer cell line SNU-407 with H2O2. Treatment of the cells with H2O2 also upregulated Akt mRNA and protein expression, and inhibited the binding of RUNX3 to the Akt promoter. The inverse correlation between the expression levels of RUNX3 and Akt in H2O2-treated cells was also associated with nuclear translocation of β-catenin and upregulation of cyclin D1 expression, which induced cell proliferation. H2O2 treatment also increased the binding of β-catenin to the cyclin D1 promoter. The results presented here demonstrate that reactive oxygen species silence the tumor suppressor RUNX3, enhance the Akt-mediated signaling pathway, and promote the proliferation of colorectal cancer cells.

Topics: beta Catenin; Blotting, Western; Cell Nucleus; Cell Proliferation; Colorectal Neoplasms; Core Binding Factor Alpha 3 Subunit; Cyclin D1; Gene Expression Regulation, Neoplastic; Humans; Hydrogen Peroxide; Immunoenzyme Techniques; Oxidants; Oxidative Stress; Promoter Regions, Genetic; Protein Transport; Proto-Oncogene Proteins c-akt; Reactive Oxygen Species; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Small Interfering; Signal Transduction; Tumor Cells, Cultured

The nonsteroidal anti-inflammatory drug tolfenamic acid has been shown to suppress cancer cell growth and tumorigenesis in different cancer models. However, the underlying mechanism by which tolfenamic acid exerts its antitumorigenic effect remains unclear. Previous data from our group and others indicate that tolfenamic acid alters expression of apoptosis- and cell-cycle arrest-related genes in colorectal cancer cells. Here, we show that tolfenamic acid markedly reduced the number of polyps and tumor load in APC(min)(/+) mice, accompanied with cyclin D1 downregulation in vitro and in vivo. Mechanistically, tolfenamic acid promotes endoplasmic reticulum (ER) stress, resulting in activation of the unfolded protein response (UPR) signaling pathway, of which PERK-mediated phosphorylation of eukaryotic translation initiation factor 2α (eIF2α) induces the repression of cyclin D1 translation. Moreover, the PERK-eIF2α-ATF4 branch of the UPR pathway plays a role in tolfenamic acid-induced apoptosis in colorectal cancer cells, as silencing ATF4 attenuates tolfenamic acid-induced apoptosis. Taken together, these results suggest ER stress is involved in tolfenamic acid-induced inhibition of colorectal cancer cell growth, which could contribute to antitumorigenesis in a mouse model.

Topics: Activating Transcription Factor 4; Adenomatous Polyposis Coli Protein; Animals; Anti-Inflammatory Agents, Non-Steroidal; Blotting, Western; Cell Transformation, Neoplastic; Colonic Polyps; Colorectal Neoplasms; Cyclin D1; eIF-2 Kinase; Endoplasmic Reticulum Stress; Humans; Immunoprecipitation; Mice; Mice, Inbred C57BL; Mice, Knockout; ortho-Aminobenzoates; Phosphorylation; Protein Serine-Threonine Kinases; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; Tumor Cells, Cultured; Unfolded Protein Response

Multidrug resistance (MDR), an unfavorable factor compromising the treatment efficacy of anticancer drugs, involves the upregulation of ATP binding cassette (ABC) transporters and induction of galectin-3 signaling. Galectin-3 plays an anti-apoptotic role in many cancer cells and regulates various pathways to activate MDR. Thus, the inhibition of galectin-3 has the potential to enhance the efficacy of the anticancer drug epirubicin. In this study, we examined the effects and mechanisms of silencing galectin-3 via RNA interference (RNAi) on the β-catenin/GSK-3β pathway in human colon adenocarcinoma Caco-2 cells. Galectin-3 knockdown increased the intracellular accumulation of epirubicin in Caco-2 cells; suppressed the mRNA expression of galectin-3, β-catenin, cyclin D1, c-myc, P-glycoprotein (P-gp), MDR-associated protein (MRP) 1, and MRP2; and downregulated the protein expression of P-gp, cyclin D1, galectin-3, β-catenin, c-Myc, and Bcl-2. Moreover, galectin-3 RNAi treatment significantly increased the mRNA level of GSK-3β, Bax, caspase-3, and caspase-9; remarkably increased the Bax-to-Bcl-2 ratio; and upregulated the GSK-3β and Bax protein expressions. Apoptosis was induced by galectin-3 RNAi and/or epirubicin as demonstrated by chromatin condensation, a higher sub-G1 phase proportion, and increased caspase-3 and caspase-9 activity, indicating an intrinsic/mitochondrial apoptosis pathway. Epirubicin-mediated resistance was effectively inhibited via galectin-3 RNAi treatment. However, these phenomena could be rescued after galectin-3 overexpression. We show for the first time that the silencing of galectin-3 sensitizes MDR cells to epirubicin by inhibiting ABC transporters and activating the mitochondrial pathway of apoptosis through modulation of the β-catenin/GSK-3β pathway in human colon cancer cells.

Topics: Apoptosis; ATP Binding Cassette Transporter, Subfamily B, Member 1; ATP-Binding Cassette Transporters; bcl-2-Associated X Protein; beta Catenin; Caco-2 Cells; Caspases; Cell Cycle; Cell Line, Tumor; Colorectal Neoplasms; Cyclin D1; Epirubicin; Galectin 3; Gene Silencing; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Humans; Mitochondria; Multidrug Resistance-Associated Protein 2; Multidrug Resistance-Associated Proteins; Proto-Oncogene Proteins c-bcl-2; Proto-Oncogene Proteins c-myc; RNA, Messenger; RNA, Small Interfering; Signal Transduction

Angiogenesis plays an important role in the progression of colorectal cancer (CRC). Studies have indicated vascular endothelial growth factor (VEGF) is the predominant angiogenic factor. Cyclin D1 (CCND1) induces production of VEGF and is required for migration of blood vessels. Our aim was to determine the roles of CCND1 and VEGF overexpression in CRC patients.. We analyzed clinicopathological features, VEGF and CCND1 expressions by immunohistochemical (IHC) staining in 100 stage I-III CRC patients (44 were postoperative relapsed; 56 were postoperative non-relapsed) to determine the correlation between clinicopathologic features and co-existence of CCND1 and VEGF. Furthermore, the clinical outcomes of co-existence of CCND1 and VEGF were investigated.. Multivariate analysis showed vascular invasion (P = 0.019), VEGF overexpression (P = 0.033), and high postoperative serum carcinoembryonic antigen (CEA) levels (P = 0.022) were independent predictors of postoperative relapse. Co-existence of CCND1 and VEGF overexpression had significantly poorer disease-free survival rates (P  = 0.004) and overall survival rates (P = 0.001) than other phenotypes.. Co-existence of CCND1 and VEGF overexpression would potentially assist in TNM staging systems to predict the prognosis of these patients who would benefit from intensive follow-up and therapeutic programs.

Topics: Adenocarcinoma; Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Colectomy; Colorectal Neoplasms; Cyclin D1; Female; Humans; Immunohistochemistry; Logistic Models; Male; Middle Aged; Multivariate Analysis; Neoplasm Metastasis; Neoplasm Recurrence, Local; Neoplasm Staging; Rectum; Survival Analysis; Treatment Outcome; Vascular Endothelial Growth Factor A

Protocatechualdehyde (PCA) is a naturally occurring polyphenol found in barley, green cavendish bananas, and grapevine leaves. Although a few studies reported growth-inhibitory activity of PCA in breast and leukemia cancer cells, the underlying mechanisms are still poorly understood. Thus, we performed in vitro study to investigate if treatment of PCA affects cell proliferation and apoptosis in human colorectal cancer cells and define potential mechanisms by which PCA mediates growth arrest and apoptosis of cancer cells. Exposure of PCA to human colorectal cancer cells (HCT116 and SW480 cells) suppressed cell growth and induced apoptosis in dose-dependent manner. PCA decreased cyclin D1 expression in protein and mRNA level and suppressed luciferase activity of cyclin D1 promoter, indicating transcriptional downregulation of cyclin D1 gene by PCA. We also observed that PCA treatment attenuated enzyme activity of histone deacetylase (HDAC) and reduced expression of HDAC2, but not HDAC1. These findings suggest that cell growth inhibition and apoptosis by PCA may be a result of HDAC2-mediated cyclin D1 suppression.

Topics: Antineoplastic Agents; Apoptosis; Benzaldehydes; Catechols; Colorectal Neoplasms; Cyclin D1; Down-Regulation; HCT116 Cells; Histone Deacetylase 2; Histone Deacetylase Inhibitors; Humans

There is increasing evidence that Special AT-rich sequence-binding protein 1 (SATB1) is aberrantly expressed in several cancers and is correlated with clinicopathologic parameters in these tumors. In this study, we showed over-expression of SATB1 in 80 cases of colorectal cancer and in 3 colorectal cancer cell lines and found expression levels were strongly associated with tumor differentiation and stage. Expression levels of SATB1 protein were higher in poorly-differentiated as compared with well-differentiated cell lines, and both quantity and distribution patterns of SATB1 were associated with tumor differentiation and pTNM stage. Strikingly, we further investigated the effect of down regulation of SATB1 expression on malignant phenotypic features in colorectal cancer cells in vitro, and showed that SABT1 down-regulation negatively affected growth potential, anchorage-independent colony formation and cancer cell invasion, and resulted in increased apoptosis. SATB1 expression was positively associated with the expression of various biological and genetic markers, including Cyclin D1, MMP-2, NF-κB, and PCNA, and was associated with loss of APC and BRAF(V600E). These findings suggest that SATB1 is involved in the carcinogenesis, development and progression of colorectal cancer.

Topics: Adult; Blotting, Western; Cell Line, Tumor; Cell Proliferation; Colorectal Neoplasms; Cyclin D1; Female; Gene Expression Regulation, Neoplastic; HT29 Cells; Humans; Immunohistochemistry; Jurkat Cells; Male; Matrix Attachment Region Binding Proteins; Matrix Metalloproteinase 2; Microscopy, Confocal; Middle Aged; Mutation; Neoplasm Staging; Proto-Oncogene Proteins B-raf; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference

Despite their pivotal roles in colorectal carcinogenesis, the interrelationship and prognostic significance of beta-catenin alterations and microsatellite instability (MSI) in colorectal cancer (CRC) needs to be further clarified. In this paper, we studied the associations between beta-catenin overexpression and MSI status with survival from CRC, and with expression of p21, p27, cyclin D1 and p53, in a large, prospective cohort study.. Immunohistochemical MSI-screening status and expression of p21, p27 and p53 was assessed in tissue microarrays with tumours from 557 cases of incident CRC in the Malmö Diet and Cancer Study. Chi Square and Spearman's correlation tests were used to explore the associations between beta-catenin expression, MSI status, clinicopathological characteristics and investigative parameters. Kaplan-Meier analysis and Cox proportional hazards modelling were used to assess the relationship between beta-catenin overexpression, MSI status and cancer specific survival (CSS).. Positive MSI screening status was significantly associated with older age, female sex, proximal tumour location, non-metastatic disease, and poor differentiation, and inversely associated with beta-catenin overexpression. Beta-catenin overexpression was significantly associated with distal tumour location, low T-stage and well-differentiated tumours. Patients with MSI tumours had a significantly prolonged CSS in the whole cohort, and in stage III-IV disease, also in multivariable analysis, but not in stage I-II disease. Beta-catenin overexpression was associated with a favourable prognosis in the full cohort and in patients with stage III-IV disease. Neither MSI nor beta-catenin status were predictive for response to adjuvant chemotherapy in curatively treated stage III patients. P53 and p27 expression was positively associated with beta-catenin overexpression and inversely associated with MSI. Cyclin D1 expression was positively associated with MSI and beta-catenin overexpression, and p21 expression was positively associated with MSI but not beta-catenin overexpression.. Findings from this large, prospective cohort study demonstrate that MSI screening status in colorectal cancer is an independent prognostic factor, but not in localized disease, and does not predict response to adjuvant chemotherapy. Beta-catenin overexpression was also associated with favourable outcome but not a treatment predictive factor. Associations of MSI and beta-catenin alterations with other investigative and clinicopathological factors were in line with the expected.. The virtual slides for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/8778585058652609.

Topics: Adult; Aged; beta Catenin; Biomarkers, Tumor; Cell Cycle Proteins; Chemotherapy, Adjuvant; Chi-Square Distribution; Colectomy; Colorectal Neoplasms; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase Inhibitor p27; Female; Humans; Immunohistochemistry; Incidence; Kaplan-Meier Estimate; Male; Microsatellite Instability; Middle Aged; Multivariate Analysis; Neoplasm Staging; Proportional Hazards Models; Prospective Studies; Registries; Risk Factors; Sweden; Time Factors; Tissue Array Analysis; Treatment Outcome; Tumor Suppressor Protein p53; Up-Regulation

The present study aimed to analyse the role of cyclin D1 A870G polymorphism in modulating the susceptibility to colorectal cancer (CRC) in the Kashmiri population. The genotype distribution of the cyclin D1 gene in 130 CRC cases in comparison with 160 healthy controls was investigated. No direct significant association between cyclin D1 genotypes and CRC was observed; however, the AG and AA genotypes were found to be associated with an increased risk of CRC compared to the GG genotype, with an almost 2-fold increase in OR. This study suggests that the cyclin D1 polymorphism is associated with an increased risk of CRC in the Kashmiri population.

Topics: Adult; Aged; Case-Control Studies; Colorectal Neoplasms; Cyclin D1; Female; Gene Frequency; Genetic Predisposition to Disease; Genotype; Humans; Interviews as Topic; Male; Middle Aged; Odds Ratio; Polymorphism, Single Nucleotide; Risk Factors

Colorectal cancer (CRC) is the second leading cause of cancer death in the Western countries. Novel approaches of treatment are needed for CRC. The purpose of the present study was to investigate cytotoxic effect of external Qi of Yan Xin Qigong (YXQ-EQ) on human colorectal cancer cells.. The effect of YXQ-EQ on viability, cell cycle progression and apoptosis in colorectal cancer HT-29 cells was investigated. Phosphorylation of Akt and Erk1/2, activation of NF-ĸB and the expression of proteins involved in regulation of cell cycle and apoptosis were examined by Western blot analysis.. YXQ-EQ markedly decreased viability and blocked colony formation of HT-29 cells. YXQ-EQ downregulated cyclin D1 expression and increased accumulation of cyclin-dependent kinase inhibitors p21(Cip1) and p27(Kip1), resulting in G1 cell cycle arrest. YXQ-EQ induced apoptosis in HT-29 cells in association with decreased expression of antiapoptotic proteins Bcl-xL, XIAP, survivin and Mcl-1 and elevated expression of proapoptotic protein Bax. YXQ-EQ significantly repressed phosphorylation of Akt and Erk1/2 and NF-ĸB activation in HT-29 cells, suggesting that YXQ-EQ may exert cytotoxic effect through regulating signaling pathways critical for cell proliferation and survival. Furthermore, YXQ-EQ treated PBS and an YXQ-EQ treated plant extract induced apoptosis in HT-29 cells.. These findings show that YXQ-EQ has potent cytotoxic effect on HT-29 cells and suggest that YXQ-EQ could be potentially used for colorectal cancer treatment either directly or indirectly via carriers.

Topics: Apoptosis; bcl-2-Associated X Protein; Cell Cycle Proteins; Colorectal Neoplasms; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase Inhibitor p27; Down-Regulation; G1 Phase Cell Cycle Checkpoints; HT29 Cells; Humans; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; NF-kappa B; Phosphorylation; Proto-Oncogene Proteins c-akt; Qi

Although dysplasia is thought to be the precursor lesion in the development of colitis-associated colorectal cancer (CRC), a significant proportion of patients with ulcerative colitis (UC) and low-grade (LGD) or indefinite (IND) dysplasia remain cancer-free during endoscopic follow-up. There is a need for biomarkers that predict neoplastic progression. We studied the value of a series of immunohistochemical markers in UC patients with flat LGD or IND with regard to neoplastic progression.. Tissue samples were collected from 12 UC patients (six flat LGD, six IND) without progression and from 10 UC patients (eight flat LGD, two IND) with documented progression to HGD and/or CRC during a median of 25 and 23 months of colonoscopic follow-up, respectively. Immunohistochemistry using monoclonal antibodies was performed for p53, CD44, Ki67, AMACR, β-catenin, cyclin D1, p21, and ALDH. Positive and negative staining patterns were compared for progression to advanced neoplasia.. When patients showed coexpression of p53 and AMACR, 6/7 patients (86%) developed advanced neoplasia, compared to 4/15 patients (27%) without p53/AMACR coexpression (P = 0.02). Patients with p53/AMACR coexpression developed advanced neoplasia in a time period of 19 months (median, range 1-101) compared to 80 months (median, range 8-169) in patients without p53/AMACR coexpression (P = 0.14). Interestingly, in three patients with progression and previous dysplasia-negative biopsies, two out of three biopsies were p53-positive a median of 12 months (range 10-14) before the LGD/IND diagnosis.. This study suggests a role for p53/AMACR coexpression as a potential marker of neoplastic progression in patients with UC.

Topics: Adolescent; Adult; Aged; Aldehyde Dehydrogenase; beta Catenin; Biomarkers; Cell Transformation, Neoplastic; Colitis, Ulcerative; Colorectal Neoplasms; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Disease Progression; Female; Humans; Hyaluronan Receptors; Immunohistochemistry; Ki-67 Antigen; Male; Middle Aged; Predictive Value of Tests; Racemases and Epimerases; Statistics, Nonparametric; Tumor Suppressor Protein p53; Young Adult

Surveillance colonoscopy is an important strategy for prevention of colorectal cancer. 5-aminosalicylate (ASA) (mesalazine) is discussed as a chemopreventive agent as it reduces the cancer risk in ulcerative colitis patients. The current study analyses the effect of 5-ASA on Wnt/β-catenin signaling in vitro and in vivo in colon epithelial cells. The effect of 5-ASA was determined using a β-catenin/T-cell factor (TCF)-reporter assay and by western blotting in cultured colon cancer cells. Formalin fixed paraffin embedded material from 227 polyps removed from a subgroup of 56 patients, who participated in a randomized placebo-controlled 3-year prevention trial with 5-ASA was evaluated according to histomorphological characteristics and expression of β-catenin and target genes Cox2, cyclin D1 and E-cadherin as well as ornithine decarboxylase (ODC). Patients were grouped into a low-risk and a high-risk group according to the number of adenomas at initial colonoscopy. ß-catenin/TCF signaling activity was significantly reduced by 5-ASA treatment possibly through a reduction in ß-catenin levels. Moreover, 5-ASA significantly reduced ß-catenin levels and nuclear localization in patients' adenomas. In addition, 5-ASA also significantly changed expression of the downstream targets Cox2, cyclin D1 and E-cadherin, correlating with ß-catenin status. Moreover, 5-ASA significantly reduced levels of ODC in vivo. Expression of p53 was unaltered by the 5-ASA treatment. Our study shows a significant in vitro and long-term in vivo effect of 5-ASA on ß-catenin signaling as a key signaling pathway in the development of colorectal adenoma. Therefore, we suggest the use of 5-ASA as a promising drug for prevention of sporadic colorectal carcinoma.

Topics: Adenoma; beta Catenin; Cadherins; Cell Line, Tumor; Colitis, Ulcerative; Colorectal Neoplasms; Cyclin D1; Cyclooxygenase 2; Disease Progression; Epithelial Cells; Female; Gene Expression Regulation, Neoplastic; Genes, Reporter; Humans; Male; Mesalamine; Ornithine Decarboxylase; Signal Transduction; TCF Transcription Factors; Tumor Suppressor Protein p53; Wnt Proteins; Wnt Signaling Pathway

Signal transducer and activator of transcription 3 (STAT3) plays a critical role in cell survival and proliferation. Constitutive activation of STAT3 is strongly correlated with pathogenesis of various types of malignant tumors including colorectal cancer (CRC), and therefore is a major focus in the development of anti-cancer agents. Pien Tze Huang (PZH), a well-known traditional Chinese formula prescribed already in the Ming Dynasty, has been demonstrated to be clinically effective in the treatment of CRC. However, the precise mechanism of its anti-cancer activity remains largely unknown. In the present study we evaluated the efficacy of PZH against tumor growth in vivo in the CRC mouse xenograft model, and investigated the underlying molecular mechanisms. We found that administration of PZH reduced tumor volume and tumor weight but had no effect on body weight gain in CRC mice, demonstrating that PZH can inhibit colon cancer growth in vivo without apparent adverse effect. We also observed that PZH treatment inhibited the phosphorylation level of STAT3 in tumor tissues. Consequently, the inhibitory effect of PZH on STAT3 activation resulted in the up-regulation of Bax/Bcl-2 ratio as well as down-regulation of Cyclin D1 and CDK4 expression, leading to the induction of apoptosis as well as the inhibition of cell proliferation. These results suggest that promotion of cancer cell apoptosis and inhibition of proliferation via suppression of STAT3 pathway might be one of the mechanisms by which PZH treats colorectal cancer.

Topics: Animals; Antineoplastic Agents, Phytogenic; Apoptosis; bcl-2-Associated X Protein; Cell Proliferation; Colorectal Neoplasms; Cyclin D1; Cyclin-Dependent Kinase 4; Drugs, Chinese Herbal; HT29 Cells; Humans; Male; Mice; Mice, Nude; Phosphorylation; Proto-Oncogene Proteins c-bcl-2; Signal Transduction; STAT3 Transcription Factor; Xenograft Model Antitumor Assays

Epidemiologic studies suggest that intake of high-fat diet (HFD) promotes colon carcinogenesis. Epithelial-mesenchymal transition (EMT) and inflammation play important roles during tumor progression of colorectal cancer (CRC). Oncogenic pathways such as phosphatidylinositol-3-kinase (PI3K)/Akt/mTOR and mitogen-activated protein kinase (MAPK)/ERK signaling cascades induce EMT and inflammation in cancer. No experimental evidence has been demonstrated regarding HFD-mediated tumor progression including EMT in CRC so far. Our results demonstrated that HFD consumption could induce tumor growth and progression, including EMT and inflammation, in a mouse xenograft tumor model. The molecular mechanisms were through activation of MAPK/ERK and PI3K/Akt/mTOR signaling pathways. HFD induced up-regulation of cyclooxygenase-2, cyclin D1 and proliferating cell nuclear antigen proteins concomitant with increases in expression of nuclear factor-κB p65 (RelA) and β-catenin proteins. Surprisingly, HFD consumption could suppress p21(CIP1/WAF1) expression through increases in nuclear histone deacetylase complex (HDAC). Moreover, HFD could mediate the disassembly of E-cadherin adherent complex and the up-regulation of Vimentin and N-cadherin proteins in tumor tissues. Taken together, our novel findings support evidence for HFD-mediated modulation of HDAC activity and activation of oncogenic cascades, which involve EMT and inflammation in CRC, playing important roles in tumor growth and progression in a mouse xenograft model.

Topics: Animals; beta Catenin; Cadherins; Cell Proliferation; Colorectal Neoplasms; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Cyclooxygenase 2; Diet, High-Fat; Disease Models, Animal; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation, Neoplastic; HT29 Cells; Humans; Mice; Mice, Inbred BALB C; Mitogen-Activated Protein Kinases; NF-kappa B; Phosphatidylinositol 3-Kinase; Proto-Oncogene Proteins c-akt; Signal Transduction; TOR Serine-Threonine Kinases; Up-Regulation; Vimentin

MicroRNAs (miRNAs) can promote or inhibit tumor growth and are therefore being developed as targets for cancer therapies. They are diverse not only in the messenger RNAs (mRNA) they target, but in their production; the same hairpin RNA structure can generate mature products from each strand, termed 5p and 3p, that can bind different mRNAs. We analyzed the expression, functions, and mechanisms of miR-28-5p and miR-28-3p in colorectal cancer (CRC) cells.. We measured levels of miR-28-5p and miR-28-3p expression in 108 CRC and 49 normal colorectal samples (47 paired) by reverse transcription, quantitative real-time polymerase chain reaction. The roles of miR-28 in CRC development were studied using cultured HCT116, RKO, and SW480 cells and tumor xenograft analyses in immunodeficient mice; their mRNA targets were also investigated.. miR-28-5p and miR-28-3p were down-regulated in CRC samples compared with normal colon samples. Overexpression of miRNAs in CRC cells had different effects and the miRNAs interacted with different mRNAs: miR-28-5p altered expression of CCND1 and HOXB3, whereas miR-28-3p bound NM23-H1. Overexpression of miR-28-5p reduced CRC cell proliferation, migration, and invasion in vitro, whereas miR-28-3p increased CRC cell migration and invasion in vitro. CRC cells overexpressing miR-28 developed tumors more slowly in mice compared with control cells, but miR-28 promoted tumor metastasis in mice.. miR-28-5p and miR-28-3p are transcribed from the same RNA hairpin and are down-regulated in CRC cells. Overexpression of each has different effects on CRC cell proliferation and migration. Such information has a direct application for the design of miRNA gene therapy trials.

Topics: Animals; Apoptosis; Case-Control Studies; Cell Movement; Cell Proliferation; Colorectal Neoplasms; Cyclin D1; G1 Phase Cell Cycle Checkpoints; Gene Expression Regulation, Neoplastic; Genetic Therapy; HCT116 Cells; Homeodomain Proteins; Humans; Mice; Mice, Inbred NOD; Mice, Knockout; Mice, SCID; MicroRNAs; Neoplasm Invasiveness; NM23 Nucleoside Diphosphate Kinases; Real-Time Polymerase Chain Reaction; Receptors, Interleukin-2; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; Time Factors; Transfection; Tumor Burden; Xenograft Model Antitumor Assays

P21-activated protein kinase1 (PAK1), a main downstream effector of small Rho GTPases, Rac1, and Cdc42, plays an important role in the regulation of cell morphogenesis, motility, mitosis, and angiogenesis. Despite its importance, the molecular mechanisms of PAK1 that contributed to colorectal carcinogenesis remain unclear. Our immunohistochemistry showed that PAK1 expression was increased with colorectal cancer (CRC) progression through the adenoma to carcinoma sequence. Furthermore, our results suggested a relationship between PAK1 nuclear localization and the Dukes staging. In the present study, we showed that PAK1 knockdown decreased proliferation and delayed the G1/S cell-cycle transition, and increased apoptosis in vivo and in vitro. In addition, PAK1 knock-down downregulated c-Jun amino terminal kinases (JNK) activity and the levels of cyclinD1, CDK4/6. Inhibition of the JNK activity by chemical inhibitor (SP600125) significantly reduced the effects of PAK1 on CRC proliferation via accumulation of phosphatase and tensin homolog deleted on chromosome 10 (PTEN). In conclusion, our results demonstrate that knockdown of PAK1 could enhance the chemosensitivity of CRCs to 5-fluorouracil through G1 arrest. The mechanism by which PAK1 induced cancer growth might involve activation of JNK as well as downregulation of PTEN. Targeting PAK1 may represent a novel treatment strategy for developing novel chemotherapeutic agents.

Topics: Animals; Anthracenes; Antimetabolites, Antineoplastic; Apoptosis; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Nucleus; Cell Proliferation; Colorectal Neoplasms; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase 6; Fluorouracil; G1 Phase; Humans; Immunoblotting; Immunohistochemistry; JNK Mitogen-Activated Protein Kinases; MAP Kinase Signaling System; Mice; Mice, Nude; p21-Activated Kinases; PTEN Phosphohydrolase; RNA Interference; Xenograft Model Antitumor Assays

We studied the effects of suberoylanilide hydroxamic acid (SAHA), a histone deacetylase (HDAC) inhibitor, on colon cancer. The expression of HDACs in colorectal cancer specimens and the effects of SAHA on colon cancer cells and tumors of nude mice were assessed. Treatment with SAHA (3 μm) for 72 h induced downregulation of different subtypes of HDAC proteins and also induced acetylation of histone 3 and histone 4. SAHA significantly inhibited the expression of the oncogenic protein c-myc and also increased the expression of the p53 and Rb proteins. The immunohistochemical staining of HDACs, including HDAC1, HDAC2, HDAC3, and HDAC4, was significantly increased in colorectal adenocarcinoma specimens compared to healthy control tissues. In addition, murine studies showed that 100 mg/kg SAHA administered by intraperitoneal injection significantly induced tumor necrosis and inhibited the growth of colon tumors. Immunohistochemistry of the tumor tissues from nude mice revealed that SAHA inhibited the expression of different subtypes of histone deacetylase, the anti-apoptotic proteins cyclin D1, survivin, and also inhibited cell proliferative as determined by Ki67 expression. SAHA inhibited the growth of colon tumors by decreasing histone deacetylases and the expression of cyclin D1 and survivin in nude mice.

Topics: Adenocarcinoma; Animals; Blotting, Western; Colorectal Neoplasms; Cyclin D1; Female; Histone Deacetylase Inhibitors; Histone Deacetylases; Humans; Hydroxamic Acids; Immunoenzyme Techniques; Inhibitor of Apoptosis Proteins; Mice; Mice, Inbred BALB C; Mice, Nude; Repressor Proteins; Survivin; Tissue Array Analysis; Tumor Cells, Cultured; Vorinostat

Our study shows that coadministration of curcumin and an orally bioactive alkylphospholipid perifosine results in a significant increase in colorectal cancer cell apoptosis and a marked inhibition of cell growth both in vitro and in vivo. This novel combinatorial regimen leads to changes of multiple cell signaling pathways including inactivation of Akt and nuclear factor-κB as well as activation of c-Jun N-terminal kinases and endoplasmic reticulum stress. Further, perifosine and curcumin synergistically increase intracellular level of reactive oxygen species and ceramide, and downregulate the expression of cyclin D1 and Bcl-2 in colorectal cancer cells. These changes at molecular level together account for the cancer cell apoptosis and growth inhibition. We conclude that perifosine sensitizes colorectal cancer cells to curcumin by modulating multiple signaling pathways. Adding perifosine with curcumin may represent an effective therapy regimen against colorectal cancers, and possible other aggressive tumors.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Caco-2 Cells; Cell Death; Cell Line, Tumor; Cell Proliferation; Ceramides; Colorectal Neoplasms; Curcumin; Cyclin D1; Down-Regulation; Drug Synergism; Endoplasmic Reticulum; Female; HCT116 Cells; HT29 Cells; Humans; I-kappa B Proteins; JNK Mitogen-Activated Protein Kinases; Mechanistic Target of Rapamycin Complex 1; Mice; Mice, SCID; Multiprotein Complexes; NF-kappa B; NF-KappaB Inhibitor alpha; Phosphorylation; Phosphorylcholine; Proteins; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species; Signal Transduction; TOR Serine-Threonine Kinases; Xenograft Model Antitumor Assays

To screen long non-coding RNA which influences radiosensitivity of colorectal carcinoma cell lines and investigate the mechanism.. Under different doses of radiation, colony formation assay and single-hit multi-target model were conducted to draw dose-survival curve and SF2 value of colorectal carcinoma cell lines(RKO, Lovo) was calculated. High-throughput lncRNA/mRNA chips were used to screen lncRNA genes and protein coding genes with expression differences more than 2 folds between RKO, Lovo cell lines and RKO cell line receiving 2Gy radiation. The main action pathway was computed by Gene Ontology analysis combined with Pathway analysis in order to explore the mechanism which induces the effect of lncRNA on radiosensitivity of colorectal carcinoma cell lines. Further experiment on P53, P21, cyclin D1 expression contents of RKO cell line was confirmed by real-time RT-PCR.. Lovo(SF2=0.47) was more sensitivity to radiation than RKO(SF2=0.53) according to the outcome of colony formation assay. High-throughput lncRNA/mRNA chips identified a total of 268 lncRNA genes and 270 protein coding genes. Gene Ontology analysis showed that the expression of genes associated with cell cycle process were significantly different (38.6%). There was a significant relationship between expression of several lncRNAs and CCND1 gene. Real-time RT-PCR showed no significant differences of P53 and P21 expression in RKO and Lovo cell lines(P>0.05), while cyclin D1 expression of RKO cell line was higher than that of Lovo cell lines(P<0.05). After exposed to 2 Gy doses of radiation, there was an obvious decrease of cyclin D1 expression in RKO cell lines(P<0.05), while P53 and P21 expressions were not different(P>0.05).. The possible mechanism is that lncRNAs compose transcription compound to combine with CCND1 gene and influence radiosensitivity of colorectal carcinoma cell lines by regulating expression of cyclin D1, which is independent of P53-P21-cyclin D1 pathway.

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

In HCT116 colorectal cancer cells, HeLa cervical cancer cells and HuH-7 hepatoma cells, miR-223 is expressed at a low level. Through infection with lentivirus containing miR-223 precursor, miR-223 [corrected] was overexpressed in all these cells. Interestingly, the expression levels of FOXO1 mRNA and protein, and phosphorylation levels became significantly lower than those of their control. FOXO1 was down-regulated mainly in the cytoplasm, while the nuclear FOXO1 level became relatively high compared to the cytoplasm. As the unphosphorylated active form of FOXO1 increased in the cells, cyclin D1/p21/p27 were up-regulated at either mRNA or protein level. Proliferation of the cells was also greatly inhibited when miR-223 was over-expressed. Therein, our data suggest that miR-223 regulates FOXO1 expression and cell proliferation.

Topics: Cell Line, Transformed; Cell Line, Tumor; Cell Nucleus; Cell Proliferation; Colorectal Neoplasms; Cyclin D1; Cyclin-Dependent Kinase Inhibitor Proteins; Cytoplasm; Female; Forkhead Box Protein O1; Forkhead Transcription Factors; Gene Expression Regulation, Neoplastic; Humans; Liver Neoplasms; Male; MicroRNAs; Neoplasm Proteins; Neoplasms; Phosphorylation; Protein Processing, Post-Translational; Protein Transport; RNA, Messenger; Uterine Cervical Neoplasms

Oxaliplatin is one of the agents used against colorectal cancer. Using PEG-liposome encapsulated oxaliplatin may enhance the accumulation of drugs in tumor cells, inducing apoptosis. However, the mechanism of action of PEG-liposome encapsulated oxaliplatin remains unclear. SW480 human colorectal cancer cells were treated with empty PEG-liposomes, free oxaliplatin or PEG-liposomal oxaliplatin. Cell cycle and apoptosis were assessed using fluorescence confocal microscopy and terminal deoxynucleotidyl transferase-mediated dUTP-fluorescein nick-end-labeling (TUNEL). Western blotting was used to analyze the expression of pro-apoptotic, anti-apoptotic and cyclin proteins. We found that PEG-liposomal oxaliplatin induced a stronger apoptotic response than empty PEG-liposomes or free oxaliplatin. Moreover, expression of Cyclin D1 increased, whereas expression of Cyclin A decreased after treatment with PEG-liposomal oxaliplatin. Furthermore, the cell cycle was arrested in the G1 phase. The results presented here indicate that PEG-liposome entrapment of oxaliplatin enhances the anticancer potency of the chemotherapeutic agent. The effect of PEG-liposomal oxaliplatin on apoptosis of SW480 human colorectal cancer cells may be through regulation of expression of Cyclin A or Cyclin D1, as well as pro-apoptotic and anti-apoptotic proteins.

Topics: Antineoplastic Agents; Apoptosis; Apoptosis Regulatory Proteins; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Colorectal Neoplasms; Cyclin A; Cyclin D1; Drug Screening Assays, Antitumor; Humans; Liposomes; Organoplatinum Compounds; Oxaliplatin; Polyethylene Glycols

In our previous study, we reported that endothelial cell specific molecule-1 (ESM-1) was increased in tissue and serum from colorectal cancer patients and suggested that ESM-1 can be used as a potential serum marker for early detection of colorectal cancer. The aim of this study was to evaluate the role of ESM-1 as an intracellular molecule in colorectal cancer. ESM-1 expression was knocked down by small interfering RNA (siRNA) in colorectal cancer cells. Expression of ESM-1 siRNA decreased cell survival through the Akt-dependent inhibition of NF-κB/IκB pathway and an interconnected reduction in phospho-Akt, -p38, -ERK1, -RSK1, -GSK-3α/β and -HSP27, as determined by a phospho-MAPK array. ESM-1 silencing induced G(1) phase cell cycle arrest by induction of PTEN, resulting in the inhibition of cyclin D1 and inhibited cell migration and invasion of COLO205 cells. Consistently, ESM-1 overexpression in HCT-116 cells enhanced cell proliferation through the Akt-dependent activation of NF-κB pathway. In addition, ESM-1 interacted with NF-κB and activated NF-κB promoter. This study demonstrates that ESM-1 is involved in cell survival, cell cycle progression, migration, invasion and EMT during tumor invasion in colorectal cancer. Based on our results, ESM-1 may be a useful therapeutic target for colorectal cancer.

Topics: Cell Cycle Checkpoints; Cell Line, Tumor; Cell Movement; Cell Proliferation; Colorectal Neoplasms; Cyclin D1; Gene Expression Regulation, Neoplastic; Humans; Matrix Metalloproteinases; Neoplasm Metastasis; Neoplasm Proteins; NF-kappa B; Proteoglycans; Proto-Oncogene Proteins c-akt; PTEN Phosphohydrolase; RNA Interference; RNA, Small Interfering; Signal Transduction

The public has paid attention to green tea due to its health benefits. Epigallocatechin-3-gallate (EGCG), the major component of green tea, is well documented to induce apoptosis and cell cycle arrest in cancer cells by targeting multiple signal transduction pathways. However, the detailed mechanism(s) of action needs to be determined.. Cell growth was evaluated by MTT assay, cell cycle analysis, and caspase 3/7 activity. Protein expression was analyzed through Western blotting. Reverse transcription polymerase chain reaction was used for examining mRNA expression of p21 and cyclin D1. The promoter activity of p21 was assessed by the luciferase reporter system.. We identified cyclin D1 and p21 as molecular targets of EGCG in human colorectal cancer cells. We observed that cyclin D1 was down-regulated, while p21 expression was up-regulated by EGCG in dose- and time-dependent manners. Furthermore, we found EGCG decreased cyclin D1 protein stability, therefore triggering ubiquitin-dependent proteasomal degradation. Meanwhile, EGCG increased p21 promoter activity, resulting in up-regulation of p21 mRNA and protein, which was likely dependent on extracellular-signal-regulated kinase (ERK), inhibitor of nuclear factor kappa-B kinase (IKK) and phosphoinositide 3-kinase (PI3 K).. The data presented here details a novel mechanism by which EGCG inhibits cell growth of colorectal cancer cells. Namely, EGCG-induced cyclin D1 degradation and p21 transcriptional activation partially contribute to growth suppression in these cells.

Topics: Apoptosis; Caco-2 Cells; Caspase 3; Caspase 7; Catechin; Cell Cycle; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Colorectal Neoplasms; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Down-Regulation; HCT116 Cells; HT29 Cells; Humans; MAP Kinase Signaling System; NF-kappaB-Inducing Kinase; Phosphatidylinositol 3-Kinases; Phosphorylation; Promoter Regions, Genetic; Protein Processing, Post-Translational; Protein Serine-Threonine Kinases; Proteolysis; Signal Transduction; Ubiquitination; Up-Regulation

Thymidylate synthase (TS) is an enzyme for DNA-synthesis and the target for 5-fluorouracil whereas cyclin-D1 plays a critical role in the progression of cells through the G1 phase of the cell-cycle. There is evidence that expression of these markers may predict the outcome of patients with colorectal cancers. The aim of this study was to examine the prognostic value of TS and cyclin D1 protein expression in patients with node negative colorectal cancers.. TS and cyclin D1 protein expression from 140 patients with UICC stage I and II colorectal cancer was analyzed by immunhistochemistry in paraffin-embedded primary tumour specimens.. The 1-, 5- and 10-year overall-survival rates were 96%, 86% and 71%, respectively. Tumour stage and recurrence were associated with overall-survival. Low- and high TS immunoreactivity was present in 68 (48%) and 72 (52%) of cancers, respectively. Low- and high cyclin D1 immunoreactivity was present in 98 (70%) and 42 (30%) of the cancers, respectively. Patients (n=72) with high TS expressing tumours had a worse overall-survival than patients (n=68) with low TS expressing colorectal cancers (p=0.011). No difference in overall-survival was seen between patients with high and low cyclin D1 expressing cancers.. TS may be helpful as a prognostic marker in lymph node negative colorectal cancer.

Topics: Adenocarcinoma; Aged; Biomarkers, Tumor; Colorectal Neoplasms; Cyclin D1; Female; Germany; Humans; Immunohistochemistry; Kaplan-Meier Estimate; Lymph Nodes; Lymphatic Metastasis; Male; Middle Aged; Multivariate Analysis; Neoplasm Staging; Predictive Value of Tests; Prognosis; Proportional Hazards Models; Retrospective Studies; Risk Assessment; Risk Factors; Survival Rate; Thymidylate Synthase; Time Factors

Increasing evidence indicates that various cancer cell types are capable of producing IgG. The exact function of cancer-derived IgG has, however, not been elucidated. Here we demonstrated the expression of IgG genes with V(D)J recombination in 80 cases of colorectal cancers, 4 colon cancer cell lines and a tumor bearing immune deficient mouse model. IgG expression was associated with tumor differentiation, pTNM stage, lymph node involvement and inflammatory infiltration and positively correlated with the expressions of Cyclin D1, NF-κB and PCNA. Furthermore, we investigated the effect of cancer-derived IgG on the malignant behaviors of colorectal cancer cells and showed that blockage of IgG resulted in increased apoptosis and negatively affected the potential for anchor-independent colony formation and cancer cell invasion. These findings suggest that IgG synthesized by colorectal cancer cells is involved in the development and growth of colorectal cancer and blockage of IgG may be a potential therapy in treating this cancer.

Topics: Animals; Apoptosis; Cell Line, Tumor; Cell Proliferation; Colorectal Neoplasms; Cyclin D1; Disease Models, Animal; Flow Cytometry; Gene Expression Regulation, Neoplastic; Humans; Immunoglobulin G; Immunohistochemistry; In Situ Hybridization; Jurkat Cells; Mice; Mice, SCID; NF-kappa B; Proliferating Cell Nuclear Antigen

Aberrant expression of Brahma-related gene-1 (BRG1), a core component of the SWI/SNF chromatin-remodelling complex, has been implicated in cancer development; however, the biological significance of BRG1 in colorectal carcinoma (CRC) remains unknown.. In CRC tissues, expression of BRG1 and Brahma (BRM) was investigated immunohistochemically. Colorectal carcinoma-derived DLD-1 cells were used for knockdown of BRG1 and PTEN with small interfering RNA (siRNA) and transduction of Akt. Complementary DNA (cDNA) microarray analysis was performed to explore the genes affected by BRG1.. Expression of BRG1, but not BRM, was frequently elevated in CRC specimens, and knockdown of BRG1 suppressed cell proliferation of DLD-1 cells. By cDNA microarray, we determined that PTEN expression was negatively regulated by BRG1 in DLD-1 cells, which subsequently influenced the cyclin D1 levels via the phosphoinositide 3-OH kinase (PI3K)-Akt signalling pathway. The interplay of BRG1 on cyclin D1 expression was confirmed by the introduction of Akt and knockdown of PTEN in the BRG1 siRNA-transduced DLD-1 cells. Interestingly, this positive correlation between BRG1 and cyclin D1 expression was also observed in CRC specimens.. Brahma-related gene-1 has an important role in the process of CRC development by activating the PI3K-Akt signalling pathway and resultant upregulation of cyclin D1 levels.

Topics: Apoptosis; Biomarkers, Tumor; Blotting, Western; Cell Proliferation; Cells, Cultured; Colon; Colorectal Neoplasms; Cyclin D1; DNA Helicases; Flow Cytometry; Fluorescent Antibody Technique; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Immunoenzyme Techniques; Nuclear Proteins; Oligonucleotide Array Sequence Analysis; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; PTEN Phosphohydrolase; Rectum; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Transcription Factors

We examined the influence that rare variants and low-frequency polymorphisms in the cancer candidate gene CCND1 have on the development of multiple intestinal adenomas and the early onset of colorectal cancer. Individuals with <100 multiple polyps and patients with colorectal cancer diagnosed before 50 years of age were recruited in UK, and screened for sequence changes in the coding and regulatory regions of CCND1. A set of about 800 UK control individuals was genotyped for the variants discovered in the cases. Variants in the promoter, intron-exon boundaries and untranslated regions of the CCND1 gene had higher frequencies in cases than in controls. Five of these variants were typed in a set of French multiple adenoma and early-onset patients, who also showed higher allele frequencies than UK controls. When pooled together, variants with frequencies lower than 1% conferred an increased risk of disease that was significant in the multiple adenoma group (odds ratio (OR) 2.2; 95% confidence interval, 1.1-4.4; P = 0.03). Most variants had a putative functional effect when assessed in silico. We conclude that rare variants of CCND1 are risk factors for colorectal cancer, with considerably larger effects than common polymorphisms, and as such should be systematically evaluated in susceptibility studies.

Topics: Adenoma; Adult; Age of Onset; Case-Control Studies; Colorectal Neoplasms; Cyclin D1; Female; France; Gene Frequency; Humans; Male; Middle Aged; Neoplasms, Multiple Primary; Polymorphism, Single Nucleotide; United Kingdom

Obesity has been reported to increase the risk of colorectal cancer, which may due to aberrant lipid metabolism. And recently findings of monoacylglycerol lipase provide a novel evidence in the correlation of obesity and cancer. So in this study, we investigated the effect of MAGL in regulation of tumor growth in colorectal cancer.. MAGL expression in tumor tissues was estimated, and then JZL184 and siRNA were used to knockdown the expression of MAGL in colorectal cancer cells. Cell viability and invasion were detected to estimate the influence of MAGL knocked down in vitro and vivo. Then cell proliferation, apoptosis, cell cycle transition and screening of candidate genes were performed for further exploring of the effect mediated by MAGL knocked down.. It was noted that the expression of MAGL was highly elevated in tumor tissues, however, it was found only significantly correlated with the BMI index. Tumor cells' growth and invasion was significantly inhibited in vitro and in vivo induced by pharmacological and siRNA mediated MAGL knocked down. Cell proliferation was reduced and apoptosis was increased. And two target genes Cyclin D1 and Bcl-2 seemed to be repressed by MAGL knocked down.. This study demonstrated colorectal cancer cells growth can be inhibited via knockdown of MAGL, which manipulate tumor cells proliferation and apoptosis by downregulation of Cyclin D1 and Bcl-2. It provides a novel therapeutic target in treatment of colorectal cancer and a further support for the correlation of obesity and colorectal cancer.

Topics: Animals; Apoptosis; Blotting, Western; Cell Adhesion; Cell Cycle; Cell Movement; Cell Proliferation; Colorectal Neoplasms; Cyclin D1; Female; Gene Knockdown Techniques; Humans; Immunoenzyme Techniques; Male; Mice; Mice, Nude; Middle Aged; Monoacylglycerol Lipases; Proto-Oncogene Proteins c-bcl-2; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; RNA, Messenger; RNA, Small Interfering; Tumor Cells, Cultured

Prognostic and predictive significance of epidermal growth factor receptor (EGFR) in colorectal carcinomas (CRCs) is still controversial. The aim of the present study was to explore and correlate membrane and nuclear EGFR and cyclin-D1 protein expression with EGFR gene status of tumor cells.. Immunohistochemical and FISH analysis was performed on 135 archival formalin fixed and paraffin embedded CRCs.. Strong membrane and strong nuclear EGFR staining was detected in 16% and 57% of cases, respectively, and strong cyclin-D1 expression in 57% samples. Gene EGFR amplification was identified in 5.9% and polysomy in 7.4% of cases, while 87% showed no EGFR gene changes. A statistically significant difference was only found between tumor grade and expression of membrane EGFR, while nuclear EGFR and cyclin-D1 expression was not associated with the clinicopathologic characteristics analyzed. Tumor cells displaying gene amplification and strong protein membrane EGFR expression overlapped, while EGFR gene status showed no correlation with nuclear EGFR and cyclin-D1. There was no association between membrane EGFR and cyclin-D1, whereas nuclear EGFR expression was strongly related to cyclin-D1 expression.. Study results revealed heterogeneity among CRCs, which could have a predictive value by identifying biologically and probably clinically different subsets of tumors with the possibly diverse response to anti-EGFR therapies.

Topics: Adenocarcinoma; Biomarkers, Tumor; Cell Nucleus; Colorectal Neoplasms; Cyclin D1; ErbB Receptors; Gene Amplification; Gene Expression; Humans; Immunohistochemistry; In Situ Hybridization, Fluorescence; Retrospective Studies; Tissue Array Analysis

We analyzed the influence of 8 germinal polymorphisms of candidate genes potentially related to EGFR signalling (EGFR, EGF, CCND1) or antibody-directed cell cytotoxicity (FCGR2A and FCGR3A) on outcome of colorectal cancer (CRC) patients receiving cetuximab-based therapy.. Fifty-eight advanced CRC patients treated with cetuximab-irinotecan salvage therapy between 2001 and 2007 were analyzed (mean age 60; 50 PS 0-1). The following polymorphisms were analyzed on blood DNA: EGFR (CA repeats in intron 1, -216 G > T, -191C > A, R497K), EGF (A61G), CCND1 (A870G), FCGR2A (R131H), FCGR3A (F158V). Statistical analyses were conducted on the total population and on patients with wt KRas tumors. All SNPs were considered as ternary variables (wt/wt vs wt/mut vs mut/mut), with the exception of -191C > A EGFR polymorphism (AA patient merged with CA patients).. Analysis of skin toxicity as a function of EGFR intron 1 polymorphism showed a tendency for higher toxicity in patients with a low number of CA-repeats (p = 0.058). CCND1 A870G polymorphism was significantly related to clinical response, both in the entire population and in KRas wt patients, with the G allele being associated with a lack of response. In wt KRas patients, time to progression (TTP) was significantly related to EGFR -191C > A polymorphism with a longer TTP in CC patients as compared to others, and to CCND1 A870G polymorphism with the G allele being associated with a shorter TTP; a multivariate analysis including these two polymorphisms only retained CCND1 polymorphism. Overall survival was significantly related to CCND1 polymorphism with a shorter survival in patients bearing the G allele, and to FCGR3A F158V polymorphism with a shorter survival in VV patients (in the entire population and in KRas wt patients). FCGR3A F158V and CCND1 A870G polymorphisms were significant independent predictors of overall survival.. Present original data obtained in wt KRas patients corresponding to the current cetuximab-treated population clearly suggest that CCND1 A870G polymorphism may be used as an additional marker for predicting cetuximab efficacy, TTP and overall survival. In addition, FCGR3A F158V polymorphism was a significant independent predictor of overall survival.

Topics: Adult; Aged; Aged, 80 and over; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Carcinoma; Cetuximab; Colorectal Neoplasms; Cyclin D1; Disease Progression; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Introns; Male; Middle Aged; Polymorphism, Genetic; Receptors, IgG; Skin Diseases; Survival Analysis

To explore the effects of sodium selenite on the expressions of beta-catenin and cyclin D1 in colorectal cancer cells HCT 116 and SW480.. HCT 116 and SW480 cells were treated by 10 micromol/L sodium selenite at different time points. The expressions and transcription of beta-catenin and cyclin D1 were detected by Western blot analysis and reverse transcriptase polymerase chain reaction (RT-PCR), respectively. Meanwhile, the impact of MG132 (a proteasome inhibitor) pretreatment on the expressions of beta-catenin and cyclin D1 was observed through Western blot analysis. The interaction between beta-catenin and T cell factor 4 (TCF4) after selenite treatment was evaluated using co-immunoprecipitation assay.. Sodium selenite inhibited the expression of beta-catenin and transcription of its target such as cyclin D1. MG132 pretreatment prevented the inhibition of beta-catenin signaling triggered by selenite in HCT 116 and SW480 cells. Furthermore, selenite treatment disrupted the interaction between beta-catenin and TCF4 in HCT 116 and SW480 cells.. Sodium selenite can lower the expression levels of beta-catenin and its target cyclin D1, during which the proteasome-mediated degradative pathway may be involved. The decreased interaction between beta-catenin and TCF4 due to sodium selenite may be also involved in the regulation of beta-catenin signaling.

Topics: beta Catenin; Cell Line, Tumor; Colorectal Neoplasms; Cyclin D1; HCT116 Cells; Humans; Sodium Selenite

It has been well elucidated that the signal transduction of cell-cycle control pathway and apoptosis pathway plays an important role in the normal growth and differentiation of organisms. To test the hypothesis that mutants of key genes involved in cell-cycle regulation and apoptosis might contribute to the increased risk of colorectal cancer (CRC), a population-based case-control study was carried out in Jiashan County, Zhejiang Province. The study population was composed of 373 CRC cases and 838 controls. Five genetic variants including CCND1 G870A, p21 codon31 C/A, p21 3'UTR C/T, caspase8 IVS12-19G/A, and caspase8 6n del/ins were genotyped. The associations of the polymorphisms with CRC were estimated by logistical regression model after adjustment for the important covariates. The interactive effect among the five selected genetic polymorphisms on CRC was explored by multifactor dimensionality reduction (MDR) software. The significant association between five single-nucleotide polymorphisms (SNPs) and CRC risk was not observed, respectively. However, caspase8 del/del showed a marginally significant association with the increased risk of rectum cancer [adjusted odds ratio (OR) (95% confidence interval, CI) = 1.92 (0.97-3.79); P = 0.06]. Furthermore, the MDR analysis indicated that the best interactive model for CRC included three factors-CCND1 G870A, caspase8 IVS12-19G/A, and caspase8 6 n del/ins-with 53.44% testing balanced accuracy and 10/10 cross-validation consistency, but the model was no longer significant after the 1000 times permutation test (P = 0.25). Our findings suggest that the selected polymorphisms of p21, CCND1, and caspase8 may not contribute to the risk of colorectal cancer.

Topics: Aged; Case-Control Studies; Caspase 8; Colorectal Neoplasms; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Female; Gene Frequency; Genetic Predisposition to Disease; Genotype; Humans; Linkage Disequilibrium; Male; Meta-Analysis as Topic; Middle Aged; Mutagenesis, Insertional; Polymorphism, Genetic; Polymorphism, Single Nucleotide; Risk Factors; Sequence Deletion

The presence of different forms of histone covalent modifications, such as phosphorylation, acetylation and methylation in localized promoter regions are markers for chromatin packing and transcription. Activation of RAS signalling pathways through oncogenic RAS mutations is a hallmark of colorectal cancer. Overexpression of Harvey-Ras oncogene induces epithelial-mesenchymal transition (EMT) in Caco-2 cells. We focused on the role of epigenetic modifications of histone H3 and its dependence on RAS signal transduction pathways and oncogenic transformation. Using cell lines stably overexpressing oncogenic Harvey-RAS with EMT phenotype, we studied the acquired changes in the H3 histone modification patterns. Two genes show inverse protein expression patterns after Ha-RAS overexpression: Cyclin D1, a cell cycle-related gene, and the EMT marker-gene E-cadherin. We report that these two genes demonstrate matching inverse histone repression patterns on their promoter, while histone markers associated with an active state of genes were affected by the RAS-activated signalling pathway MEK-ERK-MSK1. Furthermore, we show that though the level of methyltransferases enzymes was increased, the status of H3 three-methylation at lysine 27 (H3K27me(3)), associated with gene repression on the promoter of Cyclin D1, was lower. Together, these results suggest that histone covalent modifications can be affected by oncogenic RAS pathways to regulate the expression of target genes like Cyclin D1 or E-cadherin and that the dynamic balance of opposing histone-modifying enzymes is critical for the regulation of cell proliferation.

Topics: Caco-2 Cells; Cadherins; Cell Transformation, Neoplastic; Colorectal Neoplasms; Cyclin D1; Epithelial Cells; Gene Expression Regulation, Neoplastic; Histones; Humans; MAP Kinase Kinase Kinases; Mesenchymal Stem Cells; Protein Processing, Post-Translational; ras Proteins; Signal Transduction; Transgenes

Symplekin is a ubiquitously expressed protein involved in cytoplasmic RNA polyadenylation and transcriptional regulation and is localized at tight junctions (TJs) in epithelial cells. Nuclear symplekin cooperates with the Y-box transcription factor zonula occludens 1-associated nucleic acid-binding protein (ZONAB) to increase the transcription of cell cycle-related genes and also inhibits differentiation of intestinal cells. We detected high levels of nuclear symplekin in 8 of 12 human colorectal cancer (CRC) samples. shRNA-mediated reduction of symplekin expression was sufficient to decrease significantly the anchorage-independent growth and proliferation of HT-29 CRC cells as well as their tumorigenicity when injected into immunodeficient animals. Symplekin down-regulation also was found to alter ion transport through TJs, to promote the localization of ZONAB in the membrane rather than the nucleus, and strongly to enhance cell polarization in a 3D matrix, leading to the formation of spheroids organized around a central lumen. Claudin-2 expression was reduced following symplekin down-regulation, an effect mimicked when ZONAB expression was down-regulated using selective siRNA. Virus-mediated restoration of claudin-2 expression was found to restore nuclear expression of ZONAB in HT29DeltaSym cells and to rescue the phenotypic alterations induced by symplekin down-regulation of cell polarity, paracellular transport, ZONAB localization, cyclin D1 expression, proliferation, and anchorage-independent growth. Finally, siRNA-mediated claudin-2 down-regulation increased the transepithelial resistance and decreased cyclin D1 expression and ZONAB nuclear localization, similar to observations in symplekin-depleted cells. Our results suggest that nuclear overexpression of symplekin promotes tumorigenesis in the human colon and that the regulation of claudin-2 expression is instrumental in this effect.

Topics: Animals; Blotting, Western; CCAAT-Enhancer-Binding Proteins; Cell Proliferation; Claudins; Colorectal Neoplasms; Cyclin D1; Fluorescent Antibody Technique; Gene Expression Regulation, Neoplastic; Heat-Shock Proteins; HT29 Cells; Humans; Membrane Proteins; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasms, Experimental; Nuclear Proteins; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; Transplantation, Heterologous; Tumor Burden; Up-Regulation

The Wnt-pathway dominates the sporadic carcinogenesis whereas p53 plays a pivotal role in the colitis-associated counterpart. The expression of Wnt-signaling proteins and p53 during colitis-associated carcinogenesis was determined.. A tissue microarray was constructed with colonic samples from 5 groups of patients: controls (C, n=10), IBD without neoplasia (IBD, n=12), non-dysplastic IBD with neoplasia elsewhere in the colon (IBD-NE, n=12), dysplastic lesion in IBD (IBD-DYS, n=12), and IBD-associated colorectal cancer (IBD-CRC, n=10). Immunohistochemistry was performed for beta-catenin, cyclin D1 and p53. p53 sequence analysis was performed in some cases.. Nuclear beta-catenin expression was found in 0%, 0%, 50%, 55% and 100% of the patients in the C-, IBD-, IBD-NE-, IBD-DYS- and IBD-CRC-groups, respectively. Non-dysplastic IBD mucosa with neoplasia detected elsewhere showed nuclear expression in 50% of the cases compared to 0% in IBD mucosa without neoplasia (p=0.02). Cyclin D1 staining had similar expression patterns. Overexpression of p53 was only detected in the IBD-DYS (66.7%) and IBD-CRC groups (50%).. In contrast to previous findings, our results suggest activation of the Wnt-pathway in the early phase of colitis-associated carcinogenesis. Furthermore, as Wnt activation was observed in 50% of the IBD-NE cases, nuclear beta-catenin may facilitate detection of neoplasia.

Topics: Adult; beta Catenin; Biomarkers, Tumor; Carcinoma; Colitis; Colon; Colorectal Neoplasms; Cyclin D1; Disease Progression; DNA Mutational Analysis; Female; Genes, p53; Humans; Immunohistochemistry; Inflammatory Bowel Diseases; Male; Microarray Analysis; Middle Aged; Signal Transduction; Wnt Proteins

Chemoprevention is a practical and translational approach to reduce the risk of various cancers including colorectal cancer (CRC), which is a major cause of cancer-related deaths in the United States. Accordingly, here we assessed chemopreventive efficacy and associated mechanisms of long-term silibinin feeding on spontaneous intestinal tumorigenesis in the APC(min/+) mice model. Six-week-old APC(min/+) mice were p.o. fed with vehicle control (0.5% carboxymethyl cellulose and 0.025% Tween 20 in distilled water) or 750 mg silibinin/kg body weight in vehicle for 5 d/wk for 13 weeks and then sacrificed. Silibinin feeding strongly prevented intestinal tumorigenesis in terms of polyp formation in proximal, middle, and distal portions of small intestine by 27% (P < 0.001), 34% (P < 0.001), and 49% (P < 0.001), respectively. In colon, we observed 55% (P < 0.01) reduction in number of polyps by silibinin treatment. In size distribution analysis, silibinin showed significant decrease in large-size polyps (>3 mm) by 66% (P < 0.01) and 88% (P < 0.001) in middle and distal portions of small intestine, respectively. More importantly, silibinin caused a complete suppression in >3 mm sized polyps and 92% reduction in >2 to 3 mm sized polyps in colon. Molecular analyses of polyps suggested that silibinin exerts its chemopreventive efficacy by inhibiting cell proliferation, inflammation, and angiogenesis; inducing apoptosis; decreasing beta-catenin levels and transcriptional activity; and modulating the expression profile of cytokines. These results show for the first time the efficacy and associated mechanisms of long-term p.o. silibinin feeding against spontaneous intestinal tumorigenesis in the APC(min/+) mice model, suggesting its chemopreventive potential against intestinal cancers including CRC.

Topics: Adenomatous Polyposis Coli; Animals; Antioxidants; Apoptosis; beta Catenin; Cell Growth Processes; Colorectal Neoplasms; Cyclin D1; Cyclooxygenase 2; Dinoprostone; Disease Models, Animal; Intestinal Polyps; Intestine, Small; Male; Mice; Mice, Inbred C57BL; Neovascularization, Pathologic; Silybin; Silymarin; Transcriptional Activation

The significance of BRAF mutations, microsatelite instability (MSI) status and cyclin D1 expression in patients with metastatic colorectal cancer (mCRC) was evaluated.. Primary tumours from 144 patients treated for mCRC were assessed for BRAF (V600E) mutation, MSI status and cyclin D1. The data were correlated with progression-free survival (PFS) and overall survival (OS).. BRAF mutations were detected in 10 (out of 22, 45%) patients with MSI-H tumours compared with 2 (out of 122, 1.6%) in those with microsatellite stable tumours (P<0.001). The presence of BRAF mutations was correlated with cyclin D1 overexpression (7 out of 26 patients, 58% vs 5 out of 118 patients, 14%; P=0.001). Patients with BRAF-mutated primary tumours had a significantly decreased PFS (2.7 vs 9.8 months; P<0.001) and median OS (14 vs 30 months; P<0.001) than patients with wild-type (wt) tumours. Patients with MSI-H and BRAF-mutated tumours experienced significantly lower PFS (3.1 vs 11.4 months; P=0.008) and OS (14.5 vs 35.5 months; P=0.004) than patients with MSI-H and BRAF wt tumours. Similarly, BRAF mutations and cyclin D1 overexpression were correlated with decreased PFS (3.1 vs 8.6 months; P=0.03) and OS (17.8 vs 39.2 months; P=0.01).. BRAF V600E mutations are associated with MSI-H status and cyclin D1 overexpression and characterize a subgroup of patients with poor prognosis.

Topics: Adult; Aged; Aged, 80 and over; Colorectal Neoplasms; Cyclin D1; Disease-Free Survival; Female; Humans; Male; Microsatellite Instability; Middle Aged; Mutation; Neoplasm Metastasis; Prognosis; Proto-Oncogene Proteins B-raf

Polyphenol-rich longan seed extract (LSP) is a free radical scavenger and antioxidant. However, the effect of LSP on the growth of human colorectal carcinoma cells (CRC) has not yet been evaluated.. Polyphenols of longan seeds were extracted and measured by colorimetry. Four CRC cell lines (Colo 320DM, SW480, HT-29 and LoVo) were treated with LSP and assessed for viability by trypan blue exclusion, for cell cycle distribution by flow cytometry, for apoptosis by annexin V labelling and for changes in the levels of proteins involved in cell cycle control or apoptosis by immunoblotting.. Total phenol content of LSP was 695 mg g(-1) and total flavonoids were 150 mg g(-1). LSP inhibited the proliferation (25 microg mL(-1)-200 microg mL(-1)) of Colo 320DM, SW480 and HT-29, but not LoVo. LSP inhibited the proliferation by blocking cell cycle progression during the DNA synthesis phase and inducing apoptotic death. Western blotting indicated that LSP blocks the S phase, reducing the expression of cyclin A and cyclin D1. Colo 320DM and SW480 treated with LSP also showed the activation of caspase 3 and increased Bax : Bcl-2 ratio.. LSP induces S phase arrest of the cell cycle and apoptotic death in three CRC cell lines. The results indicate that LSP is a potential novel chemoprevention and treatment agent for colorectal cancer.

Topics: Apoptosis; Blotting, Western; Carcinoma; Caspase 3; Cell Line, Tumor; Cell Proliferation; Colorectal Neoplasms; Cyclin A; Cyclin D1; Flavonoids; Flow Cytometry; Humans; Phenols; Plant Extracts; Polyphenols; S Phase; Sapindaceae

Chemoprevention by dietary agents/supplements has emerged as a novel approach to control various malignancies, including colorectal cancer (CRC). This study assessed dietary grape seed extract (GSE) effectiveness in preventing azoxymethane (AOM)-induced aberrant crypt foci (ACF) formation and associated mechanisms in Fischer 344 rats. Six-week-old rats were injected with AOM, and fed control diet or the one supplemented with 0.25% or 0.5% (w/w) GSE in pre- and post-AOM or only post-AOM experimental protocols. At 16 wk of age, rats were sacrificed and colons were evaluated for ACF formation followed by cell proliferation, apoptosis, and molecular analyses by immunohistochemistry. GSE-feeding caused strong chemopreventive efficacy against AOM-induced ACF formation in terms of up to 60% (P < 0.001) reduction in number of ACF and 66% (P < 0.001) reduction in crypt multiplicity. Mechanistic studies showed that GSE-feeding inhibited AOM-induced cell proliferation but enhanced apoptosis in colon including ACF, together with a strong decrease in cyclin D1, COX-2, iNOS, and survivin levels. Additional studies showed that GSE-feeding also decreased AOM-caused increase in beta-catenin and NF-kappaB levels in colon tissues. Compared to control animals, GSE alone treatment did not show any considerable change in these biological and molecular events in colon, and was nontoxic. Together, these findings show the chemopreventive efficacy of GSE against the early steps of colon carcinogenesis in rats via likely targeting of beta-catenin and NF-kappaB signaling, and suggest its potential usefulness for the prevention of human CRC.

Topics: Animals; Anticarcinogenic Agents; Apoptosis; Azoxymethane; beta Catenin; Cell Proliferation; Colon; Colorectal Neoplasms; Cyclin D1; Cyclooxygenase 2; Gene Expression Regulation, Neoplastic; Grape Seed Extract; Microtubule-Associated Proteins; NF-kappa B; Nitric Oxide Synthase Type II; Rats; Rats, Inbred F344; Survivin

Cyclin D1, encoded by the gene CCND1, is a regulatory protein in the cell cycle transition from G(1) phase to S phase. A common polymorphism (A870G) at codon 242 affects splicing of the CCND1 transcript and may cause uncontrollable cellular growth. The present study was performed to test the association between A870G polymorphisms in the CCND1 gene and colorectal cancer risk and progression.. The 870 A>G polymorphism in the cyclin D1 gene was genotyped in a Turkish colorectal cancer case-control population including fifty-seven cases (35 male, 22 female; mean age + or - SD: 59.33 + or - 13.7 years) and 117 controls (63 male, 54 female; mean age + or - SD: 54.4 + or - 12.2 years) using polymerase chain reaction-restriction fragment length polymorphism analysis.. Genotype frequencies of our patients and controls both confirmed to the Hardy-Weinberg equilibrium. There was no difference in the distribution of CCND1 genotypes and frequencies of the alleles A (59.6% versus 49.6%) and G (40.4% versus 50.4%) in the colorectal cancer patients and controls, respectively. Women homozygous for the cyclin D1 870 GG genotype showed an increased risk for developing colorectal cancer compared to those with the AG+AA genotypes and this result was statistically significant (OR 5.568, 95% CI 1.270-24.417, p=0.02). On the other hand, the cyclin D1 GA genotype was associated with distant metastasis (p=0.016).. Our findings suggest that genetic variants of A870G might be associated with distant metastasis and also gender.

Topics: Adenocarcinoma; Adult; Aged; Aged, 80 and over; Case-Control Studies; Colorectal Neoplasms; Cyclin D1; Female; Genetic Predisposition to Disease; Humans; Male; Middle Aged; Polymerase Chain Reaction; Polymorphism, Genetic; Polymorphism, Restriction Fragment Length; Prognosis; Turkey; Young Adult

Trop2 is a cell-surface glycoprotein overexpressed by a variety of epithelial carcinomas with reported low to restricted expression in normal tissues. Expression of Trop2 has been associated with increased tumor aggressiveness, metastasis and decreased patient survival, but the signaling mechanisms mediated by Trop2 are still unknown. Here, we studied the effects murine Trop2 (mTrop2) exerted on tumor cellular functions and some of the signaling mechanisms activated by this oncogene.. mTrop2 expression significantly increased tumor cell proliferation at low serum concentration, migration, foci formation and anchorage-independent growth. These in vitro characteristics translated to increased tumor growth in both subcutaneous and orthotopic pancreatic cancer murine models and also led to increased liver metastasis. mTrop2 expression also increased the levels of phosphorylated ERK1/2 mediating cell cycle progression by increasing the levels of cyclin D1 and cyclin E as well as downregulating p27. The activation of ERK was also observed in human pancreatic ductal epithelial cells and colorectal adenocarcinoma cells overexpressing human Trop2.. These findings demonstrate some of the pathogenic effects mediated by mTrop2 expression on cancer cells and the importance of targeting this cell surface glycoprotein. This study also provides the first indication of a molecular signaling pathway activated by Trop2 which has important implications for cancer cell growth and survival.

Topics: Animals; Antigens, Neoplasm; Cell Adhesion Molecules; Cell Cycle; Cell Line; Cell Line, Tumor; Cell Proliferation; Colorectal Neoplasms; Cyclin D1; Cyclin E; Extracellular Signal-Regulated MAP Kinases; Female; HCT116 Cells; Humans; Immunohistochemistry; Mice; Mice, Nude; Neoplasms; Pancreatic Neoplasms; Proteins; Signal Transduction

β-Catenin is a critical component of the Wnt signaling pathway that regulates cell proliferation and differentiation. Wnt signaling leads to the stabilization of cytosolic β-catenin and to translocation to the nucleus, where it binds with T-cell factor and promotes the transcription and changes in target gene expression, including vascular endothelial growth factor and cyclin D1. The aim of this study was to assess the expression of cyclin D1 and vascular endothelial growth factor and to correlate them with β-catenin expression and some clinicopathologic parameters.. In this study, we analyzed paraffin-embedded specimens from 42 patients with pT3 rectosigmoid cancer for β-catenin, vascular endothelial growth factor and cyclin D1 expression using immunohistochemistry.. Thirty-six (85.7%) and 24 (57.1%) tumors expressed vascular endothelial growth factor and cyclin D1, respectively. Nuclear expression of β-catenin was detected in only 26.1% of tumors. It was revealed that cytoplasmic β-catenin expression was significantly related to vascular endothelial growth factor expression (p=0.011). No association was found between nuclear or cytoplasmic β-catenin and cyclin D1 expression. No significant association was seen between β-catenin, vascular endothelial growth factor or cyclin D1 expression and some investigated clinicopathologic features.. Our results may contribute to knowledge regarding the functional interaction between β-catenin and vascular endothelial growth factor. We suggest that the overexpression of cyclin D1 in rectosigmoid cancers may be more complicated than purely upregulation by β-catenin. Further larger studies on Wnt/β-catenin and target gene activity and protein expression are necessary to better understand and define their roles in the pathogenesis of colorectal carcinoma.

Topics: Adult; Aged; Aged, 80 and over; beta Catenin; Colorectal Neoplasms; Cyclin D1; Female; Humans; Immunohistochemistry; Male; Middle Aged; Neoplasm Staging; Neovascularization, Pathologic; Sigmoid Neoplasms; Signal Transduction; Vascular Endothelial Growth Factor A; Wnt Proteins

Laterally spreading tumors (LSTs) are considered a special subtype of superficial colorectal tumor. This study was performed to characterize the clinicopathological features and examine activation of the Wnt/beta-catenin pathway in LSTs and protruded-type colorectal adenomas (PAs). Fifty LSTs and 54 PAs were collected, and their clinicopathological characteristics were compared. The expression of E-cadherin, beta-catenin, glycogen synthase kinase-3beta (GSK-3beta), phosphorylated GSK-3beta, (phospho-GSK-3beta), cyclin D1, and c-myc was investigated by immunohistochemical staining on serial sections. Patients with LSTs were significantly older than those bearing PAs (63.4 vs 47.4 years old; p<0.001). The mean size of LSTs was significantly larger than that of PAs (27.0 mm vs 14.6 mm; p<0.01). Forty-eight percent of LSTs were located in the proximal colon, which was significantly higher than that of PAs (18.5%; p<0.05). Expression of beta-catenin, phospho-GSK-3beta, cyclin D1, and c-myc was significantly increased in LSTs compared with PAs (p<0.05). However, E-cadherin and total GSK-3beta expression was not significantly different between the two groups. The level of beta-catenin expression correlated strongly with phospho-GSK-3beta, cyclin D1, and c-myc expression in LSTs but not in PAs. Our findings suggest that activation of the Wnt/beta-catenin pathway is more prevalent in LSTs than in PAs, suggesting that phosphorylation-dependent inactivation of GSK-3beta may be involved in LST carcinogenesis.

Topics: Adenoma; beta Catenin; Biomarkers, Tumor; Colorectal Neoplasms; Cyclin D1; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Humans; Immunohistochemistry; Phosphorylation; Proto-Oncogene Proteins c-myc

The frequent occurrence of inactivating gene mutations in tumors suggests a tumor suppressor function of the mutated gene. The RNA binding motif protein 35A (RBM35A) is mutated in approximately 50% of analyzed primary colon tumors with microsatellite instability. The Tet-off regulated ectopic expression of RBM35A gene in RBM35A-null LS180 colon carcinoma cells inhibited anchorage-independent growth in vitro, suppressed tumorigenic potential in vivo and enhanced adhesiveness of these cancer cells. Using microarray hybridization we found that in response to RBM35A expression a small fraction of genes showed a decrease in polysome-associated mRNA. Experiments using cell-free in vitro translation system demonstrated that RBM35A differentially affects translation of luciferase reporter mediated by various 5'untranslated regions (UTR). We found that Gibbs energy value (DeltaG) of secondary structure formed by 5'UTRs of mRNAs can account for differential effect of RBM35A on reporter translation efficiency. Targeted mutation in the FOS 5'UTR sequence, which increased the DeltaG value of hairpin stem formation, resulted in a stronger inhibitory effect of RBM35A on reporter translation efficiency mediated by this UTR. Immunoblotting revealed that ectopic expression of RBM35A in LS180 cells caused alterations in protein levels for several cancer related genes. Our results demonstrate for the first time that RBM35A functions as a tumor suppressor in colon cancer cells. We propose that RBM35A is involved in posttranscriptional regulation of a number of genes by exerting a differential effect on protein translation via 5'UTRs of mRNAs.

Topics: 5' Untranslated Regions; Animals; Cell Line; Colorectal Neoplasms; Cyclin D1; Female; Gene Expression Regulation, Neoplastic; Humans; Mice; Mice, Nude; Neoplasm Transplantation; Nucleic Acid Conformation; Polyribosomes; RNA-Binding Proteins; Tumor Suppressor Proteins

We have investigated the expression and role of the 58-kDa microspherule protein (MSP58) in colorectal carcinoma (CRC). By immuhistochemistry and immunofluorescence, we observed MSP58 in the nucleus and cytoplasm of CRC cells, and found MSP58 to be present in CRC specimens more often than in adjacent non-tumor tissues (92.5 vs 36.3%, P < 0.01). The average staining score in adjacent non-tumor tissues was significantly lower than in CRC tissues (2.05 +/- 1.13 vs 5.23 +/- 1.38, P < 0.01). Moreover, MSP58 mRNA and protein appeared to be upregulated in six fresh CRC samples compared to their adjacent non-cancerous tissues. MSP58 expression was also detected in the human CRC-derived cell lines LoVo, CoLo205, HCT116, HT-29, SW620, and SW480. Downregulation of MSP58 inhibited in vitro growth and attenuated tumor growth in animal models by induction of cell cycle arrest, and was associated with reduced levels of cyclin D1, cyclin-dependent kinase 4, phosphorylation-Rb (p-Rb), p21, and Retino blastoma (Rb) proteins. These results indicated that MSP58 might play an important role in the carcinogenesis of CRC via regulation of the cyclin D1-cyclin-dependent kinase 4-p21 pathway.

Topics: Blotting, Western; Cell Cycle; Cell Proliferation; Colony-Forming Units Assay; Colorectal Neoplasms; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase Inhibitor p21; Down-Regulation; Enzyme-Linked Immunosorbent Assay; Flow Cytometry; Fluorescent Antibody Technique; Humans; Immunoenzyme Techniques; Nuclear Proteins; Phosphorylation; Reverse Transcriptase Polymerase Chain Reaction; RNA-Binding Proteins; RNA, Messenger; Subcellular Fractions; Tumor Cells, Cultured

Because of the poor prognosis and the development of resistance against chemotherapeutic drugs, the current treatment for advanced metastatic colorectal cancer (CRC) is ineffective. Whether curcumin (a component of turmeric) can potentiate the effect of capecitabine against growth and metastasis of CRC was investigated. The effect of curcumin on proliferation of CRC cell lines was examined by mitochondrial dye-uptake assay, apoptosis by esterase staining, nuclear factor-kappaB (NF-kappaB) by electrophoretic mobility shift assay and gene expression by Western blot analysis. The effect of curcumin on the growth and metastasis of CRC was also examined in orthotopically implanted tumors in nude mice. In vitro, curcumin inhibited the proliferation of human CRC cell lines, potentiated capecitabine-induced apoptosis, inhibited NF-kappaB activation and suppressed NF-kappaB-regulated gene products. In nude mice, the combination of curcumin and capecitabine was found to be more effective than either agent alone in reducing tumor volume (p = 0.001 vs. control; p = 0.031 vs. capecitabine alone), Ki-67 proliferation index (p = 0.001 vs. control) and microvessel density marker CD31. The combination treatment was also highly effective in suppressing ascites and distant metastasis to the liver, intestines, lungs, rectum and spleen. This effect was accompanied by suppressed expression of activated NF-kappaB and NF-kappaB-regulated gene products (cyclin D1,c-myc, bcl-2, bcl-xL, cIAP-1, COX-2, ICAM-1, MMP-9, CXCR4 and VEGF). Overall, our results suggest that curcumin sensitizes CRC to the antitumor and antimetastatic effects of capecitabine by suppressing NF-kappaB cell signaling pathway.

Topics: Animals; Antineoplastic Agents; Apoptosis; Capecitabine; Cell Line, Tumor; Cell Proliferation; Colorectal Neoplasms; Curcumin; Cyclin D1; Cyclooxygenase 2; Deoxycytidine; Drug Synergism; Fluorouracil; Gene Expression Regulation, Neoplastic; Humans; Male; Matrix Metalloproteinase 9; Mice; Neoplasm Metastasis; NF-kappa B; Receptors, CXCR4; Vascular Endothelial Growth Factor A

Cyclin D1 and cyclin-dependent kinases are commonly activated in colorectal cancer. Microsatellite instability (MSI) and CpG island methylator phenotype (CIMP) are important molecular classifiers in colorectal cancer. The aim was to clarify the relationship between cyclin D1, MSI and CIMP.. Among 865 colorectal cancers with MSI and CIMP data, 246 tumours (28.4%) showed cyclin D1 overexpression by immunohistochemistry. DNA methylation in p14 and eight CIMP-specific promoters (CACNA1G, CDKN2A (p16), CRABP1, IGF2, MLH1, NEUROG1, RUNX3 and SOCS1) was quantified by real-time polymerase chain reaction (MethyLight). Both MSI-high and CIMP-high were associated with cyclin D1 overexpression (P < 0.0001). After tumours were stratified by MSI and CIMP status, the relationship between MSI-high and cyclin D1 persisted (P < or = 0.02), whereas the relationship between CIMP-high and cyclin D1 did not. Cyclin D1 overexpression was correlated with BRAF mutation (P = 0.0001), p27 loss (P = 0.0007) and p16 loss (P = 0.02), and inversely with p53 expression (P = 0.0002) and p21 loss (P < 0.0001). After stratification by MSI status, the inverse relationship between cyclin D1 and p21 loss still persisted (P < 0.008).. Cyclin D1 activation is associated with MSI and inversely with p21 loss in colorectal cancers. Cyclin D1 may play an important role in the development of MSI-high tumours, independent of CIMP status.

Topics: Aged; Colorectal Neoplasms; CpG Islands; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; DNA Methylation; Female; Humans; Male; Microsatellite Instability; Middle Aged; Mutation; Phenotype; Proto-Oncogene Proteins B-raf; Tissue Array Analysis

Several drugs have been developed and demonstrated similar efficacy in colorectal cancer treatment therefore with choice, time comes for decision. The biologist will have to provide the tools allowing to clarify this choice. Among the tools available, those of pharmacogenetics and pharmacogenomics appear most promising and recent examples allow to illustrate their clinical interest. The pharmacogenetics of anti-cancer agents presents a clinical characteristic, which requires to hold into account the genetic variations not only of host cells but also of those of the tumor cells. Among the most conclusive examples one is that of the prediction of severe neutropenia induced by the irinotecan among patients homozygous for * 28 allele of UGT1A1 enzyme which conjugates SN38 active compound of irinotecan, the other one is the presence of a KRAS mutated allele in tumor cell to predict resistance to anti EGFR antibodies in the treatment of colorectal metastatic cancer.

Topics: Antineoplastic Agents; Camptothecin; Colorectal Neoplasms; Cyclin D1; Dihydrouracil Dehydrogenase (NADP); Epidermal Growth Factor; Fluorouracil; Glucuronosyltransferase; Glutathione Transferase; Humans; Irinotecan; Mutation; Organoplatinum Compounds; Polymorphism, Genetic; Proto-Oncogene Proteins; Proto-Oncogene Proteins p21(ras); ras Proteins; Receptors, IgG; Thymidylate Synthase

Nuclear factor-kappaB p65 (NF-kappaB p65), nuclear factor-kappaB1 p50 (NF-kappaB p50) have been shown to play a role in cell proliferation, apoptosis, cytokine production, and oncogenesis. Recently, p38 mitogen-activated protein kinase (MAPK)/ NF-kappaB/ cyclin D1 signaling pathway has been shown to play an important part in the pathogenesis of human cancers. This study was designed to investigate the expression of NF-kappaB p65, NF-kappaB p50, p38 MAPKalpha, and cyclin D1 proteins in premalignant lesions of colon and colorectal adenocarcinoma.. Paraffin sections of 20 normal mucosa, 20 low-grade tubular adenoma, 20 high-grade tubular adenoma and 64 adenocarcinoma tissues were analysed immunohistochemically for the expression of NF-kappaB p65, NF-kappaB p50, p38 MAPKalpha, and cyclin D1 proteins.. The expression of NF-kappaB p65, NF-kappaB p50, and p38 MAPKalpha proteins were significantly higher in adenocarcinoma tissue in comparison with that in normal mucosa, low-grade tubular adenoma, and high-grade tubular adenoma tissues. Expression of NF-kappaB p50 was more frequent in poorly differentiated histologic grade, presence of nodal metastasis, and advanced stage. Expression of p38 MAPKalpha protein was higher in advanced tumor stage, presence of nodal metastasis and advanced stage. Synchronous expression of NF-kappaB p65, NF-kappaB p50, p38 MAPKalpha, and cyclin D1 proteins were significantly higher in adenocarcinoma tissue.. With the increased expression of NF-kappaB p65, NF-kappaB p50, and p38 MAPKalpha proteins, p38 MAPK/ NF-kappaB/ cyclin D1 signaling pathway may play a role in the pathogenesis of colorectal carcinoma.

Topics: Adenocarcinoma; Colorectal Neoplasms; Cyclin D1; Data Interpretation, Statistical; Female; Humans; Immunohistochemistry; Intestinal Mucosa; Male; Middle Aged; Neoplasm Staging; NF-kappa B; NF-kappa B p50 Subunit; p38 Mitogen-Activated Protein Kinases; Precancerous Conditions; Transcription Factor RelA

Aberrations in the cell cycle regulators are common features of many tumours and several have been shown to have prognostic significant in colorectal cancer. The expression patterns of cyclins D1 and E as well as cyclin-dependent kinase (CDK) inhibitors p21waf1/cip1 and p27kip1 and their interrelationship with other cell cycle checkpoint proteins [p53, pRb, Ki-67 and proliferative cell nuclear antigen (PCNA)] were investigated in colorectal cancer in order to ascertain coregulation and influence on tumour behaviour or survival. These molecular markers were localisated immunohistochemically using the monoclonal antibodies anticyclin D1 (DCS-6), anticyclin E (13A3), anti-p21 (4D10), anti-p27 (1B4), anti-p53 (DO7), anti-Rb (AB-5), MIB1 and PC10 in colorectal cancer tissue from 97 patients. Data were analysed statistically using the spss software program. Overexpression of cyclin D1, cyclin E and p21waf1/cip1 proteins (>5% positive neoplastic cells) was observed in 5.9%, 30% and 7.2% of the cases respectively. Increased levels of cyclin D1 (p = 0.0001) and p21waf1/cip1 protein (p = 0.03) in tumours with mucous differentiation were observed. Overexpression of cyclin D1 was correlated with tumour stage (p = 0.03), the lymph node involvement (p = 0.02), as well as p21waf1/cip1 protein expression (p < 0.0001). Cyclin E was positively correlated with p21waf1/cip1 (p = 0.014), as well as with the cell proliferation as measured by PCNA-labelling index (p = 0.011) and Ki-67 score (p = 0.007). A positive relationship of p21waf1/cip1 expression with the proliferative-associated index Ki-67 was noted (p = 0.005). Downregulation of p27kip1 was observed in 47.4% of the cases and was correlated with downregulation of pRb (p = 0.002) and PCNA score (p = 0.004). The prognostic significance of cyclins D1, E and CDK inhibitors p21waf1/cip1, p27kip1 in determining the risk of recurrence and overall survival with both univariate (long-rang test) and multivariate (Cox regression) methods of analysis showed no statistically significance differences. In conclusion, these findings suggest that, the levels of the cell cycle regulators studied, do not seems to have a prognostic value, in terms of predicting the risk of early recurrence and overall survival. In addition, the interrelationships, probably means their contribution to the regulation of cell growth, through different pathways in colorectal carcinogenesis.

Topics: Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Cell Cycle Proteins; Colorectal Neoplasms; Cyclin D1; Cyclin E; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase Inhibitor p27; Humans; Immunohistochemistry; Ki-67 Antigen; Middle Aged; Neoplasm Proteins; Neoplasm Recurrence, Local; Nuclear Proteins; Prognosis; Proliferating Cell Nuclear Antigen; Repressor Proteins; Tumor Suppressor Protein p53; Young Adult

Cyclin D1 (CCND1) is a regulatory protein involved in the cell cycle. A common G to A polymorphism in the CCND1 gene is implicated on the splicing of the CCND1 transcript, and this protein may be associated to a deregulated cell proliferation.. Correlate the polymorphism A870G of CCND1 to the risk of colorectal cancer (CRC), to environmental risk factors and clinical aspects in Brazilian patients.. One hundred twenty-three Brazilian patients with colorectal cancer were matched by age and sex to 120 healthy individuals. PCR-RFLP was performed to investigate the A870G CCND1 genotype.. Between the cases 70 were men, the mean age was 62.6 years, 74.78% were stage II or III, and 91% were well or moderately differentiated. The patients were followed for a mean time of 37.22 months. The frequency of ethanol and fat intake was similar among the groups. Patients with a family history of CRC had a higher frequency of CRC compared with the controls (OR 4.16, CI 1.89-9.16). There was no difference in the frequency of the alleles A (43.8% versus 43.9%) and G (56.3% versus 56.1%) in the groups. In analysis of both control and cancer group, the influence of sex, smoking, alcohol, fiber, or meat intake did not differ significantly according to CCND1 genotype. The genotype AA or AG was associated with an increased risk of CRC (OR 3.63 CI 1.25-10.5) in patients with a family history of cancer. We did not find any association among the genotypes and localization of the tumor or prognosis. Although a difference on age onset of the tumor and genotype was not observed, patients with GG genotype had a mean 8 years lower than the others. This genotype was also associated to an increase risk of metastatic disease (OR 3.47, CI 1.38-8.68, p = 0.024).. We did not find a correlation among the polymorphism of CCND1 A870G and colorectal cancer risk or between this polymorphism and lifestyle habits, diet, or follow-up. GG genotype patients had an increased risk of advanced disease and between the young patients, this genotype was associated to a lower mean age. On the other hand, the genotype AA or AG had been involved to a higher risk of CRC in patients with family history of CRC.

Topics: Adult; Aged; Aged, 80 and over; Brazil; Case-Control Studies; Colorectal Neoplasms; Cyclin D1; Female; Genotype; Humans; Male; Middle Aged; Polymorphism, Genetic

About 20% of colorectal cancer (CRC) patients show some kind of familiarity, which might be caused by yet unknown combinations of low penetrance susceptibility genes. We aimed to identify genetic factors for familial CRC (fCRC) in a unique study design that includes phenotypic extremes as represented by fCRC cases and 'hyper-normal' controls without CRC history and no adenomatous polyps on colonoscopy.. Candidate gene variants were determined by allele-specific amplification (SLC10A2 c.169C>T and c.171G>T) and restriction fragment length polymorphism assays (CCND1 c.870A>G; CDH1 -160C>A; TP53 R72P; VDR T2M). In total, 98 patients with fCRC, 96 patients with sporadic CRC, and 220 hyper-normal controls were included.. The minor allele of the CDH1 -160C>A polymorphism occurred significantly more often in controls compared to fCRC cases (OR = 0.664; p = 0.042). Homozygosity of the minor allele was significantly associated with affiliation to the control group (OR = 0.577; p = 0.029), indicating that both heterozygous and homozygous carriers of the common allele are at-risk for CRC. With respect to the CCND1 c.870A>G mutation, comparison of fCRC and sporadic CRC cases showed that A/A homozygosity was more common than G/G homozygosity among fCRC patients compared to controls (OR = 2.119; p = 0.045). However, no differences in allele or genotype frequencies were detected between sporadic CRC cases and controls, and no associations were observed for SLC10A2, TP53, and VDR polymorphisms.. We report a potential association of variants in the CCND1 and CDH1 genes with fCRC using a unique study design with phenotypic extremes.

Topics: Adult; Aged; Antigens, CD; Cadherins; Case-Control Studies; Colonoscopy; Colorectal Neoplasms; Cyclin D1; Female; Gene Expression Regulation, Neoplastic; Gene Frequency; Genetic Predisposition to Disease; Heterozygote; Homozygote; Humans; Male; Middle Aged; Odds Ratio; Pedigree; Phenotype; Polymorphism, Genetic; Promoter Regions, Genetic; Prospective Studies; Risk Assessment

Polymorphisms in the 2 cell-cycle control genes Aurora A and Cyclin D1 have previously been associated with changes in the age of onset of colorectal cancer in persons harboring germline mutations in DNA mismatch repair genes associated with hereditary nonpolyposis colorectal cancer (HNPCC). In this report, we have genotyped 312 individuals, who all harbored confirmed causative mutations in either hMSH2 or hMLH1, for 2 polymorphisms, one in Aurora A (T91A) and the other in Cyclin D1 (G870A). The results reveal that the previous association with the Aurora A polymorphism could not be confirmed in our larger group of HNPCC patients. The Cyclin D1 polymorphism, however, was associated with a significant difference in the age of disease onset on patients harboring hMSH2 mutations, which was not observed in hMLH1 mutation carriers. A combined analysis of the Aurora A and Cyclin D1 polymorphisms did not reveal any obvious association. In conclusion, it appears that the polymorphic variant of Aurora A does not appear to be associated with variation in colorectal cancer risk in HNPCC, whereas there is a more complex relationship between the Cyclin D1 polymorphism and disease risk in HNPCC.

Topics: Adult; Age of Onset; Aged; Aurora Kinases; Colorectal Neoplasms; Colorectal Neoplasms, Hereditary Nonpolyposis; Cyclin D1; Disease Progression; Female; Gene Frequency; Heterozygote; Homozygote; Humans; Male; Middle Aged; Polymorphism, Single Nucleotide; Protein Serine-Threonine Kinases; Survival Analysis

6-Gingerol, a natural product of ginger, has been known to possess anti-tumorigenic and pro-apoptotic activities. However, the mechanisms by which it prevents cancer are not well understood in human colorectal cancer. Cyclin D1 is a proto-oncogene that is overexpressed in many cancers and plays a role in cell proliferation through activation by beta-catenin signaling. Nonsteroidal anti-inflammatory drug (NSAID)-activated gene-1 (NAG-1) is a cytokine associated with pro-apoptotic and anti-tumorigenic properties. In the present study, we examined whether 6-gingerol influences cyclin D1 and NAG-1 expression and determined the mechanisms by which 6-gingerol affects the growth of human colorectal cancer cells in vitro. 6-Gingerol treatment suppressed cell proliferation and induced apoptosis and G(1) cell cycle arrest. Subsequently, 6-gingerol suppressed cyclin D1 expression and induced NAG-1 expression. Cyclin D1 suppression was related to inhibition of beta-catenin translocation and cyclin D1 proteolysis. Furthermore, experiments using inhibitors and siRNA transfection confirm the involvement of the PKCepsilon and glycogen synthase kinase (GSK)-3beta pathways in 6-gingerol-induced NAG-1 expression. The results suggest that 6-gingerol stimulates apoptosis through upregulation of NAG-1 and G(1) cell cycle arrest through downregulation of cyclin D1. Multiple mechanisms appear to be involved in 6-gingerol action, including protein degradation as well as beta-catenin, PKCepsilon, and GSK-3beta pathways.

Topics: Anti-Inflammatory Agents, Non-Steroidal; Antineoplastic Agents; Apoptosis; beta Catenin; Caco-2 Cells; Catechols; Cell Line, Tumor; Cell Proliferation; Colorectal Neoplasms; Cyclin D1; Dose-Response Relationship, Drug; Fatty Alcohols; G1 Phase; HT29 Cells; Humans; Luciferases; Plasmids; Proto-Oncogene Mas; S Phase; Statistics as Topic; Transfection

BRAF kinase is a downstream target of KRAS and activates the MAPK pathway. These two molecules are prone to mutations in sporadic microsatellite unstable (MSI) colorectal carcinomas (CRC) and BRAF V600E mutations are inversely associated with oncogenic KRAS mutations. The biological significance of BRAF V600E oncogenic activation is not well established in this type of tumour. We aimed to study proliferation and survival effects induced by BRAF inhibition in MSI CRC cell lines harbouring distinct genetic backgrounds (BRAF V600E or KRAS G13D). Suppression of BRAF in BRAF V600E MSI CRC cell lines by RNA interference significantly inhibited proliferation and induced apoptosis, as demonstrated by BrdU incorporation and TUNEL assay, respectively. No significant differences were seen in proliferation and apoptosis, in cell lines harbouring KRAS G13D, after BRAF inhibition. We further analysed proliferation-associated molecules (pERK1/2, cyclin D1, p27 Kip1) and apoptosis-associated molecules (Bcl-2, Bax, pAkt, pBad, XIAP) in all cell lines. After BRAF down-regulation, we found a more pronounced decrease in ERK1/2 phosphorylation and cyclin D1 expression levels in BRAF-mutated cell lines in comparison to KRAS mutated cells. Upon BRAF inhibition, we also found an increase in p27(Kip1) levels and a more pronounced decrease in the levels of anti-apoptotic protein Bcl-2, specifically in cell lines with BRAF V600E. In conclusion, we have shown that MSI KRAS and BRAF mutant CRC cell lines respond differently to BRAF knockdown. This report provides evidence supporting BRAF as a good target for therapeutic intervention in patients with sporadic MSI CRC harbouring activating mutations in BRAF but not in KRAS.

Topics: Apoptosis; Blotting, Western; Cell Line, Tumor; Cell Proliferation; Cell Survival; Colorectal Neoplasms; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p27; DNA; Extracellular Signal-Regulated MAP Kinases; Genes, ras; Humans; Microsatellite Instability; Mutation; Proto-Oncogene Proteins B-raf; RNA Interference; RNA, Small Interfering; Signal Transduction

Alterations in the Wnt pathway play a major role in colorectal cancer with high (MSI-H) or low microsatellite instability (MSS/MSI-L). However, the differential impact of the Wnt pathway components on these tumors is poorly understood. MMP-3 (stromelysin-1) promoter is a target of the mutator phenotype in sporadic colorectal cancer. Among MMP-3 targets, we investigated E-cadherin integrity status in both groups of tumors. Because beta-catenin is the main effector of the Wnt pathway, we have also investigated the differential cellular status of beta-catenin.. Expression profiles of 114 genes related to the Wnt pathway were analyzed by oligo microarrays in 48 tumors classified by their MSI status. In addition, we analyzed 48 sporadic colorectal cancers for E-cadherin integrity status. We performed investigation of beta-catenin and cyclin D1 by immunohistochemistry using tissue arrays containing 96 tumors.. Our data show that a group of genes that negatively regulate Wnt signaling are downregulated in MSS/MSI-L as compared with MSI-H colorectal tumors. E-cadherin truncation was significantly higher in MSS/MSI-L as compared with MSI-H tumors. Moreover, MSI-H tumors showed low or null beta-catenin nuclear presence, whereas the group of tumors classified as MSS or MSI-L displayed a high content of the nuclear beta-catenin location.. Our results suggest that the differential expression of genes that negatively regulate the Wnt pathway, as well as the status of E-cadherin and beta-catenin in MSI-H or MSS/MSI-L colorectal tumors, shed some light on the different clinical behavior showed by the two groups.

Topics: beta Catenin; Blotting, Western; Cadherins; Colorectal Neoplasms; Cyclin D1; Gene Expression; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Microsatellite Instability; Oligonucleotide Array Sequence Analysis; Tissue Array Analysis; Wnt Proteins

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

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

Herein, for the first time, we investigated in vivo efficacy and associated molecular biomarkers and mechanisms of a chemopreventive agent, silibinin, against human colorectal carcinoma (CRC) HT29 xenograft growth. Nude mice were implanted with HT29 cells and fed with vehicle (carboxymethyl cellulose or phosphatidylcholine) or 200 mg/kg/d dose of silibinin or 100 and 200 mg/kg/d doses of silybin-phytosome (5 days per week) for 32 days. Silibinin inhibited tumor growth that accounted for 48% (P = 0.002) decrease in tumor volume and 42% (P = 0.012) decrease in tumor weight at the end of the experiment without any adverse health effect. A stronger antitumor efficacy was observed with silybin-phytosome preparation. Silibinin decreased proliferation index by 40% (P < 0.001), increased apoptotic index by approximately 2-fold (P = 0.001), and reduced microvessel density by 36% (P = 0.001) in tumors. Antiproliferative and proapoptotic effects of silibinin were associated with down-regulation of extracellular signal-regulated kinase 1/2 (ERK1/2) and Akt phosphorylation as well as cyclin D1 expression. Antiangiogenic effect of silibinin was coupled with a strong decrease in inducible nitric oxide synthase (NOS) and NOS3, cyclooxygenase-1 (COX-1) and COX-2, and hypoxia-inducing factor-1 alpha (HIF-1 alpha) and vascular endothelial growth factor (VEGF). These findings suggest in vivo antitumor efficacy of silibinin against CRC involving its antiproliferative, proapoptotic, and antiangiogenic activities. The inhibition of ERK1/2 and Akt signaling may account for antiproliferative and proapoptotic effects, whereas down-regulation of NOS, COX, HIF-1 alpha, and VEGF expression could lead to antiangiogenic effect of silibinin against CRC. Overall, potential use of silibinin against human CRC could be suggested.

Topics: Animals; Antioxidants; Apoptosis; Cell Growth Processes; Colorectal Neoplasms; Cyclin D1; Cyclooxygenase 2; Enzyme Activation; HT29 Cells; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Neovascularization, Pathologic; Nitric Oxide Synthase Type II; Nitric Oxide Synthase Type III; Oncogene Protein v-akt; Silybin; Silymarin; Vascular Endothelial Growth Factor A; Xenograft Model Antitumor Assays

Aberrant nuclear factor-kappaB (NF-kappaB) signaling plays a role in cancer initiation and progression; thus, it represents a potential therapeutic target. We previously identified a mechanism of repression of NF-kappaB transcriptional activity and induction of apoptosis in colon cancer cells involving nuclear/nucleolar translocation of the RelA (p65) component of NF-kappaB. This response was stimulated by cellular stress-inducing agents, including aspirin, but not by tumor necrosis factor. Here, we investigate the upstream molecular mechanisms responsible for nucleolar targeting of RelA and show that aspirin activates the p38 mitogen-activated protein kinase (MAPK) pathway in colorectal cancer cells. We also show that aspirin causes rapid, ubiquitin-dependent degradation of cyclin D1, a known p38 target. Aspirin-induced p38 activation preceded cyclin D1 degradation, which was then followed by activation of the NF-kappaB pathway, suggesting a causative link. Indeed, chemical p38 inhibition (PD169316) and small interfering RNA directed against p38 blocked aspirin-induced cyclin D1 degradation, nucleolar translocation of RelA, and apoptosis. Furthermore, chemical inhibition of the cyclin D1/cyclin-dependent kinase 4 (CDK4) kinase complex, used as a surrogate for cyclin D1 degradation, caused nucleolar translocation of RelA, repression of kappaB-driven transcription, and apoptosis, thereby reproducing the effects of aspirin. In addition, we found that aspirin and the CDK4 inhibitor induced nucleolar translocation of RelA and apoptosis through a common mechanism involving the NH(2)-terminal nucleolar localization signal. Collectively, these data suggest that aspirin causes inhibition of cyclin D1/CDK4 through the p38 MAPK pathway. This inhibition stimulates the NF-kappaB pathway to induce nucleolar translocation of RelA and apoptosis. These novel findings have considerable relevance to the rational design of novel chemotherapeutic and chemopreventative strategies.

Topics: Apoptosis; Aspirin; Cell Cycle; Cell Nucleolus; Colorectal Neoplasms; Cyclin D1; Cyclin-Dependent Kinase 4; Enzyme Activation; HT29 Cells; Humans; MAP Kinase Signaling System; p38 Mitogen-Activated Protein Kinases; Transcription Factor RelA

Rac1b is a tumor-specific splice variant of the Rac1 GTPase that displays limited functional similarities to Rac1. We have shown previously a novel cross-talk between Rac1 and beta-catenin, which induces canonical Wnt pathway activation in colorectal cancer cells. This prompted us to investigate if Rac1b, frequently overexpressed in colon tumors, contributes to Wnt pathway dysregulation. We show that Rac1b overexpression stimulates Tcf-mediated gene transcription, whereas depletion of Rac1b results in decreased expression of the Wnt target gene cyclin D1. Reconstitution experiments revealed an important difference between Rac1 and Rac1b such that Rac1b was capable of functionally interacting with Dishevelled-3 (Dvl-3) but not beta-catenin to mediate synergistic induction of Wnt target genes. In agreement, Dvl-3 but not beta-catenin caused increased activation of Rac1b levels, which may explain the functional cooperativity displayed in transcription assays. Furthermore, we show that Rac1b negatively regulates E-cadherin expression and results in decreased adhesion of colorectal cancer cells. RNA interference-mediated suppression of Rac1b resulted in reduced expression of Slug, a specific transcriptional repressor of E-cadherin, and a concomitant increase in E-cadherin transcript levels was observed. Intriguingly, mutation of the polybasic region of Rac1b resulted in complete loss of Rac1b stimulatory effects on transcription and suppressive effects on adhesion, indicating the importance of nuclear and membrane localization of Rac1b. Our results suggest that Rac1b overexpression may facilitate tumor progression by enhancing Dvl-3-mediated Wnt pathway signaling and induction of Wnt target genes specifically involved in decreasing the adhesive properties of colorectal cancer cells.

Topics: Adaptor Proteins, Signal Transducing; Amino Acid Sequence; Cadherins; Cell Adhesion; Cell Growth Processes; Cell Line, Tumor; Cell Nucleus; Colorectal Neoplasms; Cyclin D1; Dishevelled Proteins; Enzyme Activation; HCT116 Cells; HT29 Cells; Humans; Molecular Sequence Data; Phosphoproteins; Promoter Regions, Genetic; rac1 GTP-Binding Protein; Signal Transduction; Transcription, Genetic; Transcriptional Activation; Wnt Proteins

Overexpression of anti-apoptotic bcl-2 protein has been found in hematological and solid tumors and has been associated with increased resistance against cytotoxic therapy. While bcl-2 antisense (AS) treatment combined with chemotherapy has been successfully tested in clinical trials, trials evaluating the combination of bcl-2 AS with radiotherapy have not yet been performed. The aim of this study was to investigate in vivo anti-tumor effects of a combined modality treatment scheme consisting of radiation and the bcl-2 targeted AS oligonucleotide (ODN) G3139 (Oblimersen Sodium). Two human colon carcinoma cell lines, SW620, bcl-2 positive and HT-29, bcl-2 negative, were grown as xenografts and compared in their response to combined bcl-2 AS/radiation treatment. G3139 potentiated the radiation response of bcl-2 positive SW620 tumors, but had no significant effect on bcl-2 negative HT-29 tumors assayed by tumor growth delay. The profound enhancement of SW620 tumor growth delay by G3139 did not translate into effects on tumor cure, as no significant effect of G3139 was found on SW620 radiocurability (TCD50 assay). The control ODN G3622 had no effect on SW620 radiation response, indicating an ODN sequence specific effect.

Topics: Animals; Antineoplastic Agents; Carcinoma; Cell Line, Tumor; Colorectal Neoplasms; Combined Modality Therapy; Cyclin D1; Gamma Rays; Humans; Mice; Mice, Nude; Neoplasm Transplantation; Oligonucleotides, Antisense; Thionucleotides; Tumor Burden

Mutations in the serine-threonine tumor-suppressor kinase LKB1 are responsible for Peutz-Jeghers syndrome, characterized by hamartomatous proliferation and an increased risk of developing cancer. Mutations in lkb1 have also been identified in sporadic cancers, suggesting a wider role for LKB1 in cancer that is not limited to hamartomatous polyposis syndromes. Here, we show that LKB1 catalytically deficient mutants, when introduced into DLD1p21-/-p53-/- colorectal cancer cells, allowed for progression of cells through to S phase of cell cycle and elicited the expression of Rb, cyclin E, and cyclin A2 whereas the introduction of LKB1 lead to G1 cell cycle arrest independent of p21(WAF/CIP1) and/or p53 expression. Furthermore, we show that LKB1 catalytically deficient mutants activate the expression of cyclin D1 through recruitment to response elements within the promoter of the oncogene. In addition to compromising the tumor-suppressor function of LKB1, our findings highlight an emerging role for LKB1 catalytically deficient mutants, a gain of oncogenic properties.

Topics: Adenocarcinoma; AMP-Activated Protein Kinase Kinases; Blotting, Western; Cell Cycle; Cell Proliferation; Cell Transformation, Neoplastic; Colorectal Neoplasms; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Flow Cytometry; Humans; Immunoprecipitation; Mutation; Protein Serine-Threonine Kinases; Reverse Transcriptase Polymerase Chain Reaction; Transfection; Tumor Suppressor Protein p53

In immunohistochemistry, nonstandardized antigen retrieval protocols and fluids, poor-quality antibodies, and the presence of endogenous biotin frequently lead to incorrect results. Recently, advanced reagents including bifunctional SkipDewax pretreatment solution (BSPS), rabbit monoclonal (RM) antibodies, and biotin-free polymer detection systems (PDSs) have been developed, which, it is claimed, resolve these problems.. To determine whether BSPS, RM antibodies, and biotin-free PDSs improve the accuracy of immunohistochemistry; to optimize a new protocol consisting of a combination of BSPS, RM antibodies, and PDSs; and to compare it with a conventional protocol.. The efficacies of BSPS, RM antibodies, and PDSs were compared with those of their respective conventional reagents using multitissue spring-roll sections. The new protocol was compared with a conventional protocol using Ki-67 immunostaining of 49 colorectal carcinoma specimens.. For antigen retrieval, BSPS resulted in similar or better tissue staining than an EDTA solution, but the efficacy of BSPS decreased when it was reused. Most RM antibodies resulted in a greater proportion of positive cells than the corresponding non-RM antibodies, which did not produce satisfactory results in the absence of antigen retrieval. The PDSs Bond, ChemMate, and SuperPicture resulted in a high percentage of positive cells, good staining intensities, and low backgrounds. Other PDSs, except that from Ventana, resulted in high backgrounds and false positivity. The new combined protocol resulted in better Ki-67 staining than the conventional assay.. Bifunctional SkipDewax pretreatment solution, RM antibodies, and PDSs improve staining quality and diagnostic accuracy of immunohistochemistry assays and provide a foundation for standardization.

Topics: Animals; Antibodies, Monoclonal; Bacteriocins; CD3 Complex; CD5 Antigens; Colorectal Neoplasms; Cross-Linking Reagents; Cyclin D1; Humans; Immunohistochemistry; Ki-67 Antigen; Polymers; Rabbits; Reagent Kits, Diagnostic; Receptor, ErbB-2; Staining and Labeling; Synaptophysin

Although high-calcium diets have been reported to reduce the risk of colorectal cancer, our preliminary data with the adenomatous polyposis coli (Apc) Min mutation (Min/+;Apc(Min/+)) mouse shows a paradoxical increase in intestinal tumor loads (> 65%) with high calcium diets. Since we previously demonstrated that increasing dietary calcium reduces adiposity, and Apc(Min/+) mice on high calcium diets exhibited profound loss of adipose tissue, we hypothesized that loss of an adipose tissue-derived tumor suppressor factor(s) resulted in increased tumor susceptibility in animals on the high calcium diet. Accordingly, tumor prone Apc(Min/+) mice were crossed with obesity prone lethal yellow agouti (A(y)/a) mice to generate obese A(y)/Apc(Min/+) mice. Low (0.2%), normal (0.5%), and high (1.2%) calcium diets were fed to both A(y)/Apc(Min/+) mice and Apc(Min/+) mice from 35-40 days until 90 days of age (n=21/strain, n=7/diet group). The high calcium diet reduced weight gain in both strains (P < 0.01) and reduced fat pad mass by 46-57% in A(y)/Apc(Min/+)(P < 0.004) and by 65-82% in Apc(Min/+)(P < 0.03).Apc(Min/+) mice on the high calcium diet exhibited an increase in tumor number (76 vs. 29, P=0.009), but this effect was not seen in the A(y)/Apc(Min/+) mice. beta-Catenin and cyclin D1 gene expression were significantly induced with high calcium diet in intestinal tumor tissue of Apc(Min/+) mice but not in A(y)/Apc(Min/+) mice. We conclude that the differential effect of dietary calcium on intestinal tumorigenesis in lean vs. obese Apc(Min/+) may result from the loss of adipose-derived protective factor(s) due to the substantial loss of body fat in Apc(Min/+) mice fed a high calcium dairy diet, increasing beta-catenin and cyclin D1 in tumors.

Topics: Adiposity; Animals; beta Catenin; Calcium, Dietary; Colorectal Neoplasms; Cyclin D1; Dose-Response Relationship, Drug; Germ-Line Mutation; Intestinal Neoplasms; Intestines; Male; Mice; Mice, Inbred C57BL; Mice, Mutant Strains; Mice, Obese; Random Allocation; Weight Gain

The antineoplastic drug sorafenib (BAY 43-9006) is a multikinase inhibitor that targets the serine-threonine kinase B-Raf as well as several tyrosine kinases. Given the numerous molecular targets of sorafenib, there are several potential anticancer mechanisms of action, including induction of apoptosis, cytostasis, and antiangiogenesis. We observed that sorafenib has broad activity in viability assays in several human tumor cell lines but selectively induces apoptosis in only some lines. Sorafenib was found to decrease Mcl-1 levels in most cell lines tested, but this decrease did not correlate with apoptotic sensitivity. Sorafenib slows cell cycle progression and prevents irradiated cells from reaching and accumulating at G2-M. In synchronized cells, sorafenib causes a reversible G1 delay, which is associated with decreased levels of cyclin D1, Rb, and phosphorylation of Rb. Although sorafenib does not affect intrinsic radiosensitivity using in vitro colony formation assays, it significantly reduces colony size. In HCT116 xenograft tumor growth delay experiments in mice, sorafenib alters radiation response in a schedule-dependent manner. Radiation treatment followed sequentially by sorafenib was found to be associated with the greatest tumor growth delay. This study establishes a foundation for clinical testing of sequential fractionated radiation followed by sorafenib in gastrointestinal and other malignancies.

Topics: Animals; Antineoplastic Agents; Benzenesulfonates; Cell Division; Cell Growth Processes; Colorectal Neoplasms; Combined Modality Therapy; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p27; Dose-Response Relationship, Radiation; Drug Administration Schedule; Endothelial Cells; Female; G2 Phase; HCT116 Cells; Humans; Mice; Mice, Nude; Niacinamide; Phenylurea Compounds; Phosphorylation; Pyridines; Retinoblastoma Protein; Sorafenib; Xenograft Model Antitumor Assays

The abnormal oncogenes and antioncogenes in Wnt signaling transduction pathway activate downstream specific target genes, and may play important roles in tumorigenesis and tumor progression. This study was to examine the expression of adenomatous polyposis coli (APC), beta-catenin, C-myc, and Cyclin D1 in different colorectal tissues, and investigate their possible roles in the carcinogenesis of colorectal carcinoma.. The expression of APC, beta-catenin, C-myc, and Cyclin D1 in 30 specimens of normal colorectal mucosa, 30 specimens of colorectal adenoma, 10 specimens of colorectal adenoma with malignancy, and 50 specimens of colorectal carcinoma was examined by immunohistochemistry. The expression of beta-catenin on cell membrane was regarded as normal, and its expression in cytoplasm and nuclei was defined as ectopic expression.. The positive rate of APC was significantly lower in colorectal carcinoma and colorectal adenoma with malignancy than in colorectal adenoma and normal colorectal mucosa (44.0% and 40.0% vs. 86.7% and 100.0%, P<0.01). The ectopic expression rate of beta-catenin was significantly higher in colorectal carcinoma, colorectal adenoma with malignancy, and colorectal adenoma than in normal colorectal mucosa (62.0%, 50.0%, and 30.0% vs. 0%, P<0.01), and significantly higher in colorectal carcinoma than in colorectal adenoma (P<0.01). The positive rate of C-myc was significantly higher in colorectal carcinoma, colorectal adenoma with malignancy, and colorectal adenoma than in normal colorectal mucosa (56.0%, 60.0%, and 46.7% vs. 0%, P<0.01). The positive rate of Cyclin D1 was significantly higher in colorectal carcinoma, colorectal adenoma with malignancy, and colorectal adenoma than in normal colorectal mucosa (66.0%, 60.0%, and 30.0% vs. 0%,P<0.01), and significantly higher in colorectal carcinoma than in colorectal adenoma (P<0.01). The ectopic expression of beta-catenin was positively correlated to the expression of C-myc and Cyclin D1 (r=0.63,P<0.01; r=0.57, P<0.01), and negatively correlated to the expression of APC (r=-0.39, P<0.05).. The reduced expression of APC, ectopic expression of beta-catenin, overexpression of C-myc and Cyclin D1 exist in colorectal carcinoma, which may play important roles in the carcinogenesis of colorectal carcinoma.

Topics: Adenocarcinoma; Adenoma; Adenomatous Polyposis Coli Protein; beta Catenin; Colorectal Neoplasms; Cyclin D1; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Intestinal Mucosa; Precancerous Conditions; Proto-Oncogene Proteins c-myc

To analyze the cell kinetics of ulcerative colitis (UC)-associated dysplasia, cyclin A, cyclin D1, cyclin E, cdk2, cdk4, p21(Waf1), and p27(Kip1) were immunohistochemically examined, in comparison with sporadic tubular adenomas. Immunohistochemical labeling indices for each marker in formalin-fixed paraffin-embedded tissue sections were assessed in a total of 23 low-grade dysplasias, 27 high-grade dysplasias, and 14 invasive adenocarcinomas associated with UC. For comparison, 21 sporadic tubular adenomas with low-grade dysplasia, 33 with high-grade dysplasia, and 21 invasive adenocarcinomas were also examined. In UC-associated dysplasias, cyclin A and p27(Kip1) were located in the lower parts of the crypts and p21(Waf1) in the upper regions. In tubular adenomas, cyclin A, cdk4, p27(Kip1), and p21(Waf1) were all expressed in the upper parts of the crypts. The expression levels of cyclin D1, cyclin E, and cdk2 were low. The cell proliferation zone in UC-associated dysplasia is located towards the bases of the crypts with the strong expression of cyclin A and p27(Kip1), in contrast to tubular adenomas, which have their cell proliferation zone in the upper parts of neoplastic crypts. It is considered that tumorigenesis with UC-associated dysplasia is of the bottom-up type, related to altered expression of cyclin A and p27(Kip1).

Topics: Adenoma; Cell Proliferation; Colitis, Ulcerative; Colorectal Neoplasms; Cyclin A; Cyclin D1; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase Inhibitor p27; Cyclin-Dependent Kinases; Intracellular Signaling Peptides and Proteins; Models, Biological

Dysregulation of the centrosome complex and chromosomal segregation has been associated with aneuploid cells and aggressive solid tumours, but the relevance of this mechanism to the adenoma-carcinoma sequence of sporadic colorectal cancer (sCRC), especially tumours showing chromosomal instability (CIN), is still unknown. In a series of matching normal epithelial cells (n = 41), dysplastic cells (n = 18), and invasive carcinoma cells (n = 41) from cases with sCRC, mRNA levels of the centrosomal kinase Aurora-A/STK15 and the chromosomal passenger- and cell cycle-associated molecules Incenp, Survivin, Mad-2, and Cyclin-D1 were therefore measured with specific reference to the type of genetic instability. Compared with normal epithelium, significant up-regulation of mRNAs was already present for Aurora-A/STK15 (p = 0.0313) in dysplastic cells and for all investigated markers in invasive carcinoma. Whereas Aurora-A/STK15 mRNA levels were similarly up-regulated in dysplastic and invasive carcinoma cells (p = 0.0797), Survivin (p = 0.0046) and Cyclin-D1 (p = 0.0017) mRNA levels increased from dysplastic to invasive carcinoma cells. In carcinomas, Incenp mRNA correlated with T category (p = 0.0149), and Survivin (p = 0.0382) and Cyclin-D1 (p = 0.0185) were associated with tumour differentiation. Importantly, a significantly higher (p = 0.0419) fold-change of Aurora-A/STK15 mRNA (p = 0.0419), but not Incenp, Survivin, Mad-2 or Cyclin-D1, was observed in sCRC cases with CIN (n = 29) when compared with tumours showing microsatellite instability (MIN, n = 10). The present data are the first to show an early increase of the centrosomal kinase Aurora-A/STK15 in the adenoma-carcinoma sequence of sCRC. The regulation of this kinase differs in CIN- and MIN-type sCRCs and the pattern of changes is different from those of the cell-cycle-associated markers Survivin, Mad-2, and Cyclin-D1. This reinforces the concept of preferential dysregulation of the centrosome complex in CIN-type (aneuploid), compared with MIN-type, sporadic colorectal cancers and may influence the response to and efficiency of novel therapeutics targeting Aurora kinases.

Topics: Adenoma; Adult; Aneuploidy; Aurora Kinase A; Aurora Kinases; Calcium-Binding Proteins; Carcinoma; Case-Control Studies; Cell Cycle; Cell Cycle Proteins; Centrosome; Chromosomal Proteins, Non-Histone; Colorectal Neoplasms; Cyclin D1; Disease Progression; Epithelial Cells; Female; Gene Expression Regulation, Neoplastic; Genetic Markers; Humans; Immunohistochemistry; Inhibitor of Apoptosis Proteins; Mad2 Proteins; Male; Microsatellite Repeats; Microtubule-Associated Proteins; Neoplasm Proteins; Precancerous Conditions; Protein Serine-Threonine Kinases; Repressor Proteins; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Statistics, Nonparametric; Survivin

Cyclin D1 (CCND1) plays a key role in cell cycle control, particularly in the transition from G1 to S phase, which is regulated by cyclin-dependent kinases. A common adenine to guanine polymorphism (A870G) in the CCND1 gene has been associated with a longer-life protein and an increased risk of colorectal cancer and adenoma in some studies. Among subjects with hereditary nonpolyposis colorectal cancer, the A870G polymorphism has also been associated with a younger age of onset of colorectal cancer. We analysed 181 colorectal cancer cases and 475 matched controls and 524 adenoma cases and 517 matched controls within women in the Nurses' Health Study (NHS) cohort, 171 colorectal cancer cases and 347 matched controls and 372 adenoma cases and 712 matched controls nested within men in the Health Professionals' Follow-Up Study (HPFS) cohort, and 258 colorectal cancer cases and 415 matched controls within men in the Physicians' Health Study (PHS) cohort to assess the risk associated with the CCND1 A870G genotype. Moreover, we assessed whether CCND1 genotype modified the effect of a sporadic (nonsyndromic) family history of colorectal cancer as well as the effect of other dietary and lifestyle risk factors for colorectal cancer and adenoma. In all cohorts combined, the CCND1 polymorphism did not show statistically significant associations to risk of colorectal cancer (odds ratio (OR) for A allele carriers, 1.04; 95% confidence interval (95% CI), 0.82-1.32) or adenoma (OR, 0.96; 95% CI, 0.79-1.18). The CCND1 A870G genotype was associated with a modest, although nonsignificantly elevated risk of colorectal cancer (OR, 1.59; 95% CI, 0.98-2.57) in women. In contrast, the polymorphism was not associated with increased risk of adenoma in either men or women. Among participants with the A870G genotype, a family history of colorectal cancer conferred a substantially greater risk of colorectal cancer in the women (P for interaction=0.06) and adenoma in the men (P for interaction=0.02). Current postmenopausal hormone (PMH) use was associated with a significant reduction in the risk of colorectal cancer and adenoma among women with the A870G genotype, whereas there was no effect of PMH use among those with the GG genotype. The CCND1 polymorphism appeared to confer a modest elevation in the risk of colorectal cancer among women. Moreover, the A870G genotype may enhance the protective effect of postmenopausal oestrogen use on the development of colorectal neoplasia.

Topics: Adenoma; Adult; Case-Control Studies; Colorectal Neoplasms; Cyclin D1; Diet; Female; Genotype; Humans; Life Style; Middle Aged; Polymorphism, Genetic; Risk Factors

Increased NF-kappaB-mediated transcription has been extensively linked to tumorigenesis and can be stimulated by deregulated Rac1 signaling. For example, the overexpression of Rac1b, a highly activated splicing variant of Rac1 with increased expression in colorectal tumors, stimulates NF-kappaB-mediated G1/S progression and cell survival, and was shown to promote cell transformation and epithelial-mesenchymal transition. Here we show evidence of further complexity between Rac1b and Rac1 signaling toward NF-kappaB in colorectal cells. Consistent with data from other cell types we demonstrate that both Rac1 and Rac1b stimulate transcriptional activation from reporter genes driven by NF-kappaB motifs or the cyclin D1 promoter in an IkappaBalpha- and reactive oxygen species-dependent manner. However, we found that in colorectal cells Rac1, but not Rac1b, induces nuclear translocation of RelB and p52, activates transcription from a RelB-specific reporter, and can be isolated in a complex with endogenous RelB and its inhibitor NF-kappaB2/p100. In addition, Rac1 colocalizes at the plasma membrane with RelB, p100, and cullin-1, a core subunit of the E3 ubiquitin ligase that marks p100 for proteolytic processing to p52. Interestingly, this Rac1-specific pathway is not mediated via the production of reactive oxygen species. These data provide evidence that both Rac1 and Rac1b activate the canonical RelA-IkappaBalpha pathway, whereas Rac1 further stimulates NF-kappaB by inducing the RelB-NF-kappaB2/p100 pathway. The RelB pathway was reported to down-regulate canonical NF-kappaB activation during the inflammatory response, suggesting that increased levels of Rac1b in colorectal tumors may promote tumorigenesis by stimulating canonical NF-kappaB signaling while circumventing a negative feedback from the RelB pathway.

Topics: Cell Cycle Proteins; Cell Line, Tumor; Cell Membrane; Chemical Precipitation; Colorectal Neoplasms; Consensus Sequence; Cullin Proteins; Cyclin D1; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Humans; I-kappa B Kinase; NF-kappa B p52 Subunit; Promoter Regions, Genetic; Protein Transport; rac1 GTP-Binding Protein; Reactive Oxygen Species; Transcription Factor RelA; Transcription Factor RelB; Transcription, Genetic

Cyclin D1 is a regulatory protein involved in the cell cycle of both normal and neoplastic cells. Polymorphism of this gene at codon 242 in exon 4 has impacts on risk of the early-age onset in several malignant neoplasms, including colorectal cancer. This investigation was designed to evaluate the effect of cyclin D1 gene polymorphism on the risk of colorectal cancer in Chinese migrants of the Taiwanese population.. We enrolled 831 primary sporadic colorectal cancer patients as the study group and 1,052 age-gender matched healthy individuals as the control group (1,883 total cases) for present study. Cyclin D1 genotypes (AA, AG, GG) were determined using PCR-RFLP analysis on genomic DNA.. The frequency of G allele was 39.89 percent and 40.96 percent in the study group and the control group, respectively (P = 0.02). The patients were divided into three age groups for statistical analysis. The younger male patients had a higher frequency of AA/AG genotype compared with the controls (odds ratio, 2.75; 95 percent confidence interval, 1-7.9). The effect of AA/AG genotype on colorectal cancer risk was statistically significant for male patients (odds ratio, 1.34; 95 percent confidence interval, 1.04-1.72), but such phenomenon was not observed in female patients.. Our study suggests that the effect of cyclin D1 gene polymorphism on colorectal cancer risk is only observed in males and AA/AG genotype of cyclin D1 gene is associated with a higher risk of colorectal cancer in the younger patients within the Taiwanese population.

Topics: Adult; Aged; Aged, 80 and over; Alleles; Case-Control Studies; China; Colorectal Neoplasms; Cyclin D1; Female; Genetic Predisposition to Disease; Genotype; Humans; Logistic Models; Male; Middle Aged; Polymerase Chain Reaction; Polymorphism, Restriction Fragment Length; Sex Factors; Taiwan; Transients and Migrants

Colorectal carcinogenesis is initiated mainly by aberrant activation of the Wnt signaling pathway, caused by mutation of either APC or beta-catenin (CTNNB1) gene. Poly(ADP-ribose) polymerase-1 (PARP-1) is a highly conserved nuclear enzyme, which binds tightly to DNA and plays a role in DNA repair, recombination, proliferation and genomic stability. It has recently been shown that PARP-1 is a novel co-activator of TCF-4/beta-catenin-evoked gene transactivation and may play a role in colorectal carcinogenesis. The aim of this study was to examine the PARP-1 expression and determine whether it is correlated with the expression of beta-catenin and its target genes such as c-myc, cyclin D1 and matrix metalloproteinase (MMP)-7 in the early stage of sporadic colorectal carcinogenesis. Using the semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), 91 colorectal tumours, including 65 adenomas and 26 submucosal (pT1) cancers, were analysed for the expression of PARP-1, beta-catenin, c-myc, cyclin D1 and MMP-7. Immunohistochemical analysis of PARP-1 and beta-catenin was also performed. PARP-1 mRNA overexpression was detected in 64 (70.3%) of the 91 tumours. PARP-1 overexpression was significantly correlated with tumour size and histopathology. Overexpression of beta-catenin, c-myc, cyclin D1 and MMP-7 mRNA expression was observed in 39.6%, 78.0%, 83.5% and 72.5% of the 91 tumours, respectively. PARP-1 overexpression was correlated significantly with overexpression of beta-catenin, c-myc, cyclin D1 and MMP-7. Correlation of PARP-1 expression with beta-catenin overexpression was also demonstrated by immunohistochemistry. The results suggest that PARP-1, in conjunction with beta-catenin, c-myc, cyclin D1 and MMP-7, plays an important role in the early stage of colorectal carcinogenesis.

Topics: Adenocarcinoma; Adenoma; beta Catenin; Colorectal Neoplasms; Cyclin D1; Female; Genes, myc; Humans; Immunohistochemistry; Male; Matrix Metalloproteinase 7; Middle Aged; Poly (ADP-Ribose) Polymerase-1; Poly(ADP-ribose) Polymerases; Proto-Oncogene Proteins c-myc; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Neoplasm

Peptidyl prolyl cis-trans isomerase (Pin1) isomerizes only phosphorylated serine or threonine residues preceding proline in certain proteins and affects the protein function. Pin1 interacts with many signaling pathways, including Wnt signaling pathway that is crucial for colorectal tumorigenesis. Pin1 promotes cyclin D1 over-expression directly or through the stabilization of beta-catenin. Pin1 is over-expressed in some cancers such as prostate and breast cancers. This study aimed to determine whether Pin1 plays a role in colorectal tumorigenesis through the upregulation of beta-catenin and cyclin D1.. Immunohistochemical analyses were performed on 105 colorectal cancer tissue samples using anti-Pin1, anti-beta-catenin, and anti-cyclin D1 antibodies. We examined the relationships between Pin1 expression and clinicopathological factors, prognosis, and beta-catenin/cyclin D1 expression.. High Pin1 expression was observed in 40 cases (38%) and positively correlated with histological type (P=0.0240), depth of invasion (P=0.0051), and staging (P=0.0027) of colorectal tumors. High Pin1 expression was also correlated with the over-expressions of both beta-catenin (P=0.0225) and cyclin D1 (P=0.0137).. These results suggest that Pin1 plays an important role in colorectal tumorigenesis, presumably by increasing beta-catenin and cyclin D1 expressions.

Topics: Aged; beta Catenin; Colorectal Neoplasms; Cyclin D1; Disease Progression; Female; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Lymphatic Metastasis; Male; Neoplasm Invasiveness; Peptidylprolyl Isomerase; Signal Transduction; Survival Rate

Tissue microarray (TMA) holds promise as a high-throughput method for the analysis of biomarkers in tissue specimens. The validity and reliability of this method, however, may vary for different biomarkers in different tissue specimens.. In this study, we evaluated the validity and reliability of using TMA to assess biomarkers in colorectal adenomas.. Sixty-three consecutive patients with colorectal adenomas were recruited in this study. Two TMA blocks were constructed using four punches from each adenoma (one periphery, one deep, and two middle zones). The immunostaining of five markers (Ki-67, cyclin D1, beta-catenin, cyclooxygenase-2, and epidermal growth factor receptor) was analyzed, and the concordance between data obtained from TMAs and standard whole-tissue sections was evaluated by Spearman's correlation and kappa analysis.. Colorectal adenoma exhibited zonal, heterogeneous expression patterns for all five markers. The concordance rates for the semiquantitative evaluation of markers between data from TMAs and whole sections ranged from 87% to 93% with corresponding kappa statistics of 77% to 90%. In addition, both quantitative and semiquantitative methods were used to score TMA sections, and good correlations between these two methods were shown for all five markers with intraclass correlation coefficients ranging from 0.5 to 0.8.. Our study indicates that TMA can be used to reliably assess the expression levels of Ki-67, cyclin D1, beta-catenin, cyclooxygenase-2, and epidermal growth factor receptor in colorectal adenoma tissues.

Topics: Adenoma; Aged; beta Catenin; Colorectal Neoplasms; Cyclin D1; Cyclooxygenase 2; ErbB Receptors; Female; Humans; Immunohistochemistry; Ki-67 Antigen; Male; Middle Aged; Tissue Array Analysis

As gastrin may play a role in the pathophysiology of gastrointestinal (GI) malignancies, the elucidation of the mechanisms governing gastrin-induced proliferation has recently gained considerable interest. Several studies have reported that a large percentage of colorectal tumours overexpress or stabilise the beta-catenin oncoprotein. We thus sought to determine whether gastrin might regulate beta-catenin expression in colorectal tumour cells. Amidated gastrin-17 (G-17), one of the major circulating forms of gastrin, not only enhanced beta-catenin protein expression, but also one of its target genes, cyclin D1. Furthermore, activation of beta-catenin-dependent transcription by gastrin was confirmed by an increase in LEF-1 reporter activity, as well as enhanced cyclin D1 promoter activity. Finally, G-17 prolonged the tau(1/2) of beta-catenin protein, demonstrating that gastrin appears to exert its mitogenic effects on colorectal tumour cells, at least in part, by stabilising beta-catenin.

Topics: Animals; beta Catenin; Blotting, Northern; Blotting, Western; Cell Line, Tumor; Colorectal Neoplasms; Cyclin D1; Cytoskeletal Proteins; Gastrins; Mice; Promoter Regions, Genetic; Trans-Activators; Transcription, Genetic

Recent studies have implicated cyclin D1 G870A single-nucleotide polymorphism (SNP) in susceptibility to and early onset of colorectal cancers (CRC). We investigated the role of cyclin D1 G870A SNP in Singapore CRC patients without dominant family history by genotyping 254 patients and 101 controls. The risk of cancer for AA individuals was less than half that of GG individuals (odds ratio (OR) 0.41; 95% confidence interval (CI) 0.18-0.96). Furthermore, AA and AG patients whose tumours were Dukes C and D (OR 0.38; 95% CI 0.17-0.83), poorly differentiated (OR 0.28; 95% CI 0.09-0.84) and left-sided (OR 0.45; 95% CI 0.21-0.98) were associated with significantly lower risk than GG patients. Young (aged 50 years or less) GG patients had a 5-year lower mean age at onset than AA/AG patients (P=0.02). Young male GG patients had worse disease-specific survival than AA/AG patients (P=0.002). Thus, contrary to Caucasians, the GG (rather than AA) genotype is associated with increased susceptibility and advanced CRC in Singapore patients, suggesting a more complex relationship between the SNP and CRC risk, possibly modulated by population differences.

Topics: Adult; Aged; Aged, 80 and over; Colorectal Neoplasms; Cyclin D1; Female; Gene Frequency; Genetic Predisposition to Disease; Genotype; Heterozygote; Humans; Male; Middle Aged; Polymorphism, Restriction Fragment Length; Polymorphism, Single Nucleotide; Reverse Transcriptase Polymerase Chain Reaction; Risk Factors; Survival Analysis

Cyclooxygenase and its derived prostaglandin E2 (PGE2) have been shown to stimulate the growth of cancer cells and promote tumor angiogenesis. Here, we show that PGE2 activated the beta-catenin/T cell factor-dependent transcription in colon cancer cells through the cAMP/protein kinase A pathway. The expression of cyclin D1 and vascular endothelial growth factor was induced by PGE2 in LS-174T cells. Moreover, PGE2 and mutated beta-catenin stimulated the transcription of cyclin D1 and vascular endothelial growth factor in a synergistic fashion. Mechanistically, PGE2 increased the phosphorylation of glycogen synthase kinase-3 and consequently accumulated beta-catenin. In addition, PGE2 induced the expression of T cell factor-4 transcription factor, which formed transcriptionally active complex with beta-catenin. In animal experiments, administration of 16,16-dimethyl PGE2 strongly increased the expression of cyclin D1 and vascular endothelial growth factor in APC(min/+) mouse polyps. Thus, our results provide a novel mechanism, suggesting that cyclooxygenase-2/PGE2 may exert pro-oncogenic actions through stimulating the beta-catenin/T cell factor-mediated transcription, which plays critical roles in colorectal carcinogenesis.

Topics: Animals; beta Catenin; Blotting, Northern; Cell Line, Tumor; Colonic Neoplasms; Colorectal Neoplasms; Cyclin D1; Cyclooxygenase 2; Cytoskeletal Proteins; Dinoprostone; DNA-Binding Proteins; Dose-Response Relationship, Drug; Enzyme-Linked Immunosorbent Assay; Glycogen Synthase Kinase 3; Hepatocyte Nuclear Factor 1; Hepatocyte Nuclear Factor 1-alpha; Humans; Immunoblotting; Luciferases; Membrane Proteins; Mice; Models, Biological; Nuclear Proteins; Phosphorylation; Prostaglandin-Endoperoxide Synthases; Protein Binding; RNA; Time Factors; Trans-Activators; Transcription Factors; Transcription, Genetic; Transfection; Vascular Endothelial Growth Factor A

Cyclin D1 is postulated to be a target of the canonical Wnt pathway and critical for intestinal adenoma development. We show here that, unlike cyclin D1 reporter assays, endogenous cyclin D1 levels are not affected following antagonism of the Wnt pathway in vitro, nor is cyclin D1 immediately up-regulated following conditional loss of Apc in vivo. Cyclin D1 levels do, however, increase in a delayed manner in a small subset of cells, suggesting such up-regulation occurs as a secondary event. We also analyzed the immediate consequences of Apc loss in a cyclin D1(-/-) background and failed to find any cyclin D1-dependent phenotypes. However, we did observe elevated cyclin D1 expression in lesions developing 20 days after Apc loss. In these circumstances, all adenomas (but not smaller lesions) showed cyclin D1 up-regulation. Finally in a smaller study, we analyzed whether cyclin D1 deficiency affected adenoma formation 20 days following induced loss of Apc. Unlike AhCre(+) Apc(fl/fl) mice (which all developed adenomas), doubly mutant AhCre(+) Apc(fl/fl) cyclin D1(-/-) mice only developed small lesions. Taken together, this argues that cyclin D1 up-regulation in intestinal neoplasia is important for tumor progression rather than initiation.

Topics: Adenoma; Animals; beta Catenin; Cell Line, Tumor; Colorectal Neoplasms; Cyclin D1; Cytoskeletal Proteins; Gene Deletion; Gene Expression Regulation, Neoplastic; Genes, APC; Integrases; Intestinal Neoplasms; Mice; Mice, Inbred C57BL; Phenotype; Trans-Activators

To report the expression of cyclin D1 protein and its gene in a series of colorectal adenocarcinoma.. One hundred and eleven specimens of colorectal carcinomas and adjacent normal colorectal mucosa were investigated by staining with a monoclonal antibody against cyclin D1 and by RT-PCR.. Expression of CCND1 gene was found in 54 out of 111 cases of colorectal cancers, while in normal mucosa the expression of this gene was not observed. Cyclin D1 protein expression was checked in the same group of adenocarcinoma cases. Presence of this protein was observed in 69 cases and for 43 of them also expression of its gene was found. Dependence between the presence of protein and the gene expression was statistically significant (p=0.0002). In the group of cases where CCND1 gene expression was detected, high level of its protein expression was found in 20 cases. The CCND1 gene expression was associated with metastases to lymph nodes (p=0.0181) and also with distant metastasis (p=0.0204).. The combined measurement of both the gene and its protein product, is an important contribution to the study of molecular markers in histological material.

Topics: Adenocarcinoma; Aged; Antibodies, Monoclonal; Biomarkers, Tumor; Case-Control Studies; Colorectal Neoplasms; Cyclin D1; Female; Gene Expression Profiling; Humans; Immunohistochemistry; Male; Middle Aged; Neoplasm Metastasis; Reverse Transcriptase Polymerase Chain Reaction

Colorectal signet-ring cell carcinoma (SRCC) is a rare cancer and the prognosis is usually very poor. The biologic pathways involved in its oncogenesis are unknown. beta-catenin, a key target in the Wnt-signaling pathway, is recognized to play an important role in the carcinogenesis in conventional colorectal carcinoma. This study explores the involvement of Wnt-signaling molecules beta-catenin and cyclin D1, cell cycle regulators cyclin D3, proliferative index Ki-67, apoptotic index, and angiogenic indicator CD31 in 20 colorectal SRCC paraffin-embedded specimens. Results showed that there were 2 specimens with nuclear beta-catenin and higher expression of cyclin D1 than the remaining 18 specimens. Surprisingly, those 2 patients had a much shorter survival of 6 months than the remaining 15 patients, who had around 24 months. Moreover, all colorectal SRCC specimens had an overexpression of cyclin D1, cyclin D3, and Ki-67, as well as much more angiogenesis and apoptosis than adjacent normal epithelial tissues. The authors make the preliminary comment that nuclear beta-catenin is a rare phenomenon in colorectal SRCC, but the involvement of it may indicate a worse prognosis with shorter survival than colorectal SRCC without nuclear beta-catenin expression. Besides, overexpression of cyclin D1, cyclin D3, Ki-67, and increased angiogenesis and apoptosis may play a vital role in promoting colorectal SRCC development.

Topics: Apoptosis; Carcinoma, Signet Ring Cell; Cell Nucleus; Colorectal Neoplasms; Cyclin D1; Cyclin D3; Cyclins; Gene Expression Regulation, Neoplastic; Humans; Ki-67 Antigen; Neovascularization, Pathologic; Prognosis; Survival Rate

Elevated beta-catenin levels in human colorectal cancer (CRC) cells lead to increased trans-activation of 'protumorigenic' beta-catenin/T-cell factor (TCF) target genes such as cyclin D1. Therefore, possible targets for the anti-CRC activity of nonsteroidal anti-inflammatory drugs (NSAIDs) are beta-catenin and catenin-related transcription (CRT). We tested the antiproliferative activity and the effects on levels of beta-catenin and cyclin D1 protein, as well as CRT (measured using a synthetic beta-catenin/TCF-reporter gene [TOPflash]), of a panel of NSAIDs (indomethacin, diclofenac, sulindac sulphide and sulphone, rofecoxib; range 10-600 microM) on SW480 human CRC cells in vitro. Following NSAID treatment, there was no consistent relationship between reduced cell proliferation, induction of apoptosis and changes in beta-catenin protein levels or CRT. All the NSAIDs, except rofecoxib, decreased nuclear beta-catenin content and cyclin D1 protein levels in parallel with their antiproliferative activity. However, cyclin D1 downregulation occurred prior to a decrease in total beta-catenin protein levels and there was no correlation with changes in CRT, suggesting the existence of CRT-independent effects of NSAIDs on cyclin D1 expression. In summary, NSAIDs have differential effects on beta-catenin protein and CRT, which are unlikely to fully explain their effects on cyclin D1 and their antiproliferative activity on human CRC cells in vitro. British Journal of Cancer (2004) 91, 153-163. doi:10.1038/sj.bjc.6601901 www.bjcancer.com Published online 8 June 2004

Topics: Anti-Inflammatory Agents, Non-Steroidal; Apoptosis; beta Catenin; Cell Division; Colorectal Neoplasms; Cyclin D1; Cytoskeletal Proteins; Down-Regulation; Humans; Trans-Activators; Transcription, Genetic; Tumor Cells, Cultured

High expression of thymidylate synthase (TS) is allegedly associated with the chemoresistance to 5-fluorouracil (5-FU) in colorectal cancers. However, low TS expression does not necessarily imply chemosensitivity. Inactivation of p16(INK4a) correlates with poor prognosis in various cancers. We immunohistochemically evaluated the relationship between the expression of TS, p16(INK4a), CDK4 and cyclin D1 and the effect of 5-FU-based chemotherapy in colorectal cancers. After antigen retrieval, immunoperoxidase staining was performed on the paraffin-embedded, biopsy and surgical specimens of 37 advanced colorectal cancers preoperatively treated with peroral administration of 5-FU derivatives. As a control group, 31 colorectal cancers without preoperative treatment were analyzed. High TS expression was found in 23 (74%) of 31 tumors resected from histological non-responders and in 19 (61%) of 31 controls but in none of six responders. High p16(INK4a) expression was seen in 83% of the responders, 52% of the non-responders and 32% of the controls. The TS-low/p16(INK4a)-high phenotype was noted in 83% of the responders, but only in 3% of the non-responders (P = 0.0001). Induction of p16(INK4a) expression after chemotherapy was predominantly seen in the responders. Neither CDK4 nor cyclin D1 expression was related to the chemotherapeutic effects. In conclusion, the combination of low expression of TS and induction of p16(INK4a) after chemotherapy can be important indicators of the sensitivity to 5-FU-based chemotherapy in colorectal cancers.

Topics: Adenocarcinoma; Administration, Oral; Adult; Aged; Aged, 80 and over; Antimetabolites, Antineoplastic; Biomarkers, Tumor; Colorectal Neoplasms; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase Inhibitor p16; Cyclin-Dependent Kinases; Female; Fluorouracil; Humans; Male; Middle Aged; Preoperative Care; Proto-Oncogene Proteins; Thymidylate Synthase; Treatment Outcome

A striking feature of colon tumors is the significant reduction of goblet cells. Although targeted deletion of Math1 in mice leads to a loss of intestinal secretory cells, including goblet cells, the role of Hath1 in colon tumorigenesis remains unknown. Here we report that Hath1, the human ortholog of Math1, was dramatically down-regulated in colon tumor samples and colon cancer cell lines. Overexpression of Hath1 in HT29, an aggressive colon cancer cell line, resulted in a significant inhibition on cell proliferation, anchorage-independent growth in soft agar and, more importantly, growth of human colon cancer cell xenografts in athymic nude mice. Such inhibition was accompanied by altered expression of a goblet cell differentiation marker, MUC2, and cell cycle regulators cyclin D1 and p27kip1. Hath1 expression also was up-regulated on inhibition of the Wnt pathway, which has been well implicated in colon tumorigenesis. Hence, this study suggests that Hath1 may be a novel factor downstream of the Wnt pathway capable of suppressing anchorage-independent growth of colon cancer cell lines. More importantly, this study is the first to establish a link between down-regulation of Hath1 expression and colon tumorigenesis.

Topics: Adenocarcinoma; Animals; Basic Helix-Loop-Helix Transcription Factors; Cell Adhesion; Cell Cycle Proteins; Cell Differentiation; Cell Division; Colorectal Neoplasms; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p27; DNA-Binding Proteins; Down-Regulation; Gene Expression Regulation, Neoplastic; Goblet Cells; HT29 Cells; Humans; Mice; Mice, Nude; Mucin-2; Mucins; Proto-Oncogene Proteins; Signal Transduction; Transfection; Transplantation, Heterologous; Tumor Suppressor Proteins; Wnt Proteins

Specific ligands of the peripheral benzodiazepine receptor (PBR) have been shown to induce both apoptosis and G1/G0 cell cycle arrest in colorectal cancers. The signaling pathways leading to cell cycle arrest are still unknown. Using cDNA array technology, we identified signaling molecules involved in cell cycle arrest induced by the PBR ligands FGIN-1-27 and PK 11195. Differential gene expression was confirmed by semi-quantitative RT-PCR or Western blot analysis of gene products. The PBR ligand-mediated signaling involved the upregulation of the cyclin-dependent kinase inhibitors p21WAF1/CIP1 and p27Kip1, cdc16, and the cell cycle inhibitors gadd45 and gadd153, the downregulation of the cyclins D1 and B1, as well as the inactivation of ERK1/2. The p21-deficient colorectal cancer cell line HCT116 p21-/- was significantly less sensitive to PBR ligands than the parental HCT116 wild-type cells, demonstrating the functional involvement of p21WAF1/CIP1 in PBR ligand-mediated G1 arrest. This study thus revealed PBR ligand-triggered signaling pathways leading to cell cycle arrest. Moreover, we showed the functional implication and interaction of differentially expressed gene products and provided a model of signaling pathways involved in PBR ligand-induced G1 arrest. These results form the basis for future PBR ligand-mediated therapeutic approaches.

Topics: Blotting, Western; Cell Cycle; Cell Cycle Proteins; Cell Line, Tumor; Colorectal Neoplasms; Cyclin B; Cyclin B1; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase Inhibitor p27; DNA, Complementary; Dose-Response Relationship, Drug; Down-Regulation; G1 Phase; Gene Expression Regulation; Humans; Ligands; MAP Kinase Signaling System; Models, Biological; Oligonucleotide Array Sequence Analysis; Poly A; Receptors, GABA-A; Resting Phase, Cell Cycle; Reverse Transcriptase Polymerase Chain Reaction; RNA; RNA, Messenger; Signal Transduction; Time Factors; Tumor Suppressor Proteins; Up-Regulation

Germline mutations in APC tumor suppressor gene are responsible for familial adenomatous polyposis (FAP). A major role of these genetic changes is the constitutive activation of beta-catenin-Tcf-4 mediated transcription of nuclear target genes, but other cellular functions can be misregulated. To assess how different APC mutations can drive the early steps of colonic tumorigenesis, we studied the effect of 10 different germline-truncating alterations on the phenotype of the corresponding adenomas. A significant reduction of apoptosis, uncoupled with an increased c-myc and cyclin-D1 expression, was seen with a frameshift mutation on codon 1383, in the 20-aa repeats of the beta-catenin degradation domain, independent of a somatic alteration on the wild-type allele. The decreased apoptotic level was associated with a higher incidence of cancerization. No other APC mutation was linked with a similar effect, even in presence of a somatic allelic loss. These findings suggest that mutations in critical sites of the beta-catenin degradation domain of APC gene can convey a selective advantage to the colonic neoplastic clones by altering the apoptotic surveillance rather than enhancing the beta-catenin-Tcf-4 transcription of growth-promoting genes.

Topics: Adenoma; Adenomatous Polyposis Coli; Adult; Apoptosis; beta Catenin; Colorectal Neoplasms; Cyclin D1; Cytoskeletal Proteins; DNA Mutational Analysis; DNA Primers; DNA, Neoplasm; Female; Genes, APC; Germ-Line Mutation; Humans; Immunohistochemistry; In Situ Nick-End Labeling; Male; Proto-Oncogene Proteins c-myc; Reverse Transcriptase Polymerase Chain Reaction; RNA, Neoplasm; Trans-Activators

A large number of epidemiological studies have shown that regular use of aspirinor other nonsteroidal anti-inflammatory drugs (NSAIDs) results in a 40-50% reduced risk of colorectal cancer (CRC). Furthermore, NSAIDs cause the regression of preexisting adenomas in patients with familial adenomatous polyposis and significantly inhibit tumor growth in animal models of CRC. To establish a CRC liver metastasis model, we implanted mouse colon tumor MC-26 cells into the splenic subcapsule of BALB/c mice, after which mice were given either standard chow or chow containing the cyclooxygenase (COX)-2-specific inhibitor rofecoxib, alone or in combination with the standard antineoplastic agents, 5-fluoruracil or irinotecan. After 14 days, mice that were given rofecoxib or irinotecan, but not 5-fluoruracil, had significantly smaller primary tumors and fewer metastases. Rofecoxib, at clinical anti-inflammatory plasma concentrations, enhanced the effects of both antineoplastic agents when used in combination. Biochemical analyses of the primary splenic tumor in rofecoxib-treated mice showed no alteration in COX-1 expression, but significant decreases in the expression of the tumor-promoting proteins COX-2, cyclin D1, cytosolic beta-catenin, matrix metalloproteinases-2 and -9, and vascular endothelial cell- derived growth factor. Rofecoxib also decreased growth-enhancing prostaglandin E(2) and tumor-suppressive interleukin-10, whereas antineoplastic interleukin-12 was increased. Two separate survival studies were performed. When mice were fed chow containing 0.01% rofecoxib beginning on day 0 after tumor cell implantation, which achieved clinical anti-inflammatory plasma concentrations, survival time was significantly longer compared with mice given control chow. After 30 days, mortality in the control group was 90%, whereas only one mouse (5%) treated with rofecoxib had died after 30 days. In the second survival study, all of the mice were initially fed with regular chow after tumor cell implantation. On day 7, mice were randomly divided into three dietary groups: control chow, low-dose (0.01%) rofecoxib chow, and high-dose (0.025%) rofecoxib chow. After 28 days, mortality was 100%, 20%, and 10% in control, low-, and high-dose rofecoxib fed groups, respectively. These studies demonstrate that rofecoxib decreases the growth and metastatic potential of CRC in mice through multiple mechanisms. These studies in mice also provide important information that supports the benef

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Antineoplastic Combined Chemotherapy Protocols; beta Catenin; Camptothecin; Cell Division; Colorectal Neoplasms; Cyclin D1; Cyclooxygenase 1; Cyclooxygenase 2; Cyclooxygenase 2 Inhibitors; Cyclooxygenase Inhibitors; Cytoskeletal Proteins; Dinoprostone; Drug Synergism; Endothelial Growth Factors; Fluorouracil; Intercellular Signaling Peptides and Proteins; Interleukin-10; Interleukin-12; Irinotecan; Isoenzymes; Lactones; Liver Neoplasms, Experimental; Lymphokines; Male; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Membrane Proteins; Mice; Mice, Inbred BALB C; Neoplasm Transplantation; Prostaglandin-Endoperoxide Synthases; Sulfones; Trans-Activators; Tumor Cells, Cultured; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

Dietary zinc is an important trace element in the body and is related to both cell proliferation and growth arrest. A recent study found that extracellular zinc-sensing receptors trigger intracellular signal transduction in HT-29 human colorectal cancer cells. However, the signaling mechanism causing this growth regulation by extracellular zinc is not clearly understood. At 10- and 100-microM levels of ZnCl2 treatment, HT-29 cell growth and proliferation increased and decreased, respectively, in a minimally serum-starved medium (MSSM). A lack of significant increase in intracellular zinc levels after zinc treatment suggested that this differential growth regulation of HT-29 cells by extracellular zinc is acquired by receptor-mediated signal transduction. Moreover, this zinc-induced growth regulation was differentially affected by PD-98059, suggesting the involvement of the ERK pathway. Transient ERK activation and subsequent cyclin D1 induction were observed on adding 10 microM ZnCl2 in MSSM in the presence of cell proliferation. On the other hand, prolonged ERK activity was observed with a subsequent increase of cyclin D1 and p21(Cip/WAF1) on adding 100 microM ZnCl2 in MSSM, and this was associated with nonproliferation. Moreover, this ERK activation and cyclin D1 and p21(Cip/WAF1) induction were abolished by PD-98059 pretreatment. The differential regulations of cell growth, ERK activities, and cyclin D1 and p21(Cip/WAF1) inductions were also observed in serum-enriched medium containing higher zinc concentrations. Therefore, differential cell cycle regulator induction occurs by a common ERK pathway in the differential growth regulation of HT-29 cells by extracellular zinc.

Topics: Animals; Cattle; Cell Division; Chlorides; Colorectal Neoplasms; Culture Media, Serum-Free; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Dose-Response Relationship, Drug; Enzyme Activation; Enzyme Inhibitors; Extracellular Fluid; Fetal Blood; Flavonoids; HT29 Cells; Humans; Mitogen-Activated Protein Kinases; Osmolar Concentration; Zinc; Zinc Compounds

It is unclear whether and how cyclin D1 and/or p21(WAF1/CIP1) dysregulation contribute to ulcerative colitis (UC)-related inflammation and colorectal carcinogenesis. Cases of quiescent UC (QUC; n = 15), active UC (AUC; n = 23), UC-related dysplasia (n = 35) and UC-related colorectal adenocarcinomas (CRCs; n = 11) were studied with cyclin D1 and p21(WAF1/CIP1) immunohistochemistry. The CRCs were also studied with beta-catenin, bcl2, and p53 immunohistochemistry, p53 and k-ras mutation analyses, and cyclin D1 gene fluorescence in situ hybridization. QUC showed cyclin D1 (negative/weak staining) and p21(WAF1/CIP1) (surface epithelial and upper-third crypt staining) expression similar to that of normal colorectum. Moderate or strong cyclin D1 immunostaining was seen in 9% of AUC cases, 40% of dysplasia cases, and 36% of UC-related CRCs. Although these carcinomas showed neither cyclin D1 gene amplification nor any association between k-ras mutation and cyclin D1 overexpression, the latter was closely related to nuclear beta-catenin expression. Increased lower-third crypt p21(WAF1/CIP1) staining was seen in 57% of AUC cases; decreased upper-third crypt p21(WAF1/CIP1) staining, in 23% of dysplasia cases; and absent or weak p21(WAF1/CIP1) staining, in 55% of UC-related CRCs. The latter change was always associated with p53 mutation but could not be related to p53 or bcl2 expression. In conclusion, AUC shows up-regulated cyclin D1 and p21(WAF1/CIP1) expression. Cyclin D1 up-regulation and p21(WAF1/CIP1) down-regulation occur early in UC-related carcinogenesis. Cyclin D1 up-regulation is less common in UC-related CRCs than in sporadic CRCs, and is related to beta-catenin nuclear signaling. p21(WAF1/CIP1) down-regulation is seen at an equal or higher frequency among UC-related CRCs compared with sporadic CRCs and is attributable to p53 mutation.

Topics: Adenocarcinoma; Biomarkers, Tumor; Cell Division; Colitis, Ulcerative; Colorectal Neoplasms; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; DNA Mutational Analysis; DNA, Neoplasm; Down-Regulation; Humans; Immunohistochemistry; Mutation; Tumor Suppressor Protein p53

Increased expression and/or activity of c-Met, the receptor protein tyrosine kinase for hepatocyte growth factor/scatter factor, occurs commonly during colon tumor progression. To examine potential roles for c-Met in promoting metastasis, we compared the colon tumor cell line KM12C with low metastatic potential to the isogenic variants KM,12L4 and KM12SM with high metastatic potential. KM12C cells express c-Met with low levels of tyrosine phosphorylation in the absence of HGF. The high metastatic cells express a c-Met that is constitutively tyrosine phosphorylated, they have increased colony formation, and are minimally responsive to HGF relative to the parental cells. Tyrosine-phosphorylated beta-catenin was constitutively associated with c-Met in the more metastatic cells, but was inducible only after HGF addition in the less metastatic cells. Functions mediated by beta-catenin, including cell-cell adhesion and migration, and activation of the tcf (T-cell factor) family of transcription factors, were also elevated in the more metastatic KM12SM and L4 cells. Furthermore, analysis of the known tcf transcriptional target genes, cyclin D1, c-Myc, and uPAR, demonstrated increased expression in the high metastatic cells, correlating with the levels of tcf activity. Collectively, these results suggest that endogenous activation of c-Met in highly metastatic KM12SM CRC cells results in increased survival and growth under anchorage independent conditions, increased in vitro migration, and elevated levels of tcf target genes. Thus, beta-catenin association with activated c-Met may contribute to a more aggressive liver metastatic phenotype of these cells.

Topics: beta Catenin; Blotting, Western; Cell Adhesion; Cell Movement; Colony-Forming Units Assay; Colorectal Neoplasms; Cyclin D1; Cytoskeletal Proteins; Gene Expression Regulation, Neoplastic; Hepatocyte Growth Factor; Humans; Luciferases; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Mutation; Phosphorylation; Promoter Regions, Genetic; Proto-Oncogene Proteins c-met; Trans-Activators; Transcriptional Activation; Tumor Cells, Cultured; Tyrosine

Signal transducers and activators of transcription 3 (Stat3) pathway can be activated by cytokines and growth factors, and activation of Stat3 is involved in modulating cell proliferation, differentiation, and apoptosis. Stat3 has been classified as an oncogene because Stat3 can mediate malignant transformation of cultured cells. This study was conducted to investigate the expression of Stat3 and its target gene products including Cyclin D1 and Bcl-x(L) in human colorectal carcinoma (CRC) tissues and cells, and to explore the mechanisms in tumorigenesis of CRC.. The expression of Stat3, p-Stat3, Cyclin D1, and Bcl-x(L) in 45 cases of cancerous tissues, adjacent normal tissues, and two colon cancer cell lines including SW480 and HCT116 was measured by Western blot analysis. The expression pattern of Stat3 and its activated form p-Stat3 was determined by immunohistochemical staining. The relationship of the expression of p-Stat3, Cyclin D1, and Bcl-x(L) in CRC with various clinicopathological characteristics was analyzed statistically.. The protein expression rates of p-Stat3, Cyclin D1, and Bcl-x(L) in colorectal cancer and adjacent normal mucosa were 57.8%, 64.4%, 68.9%, and 42.2%, 35.6%, 31.1%,respectively; and their protein levels (A value) were 114263+/-53598, 58321+/-24872, 71032+/-43425 in colorectal cancer and 55971+/-28762, 22563+/-11160, 37281+/-14622 in adjacent normal mucosa (P< 0.05). Overexpression of p-Stat3 was correlated with clinical stage and nodal metastasis in colorectal cancer (P = 0.026 and P= 0.018, respectively). Elevated levels of Cyclin D1 were associated with nodal metastasis (P= 0.041). It was found that Cyclin D1 was in a positive linear correlation fashion with p-Stat3 in tumor (r = 0.382, P< 0.05). Activated Stat3 was also detected in both colon cancer cell lines SW480 and HCT116.. Stat3 signaling pathway may play an important role in the tumorigenesis of colorectal carcinoma. Determination of Stat3 and its target gene products can be used to indicate the malignancy degree of colorectal carcinoma.

Topics: Adult; Aged; Aged, 80 and over; bcl-X Protein; Colorectal Neoplasms; Cyclin D1; DNA-Binding Proteins; Female; Humans; Male; Middle Aged; Proto-Oncogene Proteins c-bcl-2; Signal Transduction; STAT3 Transcription Factor; Trans-Activators

Cyclin D1, encoded by the CCND1 gene and activated by the adenomatous polyposis coli-beta-catenin-T-cell factor/lymphoid enhancing factor pathway, induces G(1) to S-phase cell cycle transition, promoting cell proliferation. A recently described codon 242, exon 4, G to A single nucleotide polymorphism (A870G) produces a longer half-life cyclin D1. To investigate whether CCND1 genotype influences risk for colorectal adenoma, we genotyped CCND1 by PCR/RFLP on 161 incident sporadic adenoma cases and 213 controls ages 30-74 years in a North Carolina colonoscopy-based case-control study. At least one polymorphic A allele was found in 68% of cases and 60% of controls. Having an A allele was associated with increased risk for adenoma: the age- and sex-adjusted odds ratio (OR) was 1.5 [95% confidence interval (CI) 1.0-2.4], a finding that was stronger for those whose adenomas were multiple (OR 2.9, 95% CI 1.4-6.0), larger (>or=1 cm; OR 2.4, 95% CI 1.2-4.8), had moderate to severe dysplasia (OR 2.1, 95% CI 1.1-3.8), or were in the right side of the colon (OR 3.6, 95% CI 1.3-10.0). Joint risk factor multivariate analyses revealed stronger positive associations among those who were older (>57 years; OR 2.8, 95% CI 1.4-5.5), male (OR 2.8, 95% CI 1.3-5.7), currently smoked (OR 2.7, 95% CI 1.3-5.7), or currently drank alcohol (OR 2.2, 95% CI 1.2-4.2) if they had an A allele and stronger inverse associations among those who used nonsteroidal anti-inflammatory drugs (OR 0.4, 95% CI 0.2-0.9) or had higher calcium intakes (OR 0.4, 95% CI 0.2-0.9) if they had no A allele. These data support the hypothesis that the CCND1 A870G polymorphism may increase risk for colorectal neoplasms.

Topics: Adenoma; Alleles; Cell Division; Colorectal Neoplasms; Cyclin D1; Epithelial Cells; Female; Genetic Predisposition to Disease; Humans; Intestinal Mucosa; Male; Middle Aged; Molecular Epidemiology; Polymorphism, Genetic

To evaluate and compare differences in the molecular genetics among high-risk (Ashkenazi Jews), intermediate-risk (Sephardic Jews) and low-risk (Palestinians) groups for colorectal cancer who live in the same geographical region.. The 1995-1996 records from the Tel Aviv Medical Center and Muqased hospital (East Jerusalem) randomly identified patients with colorectal cancer. There were 25 patients from each ethnic group. Epidemiological data were obtained from interviews with the patients and from their hospital charts. The levels of cyclin D1, beta-catenine, p27, p53, Ki-67 and Her-2/neu proteins were determined by immunohistochemistry. The main outcome measures were the association between gene expression and colorectal incidence in the different ethnic groups.. Ashkenazi Jews have the highest rate of colorectal cancer, and are diagnosed at an early stage compared with Palestinians (72% and 33% of the cases are in Dukes' A and B, respectively), and, hence, this may explain the better 5-year survival rate among this group. Sephardic Jews are diagnosed at a more advanced stage, the tumors are poorly differentiated and they lack p27. Palestinians have significantly higher cyclin D1 levels. There was a statistically significant inverse correlation between the expression of beta-catenine and cyclin D1, as well as p53 and p27 (P <0.05).. Increased expression of cyclin D1, p53, Ki-67, beta-catenine and Her-2/neu, and decreased expression of p27 may be important events in the three ethnic groups with colorectal cancer. The lower mortality rate among Ashkenazi Jews may be partially explained by their better molecular biology profile.

Topics: Aged; Aged, 80 and over; Arabs; beta Catenin; Biopsy, Needle; Colorectal Neoplasms; Cyclin D1; Cytoskeletal Proteins; Female; Genes, p53; Genetic Markers; Genetic Predisposition to Disease; Humans; Immunohistochemistry; Israel; Jews; Ki-67 Antigen; Male; Middle Aged; Molecular Epidemiology; Receptor, ErbB-2; Registries; Retrospective Studies; Sensitivity and Specificity; Trans-Activators

Mutations of the adenomatous polyposis coli tumor suppressor gene, or its downstream target beta-catenin, have been implicated in the initiation of most sporadic human colorectal epithelial neoplasms. These mutations, in turn, lead to aberrant nuclear accumulation of beta-catenin and subsequent activation of the beta-catenin/Tcf transcription factor complex. In vitro studies utilizing cultured human colon cancer cell lines have identified c-myc, cyclin D1 and fra-1 as target genes of beta-catenin/Tcf signaling. In our study, 12 cases of human colorectal adenocarcinomas were examined by Western immunoblotting analysis and immunohistochemical staining to specifically investigate whether the protein expression of these target genes was indeed altered in vivo by beta-catenin dysregulation. The results show that the protein level of beta-catenin was significantly increased in all 12 tumors (3.4 +/- 1.0-fold increase compared to the control normal mucosa by Western immunoblotting, p < 0.05), and this increase was associated with positive nuclear staining by immunohistochemistry in 10 cases. Increased levels of expression of cyclin D1 and Fra-1 proteins were also demonstrated in every tumor (9.0 +/- 2.7 and 3.3 +/- 0.9-fold increases compared to normal mucosa, respectively). Surprisingly, the protein level of c-Myc was significantly decreased in all tumors examined by 49 +/- 19% (p < 0.05), but the c-myc mRNA level was increased in 8 of 12 tumors when compared to that in normal mucosa by RT-PCR. Immunohistochemical staining performed on these carcinomas and additional 27 colorectal carcinomas further demonstrated that the protein expression level of c-Myc and beta-catenin nuclear localization were not correlated. Moreover, 15 of 20 colorectal adenomas exhibited positive nuclear beta-catenin immunostaining, among which 11 also exhibited increased c-Myc protein expression. These data thus support the notion that upregulation of cyclin D1 and Fra-1 in human colorectal adenocarcinomas is driven by abnormally expressed beta-catenin. However, the regulation of c-myc expression in colorectal tumors appears to be more complex. While dysregulated beta-catenin may cause a transcriptional upregulation of the c-myc gene, the c-Myc protein expression appears to be further regulated by a posttranscriptional mechanism(s) during the process of neoplastic progression.

Topics: Adenocarcinoma; Adult; Aged; beta Catenin; Blotting, Western; Cell Nucleus; Colorectal Neoplasms; Cyclin D1; Cytoskeletal Proteins; Female; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Male; Middle Aged; Mutation; Proto-Oncogene Proteins c-fos; Proto-Oncogene Proteins c-myc; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Trans-Activators

The stabilization and nuclear translocation of beta-catenin are early events in the majority of sporadic colorectal carcinomas (CRC). beta-catenin up-regulates c-Myc and cyclin D1, which antagonize the association of the cyclin-dependent kinase (Cdk) inhibitor, p27(kip1), with Cdk2, thus allowing cell cycle progression through G1 to S-phase. Lack of p27 is a significant predictor of poor survival in a series of 136 CRC specimens. A combination of molecules in the same pathway may be a better prognostic factor.. The expression of beta-catenin, c-Myc, and cyclin D1 in relation to patients' survival and clinicopathologic parameters in the same series was evaluated by immunohistochemistry.. Intense nuclear overexpression of beta-catenin, but not a lack of cell membrane or cytoplasmic expression, is a significant predictor of poor survival by both univariate (P = 0.0029) and multivariate analyses (P = 0.004, risk ratio =3.8), suggesting that beta-catenin is retained in the nucleus to function as an oncogene. None of the patients with high nuclear beta-catenin and low p27 expression survived 5 years or more whereas 65% of patients with all other combinations of the two markers survived (P < 0.0001). This combination is also a significant and independent prognostic factor (P = 0.001; risk ratio = 9.7). Overexpression of c-Myc is associated with higher mortality rates, but the expression of cyclin D1 has no prognostic significance.. The combined expression of beta-catenin and p27 can stratify patients into markedly different survival groups, possibly via their antagonistic effects on metastasis promotion.

Topics: Adenocarcinoma; beta Catenin; Biomarkers, Tumor; Cell Cycle Proteins; Cell Differentiation; Colorectal Neoplasms; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p27; Cytoskeletal Proteins; Enzyme Inhibitors; Female; Gene Expression Regulation, Neoplastic; Humans; Immunoenzyme Techniques; Male; Middle Aged; Neoplasm Staging; Prognosis; Proto-Oncogene Proteins c-myc; Retrospective Studies; Survival Rate; Trans-Activators; Tumor Suppressor Proteins

Indomethacin-induced G(1) arrest and apoptosis of human colorectal cancer (CRC) cells is associated with a dose-dependent decrease in beta-catenin protein levels. Beta-catenin plays a pivotal role in the WNT signalling pathway and its expression is frequently dysregulated at early stages of colorectal carcinogenesis. The objective of this study was to investigate the effect of indomethacin on catenin expression and downstream WNT signalling events in human CRC cells. Beta-catenin, gamma-catenin and T-cell factor (TCF) target gene (cyclin D1, c-MYC and PPARdelta) expression was studied following indomethacin treatment of SW480 and HCT116 cells. Cyclin D1 was used as a model TCF target gene for analysis of beta-catenin-TCF-4 DNA binding and trans-activation. Indomethacin treatment was associated with a specific decrease in beta-catenin (but not gamma-catenin) expression. Resulting TCF target gene expression was gene specific (cyclin D1, decreased; c-MYC, increased; PPARdelta, no significant change). Cyclin D1 promoter analysis revealed that indomethacin disrupted formation of a beta-catenin-TCF-4-DNA complex. Indomethacin-induced G(1) arrest and apoptosis is associated with specific beta-catenin down-regulation in human CRC cells in vitro. Differential expression of TCF target genes following indomethacin treatment implies complex effects on multiple genes which play an important role in colorectal carcinogenesis.

Topics: Acetylcysteine; beta Catenin; Blotting, Western; Colorectal Neoplasms; Cyclin D1; Cytoskeletal Proteins; Desmoplakins; DNA; Dose-Response Relationship, Drug; Down-Regulation; Electrophoretic Mobility Shift Assay; gamma Catenin; Gene Expression Regulation, Neoplastic; Humans; Indomethacin; Interleukin-2; Isoenzymes; Nitrobenzenes; Promoter Regions, Genetic; Protein Kinase C; Protein Kinase C beta; Proto-Oncogene Proteins c-myc; Receptors, Cytoplasmic and Nuclear; RNA, Messenger; Sulfonamides; Trans-Activators; Transcription Factors; Tumor Cells, Cultured

The tumor suppressor p53 gene product is an essential component of the cytotoxic pathway triggered by DNA-damaging stimuli such as chemotherapeutic agents and ionizing radiation. We previously demonstrated that adenovirus-mediated wild-type p53 gene transfer could enhance the cytotoxic actions of chemotherapeutic drugs both in vitro and in vivo; however, the molecular mechanism of this chemosensitization is still unclear. Cyclin D1 is a major regulator of the progression of cells into the proliferative stage of the cell cycle. Here we show that infection with an adenovirus vector expressing the wild-type p53 gene (Ad-p53) caused an increase in cyclin D1 protein levels in human colorectal cancer cell lines DLD-1 and SW620; treatment with the anti-cancer drug adriamycin, however, down-regulated their cyclin D1 protein expression in a dose-dependent manner. The suppression of cyclin D1 expression following adriamycin treatment could be blocked by simultaneous Ad-p53 infection. Furthermore, DLD-1 and SW620 cells transfected with the cyclin D1 expression construct displayed increased sensitivity to adriamycin compared to that of the vector-transfected control. Our results suggest that ectopic wild-type p53 gene transfer results in increased cyclin D1 expression and, consequently, sensitizes human colorectal cancer cells to chemotherapeutic agents.

Topics: Adenoviridae; Antineoplastic Agents; Blotting, Western; Colorectal Neoplasms; Cyclin D1; Dose-Response Relationship, Drug; Down-Regulation; Doxorubicin; Genetic Vectors; Humans; RNA, Neoplasm; Transfection; Tumor Cells, Cultured; Tumor Suppressor Protein p53

Activation of the extra cellular signal regulated kinase (ERK) pathway is involved in both proliferation and growth arrest of cells depending on intensity and duration of stimuli. In this study, we have elucidated differential regulation of the zinc-stimulated p21(CiP/WAF1) and cyclin D1 activation by inhibition of phosphoinositide 3-kinase (PI3K). In HT-29 colorectal cancer cells, the ERK activities were increased by zinc, which was accompanied by the induction of p21(Cip/WAF1) and cyclin D1. However, in the HT-29 cells pre-treated with PI3K inhibitor, LY294002, zinc induced further the p21(CiP/WAF) induction whereas abrogated cyclin D1 induction. In addition, the induction of p21(Cip/WAF1) expression completely inhibited the incorporation of bromodeoxyuridine (BrdU) into the nucleus, indicating that p21(CiP/WAF1) is an important indicator for ERK-dependent growth arrest. These studies suggest presence of an inter-related regulatory mechanism of cell proliferation by ERK and PI3K pathways.

Topics: Chromones; Colorectal Neoplasms; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Enzyme Activation; Enzyme Inhibitors; Flavonoids; HT29 Cells; Humans; Immunohistochemistry; MAP Kinase Signaling System; Mitogen-Activated Protein Kinases; Morpholines; Phosphatidylinositol 3-Kinases; Zinc

Cyclin D1 protein overexpression is commonly found in colorectal carcinomas (CRCs) and is associated with a poorer prognosis, but the mechanism underlying overexpression remains uncertain. Both dysregulation of beta-catenin protein expression and k-ras mutation have recently been shown to promote cyclin D1 expression in human in vitro and rodent in vivo studies. In this study, 53 sporadic CRCs were examined by immunohistochemistry for cyclin D1 and beta-catenin protein expression, and with PCR and direct DNA sequencing for k-ras gene status. The study also addressed whether cyclin Dl overexpression might associate with poorer prognosis because of a relationship with poorer response to 5-fluorouracil (5FU) chemotherapy. Cyclin D1 overexpression was demonstrated in 34/53 (64%) CRCs, was significantly associated with higher Dukes' stage, and was particularly prominent at the invasive edges of carcinomas. Furthermore, cyclin D1 overexpression was always and only seen in association with nuclear expression of beta-catenin. There were no significant associations between cyclin D1 overexpression and k-ras mutation or response to 5FU. Amongst 17 microsatellite unstable CRCs, a smaller proportion of tumours showed cyclin D1 overexpression (18%), but again cyclin D1 overexpression was only seen in cases showing nuclear beta-catenin expression. In conclusion, beta-catenin protein dysregulation, but not k-ras mutation, appears to be required for cyclin D1 overexpression in colorectal carcinoma in vivo.

Topics: Adult; Aged; Antimetabolites, Antineoplastic; beta Catenin; Biomarkers, Tumor; Cell Division; Colorectal Neoplasms; Cyclin D1; Cytoskeletal Proteins; Female; Fluorouracil; Genes, ras; Humans; Male; Microsatellite Repeats; Middle Aged; Mutation; Neoplasm Proteins; Prognosis; Trans-Activators; Treatment Outcome

Kirsten-ras is frequently mutated in colorectal cancers and may be an important therapeutic target, particularly because we have previously shown that acquisition of a mutation is associated with a poorer outcome. Understanding the role of Kirsten-ras and the consequences of inhibiting its activity or expression will contribute to our comprehension of colorectal cancer biology and may help to rationalize the choice of molecular targets suitable for therapeutic manipulation. Therefore we undertook a simple screen, incubating a library of oligonucleotides with Kirsten-ras mRNA and RNase H to identify an antisense oligonucleotide that effectively inhibited Kirsten-ras expression. We show for the first time in a human colon cancer cell line that inhibition of Kirsten-ras expression inhibits constitutive phosphorylation of Erk1/2, but not c-Akt, suggesting that in these cells constitutive phosphorylation of Erk 1/2 is dependent upon Kirsten-ras. Successful inhibition of Kirsten-ras had little effect on cell number or cell death and there was no evidence for accumulation of cells in any particular phase of the cell cycle. Kirsten-ras inhibition significantly reduced secretion of VEGF-A165 into the culture medium. Gene expression profiling by microarray detected altered expression of a number of genes. Of particular interest for future studies was the altered expression of genes encoding products involved in protein trafficking and the potential effects of these changes on cell adhesion. Our results suggest that, at least in this model, Kirsten-ras may contribute to malignancy predominantly through effects on angiogenesis, invasion, and metastasis, and that therapies directed at Kirsten-ras, including antisense approaches, may have particular utility through these mechanisms.

Topics: Adenocarcinoma; Angiogenesis Inducing Agents; Blotting, Western; Colorectal Neoplasms; Cyclin D1; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Oligonucleotide Array Sequence Analysis; Oligonucleotides, Antisense; Phosphorylation; Point Mutation; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins p21(ras); ras Proteins; RNA, Messenger; Tumor Cells, Cultured; Vascular Endothelial Growth Factor A

Increasing epidemiological and experimental evidence implicates non-steroidal anti-inflammatory drugs (NSAIDs) as anti-tumorigenic agents. The precise mechanisms whereby NSAIDs exert their anti-neoplastic effects remain poorly understood. Studies from hereditary and sporadic colorectal cancer (CRC) patients suggest that NSAIDs may interfere with initiating steps of carcinogenesis, i.e. disturbances within the beta-catenin signaling pathway. We therefore investigated beta-catenin/TCF signaling in response to aspirin or indomethacin, respectively, in four CRC cell lines (SW948, SW480, HCT116, LoVo). Both, aspirin and indomethacin inhibited transcription of a beta-catenin/TCF-responsive reporter gene in a dose dependent manner. In addition, the beta-catenin/TCF transcriptional target cyclin D1 was downregulated by both drugs. Endogenous beta-catenin levels remained unaffected by either drug. Moreover, indirect immunofluorescence studies revealed no significant changes of subcellular beta-catenin localization in either cell line after NSAID treatment. Likewise, binding of the beta-catenin/TCF complex to its specific DNA-binding sites was not altered, as demonstrated by electrophoretic mobility shift assay (EMSA) of nuclear extracts derived from NSAID treated cells. These results strongly suggest that aspirin and indomethacin attenuate the transcription of beta-catenin/TCF-responsive genes, by modulating TCF activity without disrupting beta-catenin/TCF complex formation.

Topics: Anti-Inflammatory Agents, Non-Steroidal; Aspirin; beta Catenin; Colorectal Neoplasms; Cyclin D1; Cyclooxygenase 2; Cytoskeletal Proteins; DNA, Neoplasm; Down-Regulation; Humans; Indomethacin; Isoenzymes; Membrane Proteins; Prostaglandin-Endoperoxide Synthases; Response Elements; Signal Transduction; Subcellular Fractions; TCF Transcription Factors; Trans-Activators; Transcription Factor 7-Like 2 Protein; Transcription Factors; Transcription, Genetic; Tumor Cells, Cultured

Topics: Adult; Age of Onset; Case-Control Studies; Colorectal Neoplasms; Cyclin D1; Female; Genotype; Humans; Logistic Models; Male; Polymorphism, Genetic; Risk; Risk Factors

The vitamin D receptor knockout (VDR-KO) mouse presents with a skeletal phenotype typical for complete lack of genomic 1,25-dihydroxycholecalciferol effects. Our previous data from human colorectal tissue suggest that the steroid hormone and its receptor may have protective function against tumour progression. In order to investigate the relevance of the vitamin D system for pre-malignant site-directed changes in the colon, we characterized the amount and site-specific distribution of the VDR along the large intestine in wild-type (WT), heterozygote (HT) and KO mice. We also evaluated expression of proliferating cell nuclear antigen (PCNA), of cyclin D1 and the levels of 8-hydroxy-2'-deoxyguanosine (8-OHdG), a marker of oxidative stress. In colon ascendens, proliferative cells were dispersed all along the crypt and expression levels of all three markers were high in WT mice. A decrease of VDR expression did not affect expression significantly. In colon descendens, however, fewer proliferative cells were solely located in the lower third of the crypt, and an inverse relationship between VDR reduction, PCNA positivity and cyclin D1 expression was found in HT and KO mice. In parallel to enhanced proliferation a highly significant increase of 8-OHdG positivity occurred. Therefore, the sigmoid colon of VDR-KO mice, fed on an appropriate lactose/calcium-enriched diet to alleviate impaired calcium homeostasis-related phenotypic changes, is an excellent model for investigating induction and prevention of pre-malignant changes in one of the hotspots for human colorectal cancer incidence.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Animals; Calcium; Colon; Colorectal Neoplasms; Cyclin D1; Deoxyguanosine; Disease Models, Animal; DNA Damage; Female; Homeostasis; Immunohistochemistry; Male; Mice; Mice, Knockout; Precancerous Conditions; Proliferating Cell Nuclear Antigen; Receptors, Calcitriol

P21 (WAF1), P53 and cyclin D1 belong to the cell cycle-regulating family of proteins, and the loss of activity of proteins P53 and P21 (WAF1) seems to be one of the most important regulatory mechanisms of carcinogenesis in colorectal cancer. The purpose of this study was to assess the relationship between P21 (WAF1), P53 and cyclin D1 immunoreactivity, and to evaluate the prognostic significance of their expression. Tissue sections from 122 paraffin-embedded colorectal carcinomas were immunostained with monoclonal antibodies. Positivity for P21 (WAF1) was found in 48 cases (39%), positivity for P53 in 96 cases (70%) and positivity for cyclin D1 in all the cases (100%). Statistical analyses revealed a statistically significant inverse correlation between P53 and P21 (WAF1)-immunopositivity and between P21 (WAF1)-immunopositivity and the degree of cyclin D1-immunopositivity, as well as an inverse correlation between P21 (WAF1) expression and clinical stage. In univariate analysis, down-regulation of P21 (WAFI) expression was associated with poor prognosis, but multivariate analysis did not confirm its independent prognostic significance. In Cox's analysis only regional lymph node invasion and hepatic metastases were proven as independent prognostic parameters. Our investigation results suggest that in colorectal cancer, the induction of P21 (WAF1) may occur mostly in a P53-dependent pathway. P21 (WAF1), as the main cyclin-dependent kinase (CDK)-inhibitor, may also inhibit the activity of cyclins such as cyclin D1.

Topics: Adult; Aged; Aged, 80 and over; Carcinoma; Colorectal Neoplasms; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Female; Follow-Up Studies; Humans; Immunohistochemistry; Male; Middle Aged; Prognosis; Survival Analysis; Survival Rate; Tumor Suppressor Protein p53

Analysis of tumor markers focuses on expression in primary tumors with the assumption that this is representative of metastatic tumor, against which treatment is targeted. Few studies have compared the expression of such markers in primary and secondary tumors. In this study, several key genes involved in cell cycle regulation were investigated in colorectal tumors and corresponding lymph node metastases. The cell cycle regulators p53, cyclin D1, p21, p27, retinoblastoma protein (Rb), and proliferating cell nuclear antigen (PCNA) were examined in a series of 42 paired samples of primary colorectal and secondary lymph node tumors by immunohistochemistry. Expression of p53, p27, and Rb was similar in virtually all paired samples (p53, 38 of 42; p27, 39 of 42; Rb, 40 of 42), indicating that the pattern of these proteins in colorectal tumors may be used to predict that in lymph node tumors. It also suggests a lack of direct involvement in the metastatic process. A lower concordance for p21 and cyclin D1 staining was observed between primary and secondary tumors (p21, 19 of 42; cyclin D1, 22 of 42). p21 expression was more often observed in primary colorectal cancers, whereas cyclin D1 expression was more frequently seen in lymph node metastases, in keeping with the contrasting roles of these proteins as a cell cycle inhibitor (p21) and activator (cyclin D1). The PCNA-labeling index was found to vary considerably in a number of cases, thus limiting the ability to predict expression of this protein in lymph node metastases from the primary tumor. In addition, PCNA-labeling indices between paired samples were neither consistently higher nor lower, suggesting that the proliferative capacity of tumor cells is not directly related to their ability to metastasize.

Topics: Adult; Aged; Aged, 80 and over; Cell Cycle Proteins; Colorectal Neoplasms; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase Inhibitor p27; Cyclins; Female; Humans; Immunohistochemistry; Lymph Nodes; Lymphatic Metastasis; Male; Microtubule-Associated Proteins; Middle Aged; Predictive Value of Tests; Prognosis; Proliferating Cell Nuclear Antigen; Retinoblastoma Protein; Tumor Suppressor Protein p53; Tumor Suppressor Proteins

Cyclin D1 is a key cell cycle regulatory protein, the expression and subcellular localization of which is often altered in human tumor cells. A common A/G single nucleotide 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 prognosis in lung cancer and head and neck carcinoma. We have investigated both the expression of cyclin D1 protein and the CCND1 A870G polymorphism in 100 colorectal cancer patients. Immunohistochemistry demonstrated cyclin D1 protein expression in 55% of tumors, and while the absence of cyclin D1 protein was not associated with outcome (p=0.81), high levels of protein expression (>50% of tumor cells expressing cyclin D1) correlated with significantly shortened overall survival (p=0.01). Using polymerase chain reaction restriction fragment length polymorphism analysis, we determined the frequency of each genotype and found that CCND1 genotype was not related to overall survival (p>0.05). In addition, genotype was unrelated to the level of expression and localization of cyclin D1 protein, as well as other key G1/S checkpoint proteins (p21, p27, p53, retinoblastoma) and tumor proliferation markers (proliferating cell nuclear antigen). However, higher levels of p27, and to a lesser extent p21, were associated with reduced cytoplasmic cyclin D1 protein (p=0.029 and p=0.054, respectively). In conclusion, we have demonstrated that high levels of cyclin D1 protein expression are related to outcome in colorectal cancer; however, the CCND1 A870G polymorphism is unrelated to either cyclin D1 protein expression or patient survival.

Topics: Adenocarcinoma; Adult; Aged; Aged, 80 and over; Cell Cycle Proteins; Colorectal Neoplasms; Cyclin D1; Female; Genotype; Humans; Immunohistochemistry; Male; Middle Aged; Polymerase Chain Reaction; Polymorphism, Genetic; Polymorphism, Restriction Fragment Length; Prognosis; Subcellular Fractions; Survival Analysis

Cyclin D1, P53 and P21 (WAF1) are cell cycle regulating proteins, playing a crucial role in oncogenesis of a large number of human malignancies, and loss of activity of P53 and P21 (WAF1) proteins seems to be one of the most important regulatory mechanisms of carcinogenesis in colorectal cancer. To find out their mutual relations we investigated the expression of cyclin D1, P53 and P21 (WAF1) in 122 colorectal cancers by immunohistochemistry. Positivity for cyclin D1 was found in all the cases (100%), positivity for P53 in 96 cases (70%) and positivity for P21 in 48 cases (39%). Statistical analysis revealed a statistically significant inverse correlation between P53 and P21 (WAF1)-immunopositivity and also between P21 (WAF1)-immunopositivity and the degree of cyclin D1-immunopositivity. These data suggest that in colorectal cancer induction of P21 (WAF1) may occur mostly in a P53-dependent pathway. Wild-type P53, which is undetectable by immunohistochemistry, induces transcriptionally P21 (WAF1) and in tumours it may cause its accumulation, while mutations of the P53 may result in a sufficient increase of intracellular protein having no ability to transactivate P21 (WAF1). Moreover P21 (WAF1) as the main cyclin-dependent kinases (CDKs) inhibitor may also inhibit the activity of the cyclins, thus overexpression of P21 (WAF1) may result in reduced level of cyclin D1.

Topics: Adult; Aged; Aged, 80 and over; Cell Cycle Proteins; Colorectal Neoplasms; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinases; Cyclins; Female; Gene Expression; Humans; Immunohistochemistry; Male; Middle Aged; Point Mutation; Tumor Suppressor Protein p53

A systematic spatial heterogeneity with high proliferative activity at the luminal border and low activity at the invasive margin is an unexpected behavior that has been observed in colorectal cancer (CRC). To clarify this phenomenon and possible underlying regulatory mechanisms, we have by immunohistochemistry elucidated the proliferative activity and the expression of G1/S regulatory proteins in small and large tumor cell clusters at the invasive margin in 97 CRCs. By identifying small tumor clusters at the tumor front, actually invading cancer cells could be characterized and analyzed separately. These cells could then be compared with the main tumor mass represented by the larger tumor clusters. The proliferation was significantly lower in small tumor clusters compared with larger clusters (P < 0.001) and the decrease in proliferation was correlated with a p16 up-regulation (r(s) = -0.41, P < 0.001). Interestingly, CRCs lacking p16 expression (18%) or tumors with other aberrations in the p16/cyclin D1/pRb pathway had a less pronounced decrease in proliferation between large and small clusters (P < 0.001), further strengthening the association between p16 and ceased proliferation at the invasive margin. This contrasts to tumors with low p27 or abnormal p53 levels showing sustained proliferation in small tumor clusters. Our findings imply that invading CRC cells generally have low proliferative activity, and this phenomenon seems to be mediated through p16 and the p16/cyclin D1/pRb pathway.

Topics: Cell Division; Colorectal Neoplasms; Cyclin D1; G1 Phase; Gene Expression; Genes, p16; Humans; Neoplasm Invasiveness; Retinoblastoma Protein; Retrospective Studies; S Phase; Tissue Distribution; Up-Regulation

Topics: Adult; Colonic Neoplasms; Colorectal Neoplasms; Cyclin D1; Female; Humans; Incidence; Japan; Kidney Transplantation; Male; Middle Aged; Neoplasm Staging; Neoplasms; Postoperative Complications; Rectal Neoplasms; Retrospective Studies; Tumor Suppressor Protein p53

To explore the relationship between the expression of cell cycle regulators and microsatellite instability (MSI) in colorectal carcinomas(CRC).. The expression of cell cycle regulators cyclin D1, P16, P21WAF1/CIP1, P53, Rb and PCNA were detected by immunohistochemical staining, and MSI at six microsatellite loci (D18S34, D17S799, D5S409, TCF-2, Rb and P53) were examined by PCR-SSLP in 56 cases of colorectal carcinomas.. The overall positive rate of MSI of CRC was 44.6% (25/56). MSI was positive at 1 locus in 7 cases, at 2 and more than 2 loci in 18 cases. In CRC with MSI, the positive rate of cyclin D1 and P53 protein was significantly lower, while that of P21 protein was significantly higher than those without MSI (P < 0.05). The positive rate of P16 in CRC with MSI was higher than those without MSI. The expression of Rb proteins was similar in frequency in CRC with or without MSI.. Overexpression of P21 may play an important role in MSI genesis in CRC. It may also be related to decreased expression of cyclin D1 and P53 and overexpression of P16.

Topics: Cell Cycle; Cell Cycle Proteins; Colorectal Neoplasms; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p16; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Gene Expression; Humans; Immunohistochemistry; Microsatellite Repeats; Tumor Suppressor Protein p53

Cyclin D1 is a cell cycle regulator which is overexpressed in a variety of human cancers. We examined overexpression of cyclin D1 in several stages of rat colorectal carcinogenesis induced by azoxymethane (AOM) treatment. The level of cyclin D1 in 13 aberrant crypt foci (ACF) (atypical hyperplasias), 22 colorectal tumors (14 non-invasive adenocarcinomas and eight invasive adenocarcinomas) was assessed by immunostaining using a polyclonal antibody. Cell proliferation of these samples was investigated by measurement of 5-bromo-2'-deoxyuridine-labeling index. Indices of cyclin D1-positive cells in adenocarcinomas and atypical hyperplasias were significantly higher than that in normal crypts (P < 0.05). Moreover, cyclin D1-positive rates in the two types of adenocarcinomas were significantly higher than that in atypical hyperplasias (P < 0.05). Staining of nuclear cyclin D1 was very strong in almost all adenocarcinomas and four ACF. Comparisons of BrdU-positive indices in colorectal lesions showed similar results to the cyclin D1-positive indices. These results suggested that overexpession of cyclin D1 occurs early in the multistep carcinogenesis, and plays an important role in rat colorectal carcinogenesis.

Topics: Adenocarcinoma; Animals; Azoxymethane; Bromodeoxyuridine; Cell Division; Cell Nucleus; Colorectal Neoplasms; Cyclin D1; Cytoplasm; Gene Expression; Hyperplasia; Intestinal Mucosa; Male; Rats; Rats, Inbred F344; Time Factors

Cyclin D1 is a G1 cyclin that controls the transition of the cell cycle from G1 phase to S phase, and its gene is located on chromosome 11q13. We evaluated the expression of cyclin D1 mRNA in surgically resected specimens of gastric and colorectal cancers using quantitative RT-PCR. In this method, cDNA derived from cyclin D1 mRNA was amplified in a tube together with an internal control. The expression of cyclin D1 mRNA was high in 8 of 36 gastric cancer tissues (22%) and 9 of 27 (33%) colorectal cancer tissues, compared to normal mucosal tissues. In gastric cancers, the rate of cyclin D1 mRNA expression (an index of the density of DNA bands) was significantly higher in patients with tumors invading beyond the submucosal layer, regional lymph nodes and lymphatic vessels (i.e., patients with stage III or IV). In colorectal cancers, the rate of cyclin D1 mRNA expression was significantly higher in patients with venous invasion. Moreover, in patients with colorectal cancer, the survival rate of high-expression group was significantly lower than in low-expression group. Our results suggested that overexpression of cyclin D1 mRNA reflected the severity of gastric cancer and poor prognosis of colorectal cancer.

Topics: Adult; Aged; Aged, 80 and over; Base Sequence; Cell Cycle; Colorectal Neoplasms; Cyclin D1; DNA Primers; Female; Gene Expression; Humans; Male; Middle Aged; Neoplasm Invasiveness; Prognosis; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Neoplasm; Stomach Neoplasms

We describe and discuss a method of protein extraction for Western blot analysis from formalin-fixed, paraffin-embedded tissue sections. From 5-mm2 50-micron-thick tissue sections, an abundance of proteins could be extracted by incubating the sections in lysis buffer containing 2% sodium dodecyl sulfate (SDS) at 100C for 20 min followed by incubation at 60C for 2 hr. Extracts yielded discernible protein bands ranging from 10 kD to 120 kD as identified by SDS-polyacrylamide gel electrophoresis (PAGE). Western blot analysis successfully detected membrane-bound protein such as E-cadherin, cytosolic protein such as beta-catenin, and nuclear proteins including proliferating cell nuclear antigen (PCNA), mutant-type p53, cyclin D1, cyclin E, and cyclin-dependent kinases (CDKs). With this technique, we could examine cyclin D1 and CDK2 expression in small adenomas compared with cancer tissues and normal mucosa. The simple method of protein extraction described here should make it possible to use large-scale archives of formalin-fixed, paraffin-embedded samples for Western blot analysis, and its application could lead to detailed analysis of protein expression. This new technique should yield valuable information for molecular biology.

Topics: Adenoma; Blotting, Western; CDC2-CDC28 Kinases; Colorectal Neoplasms; Cyclin D1; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinases; Formaldehyde; Humans; Nuclear Proteins; Paraffin; Proliferating Cell Nuclear Antigen; Protein Serine-Threonine Kinases; Proteins; Tissue Fixation

Abnormal expression of the retinoblastoma protein (pRb) and cyclin D1 have been reported in a variety of malignancies, but the frequencies of these deregulations and their relation to prognosis in colorectal cancer has not been clarified. We characterised 90 colorectal cancers with respect to immunohistochemical expression of cyclin D1, pRb and Ki-67. Two of 90 (2%) tumours lacked nuclear pRb staining, indicating inactivation of the protein, while 10 (11%) expressed high levels of pRb. Abnormal expression of pRb was significantly correlated to low levels of nuclear cyclin D1 observed in 32% of the tumours. Strong nuclear cyclin D1 expression was detected in 12% of the tumours. Cytoplasmic staining of cyclin D1 was observed in 17% of the tumours, showing an inverse relationship (P = 0.006) to the Ki-67 labelling index. Eight of 11 tumours with high nuclear overexpression of cyclin D1 and both tumours with pRb defects were located in the right colon in comparison with zero of 25 in the rectum (P = 0.009). Regarding prognosis, neither pRb nor cyclin D1 expression correlated with patient survival.

Topics: Adult; Aged; Aged, 80 and over; Cell Division; Colorectal Neoplasms; Cyclin D1; Female; Humans; Immunohistochemistry; Ki-67 Antigen; Male; Middle Aged; Neoplasm Proteins; Neoplasm Staging; Prognosis; Retinoblastoma Protein; Retrospective Studies; Survival Analysis

The expression of cyclin-dependent kinase inhibitor p27(kip1) in human tumors and normal tissues was investigated using a panel of novel anti-p27(kip1) mAbs. An inverse correlation between expression of p27(kip1) and cell proliferation was generally observed after analyzing its expression in 25 different normal human tissues. In some highly proliferative human breast cancer cells, however, high level p27(kip1) expression was seen, indicating the existence of a mechanism by which some growing tumor cells may tolerate this inhibitor of cell cycle progression. Detailed studies demonstrated a correlation between the high level expression of p27(kip1) and cyclin D1 in human breast cancer cells. There was also an inverse correlation between the expression of p27(kip1) and the degree of tumor malignancy in human breast and colorectal cancers, indicating that p27(kip1) may be a useful prognostic marker in these cancers.

Topics: Biomarkers, Tumor; Breast Neoplasms; Cell Cycle; Cell Cycle Proteins; Cell Division; Colorectal Neoplasms; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p27; Cyclin-Dependent Kinases; Cyclins; Female; Genes, Tumor Suppressor; Humans; Immunohistochemistry; Kinetics; Male; Microtubule-Associated Proteins; Oncogene Proteins; Organ Specificity; Prognosis; Reference Values; Tumor Cells, Cultured; Tumor Suppressor Proteins

Multiple genetic changes occur during the evolution of normal cells into cancer cells. It has been reported that both cyclin D1 and p53 genes play major roles in oncogenesis and/or cell cycle control in various cancers. In this study, we examined the overexpression of cyclin D1 and p53 by the immunohistochemical method and investigated the correlation between expression of these antigen and prognosis in patients with colorectal adenocarcinoma. Disease-free survival was significantly lower in the patients with cyclin D1-strongly positive tumors than in those with cyclin D1-negative tumors. Similarly, disease-free survival of the patients with p53-strongly positive tumors was significantly lower than that of those with p53-negative tumors. Moreover, multivariate analysis indicated that both cyclin D1 and p53 overexpression are independent prognostic factors in patients with colorectal adenocarcinoma. In conclusion, both cyclin D1 and p53 overexpression may be useful predictors of disease recurrence in patients with colorectal adenocarcinoma.

Topics: Adenocarcinoma; Adult; Aged; Biomarkers, Tumor; Colorectal Neoplasms; Cyclin D1; Cyclins; Disease-Free Survival; Female; Humans; Male; Middle Aged; Neoplasm Recurrence, Local; Oncogene Proteins; Proliferating Cell Nuclear Antigen; Retrospective Studies; Tumor Suppressor Protein p53

We evaluated the clinical significance of cyclin D1 expression in colorectal adenocarcinoma. One hundred twenty-three specimens resected from patients with colorectal cancers were investigated by staining with a monoclonal antibody against cyclin D1. The possible correlations among cyclin D1 expression, clinicopathologic factors and prognosis were studied. There was no significant association between cyclin D1 expression and various clinicopathological factors. However, disease-free survival was significantly worse in the patients with cyclin D1 strongly positive tumors than in those with cyclin D1 negative tumors. Cyclin D1 overexpression may be useful as a predictor of disease recurrence in patients with colorectal adenocarcinoma.

Topics: Adenocarcinoma; Adult; Aged; Biomarkers, Tumor; Colorectal Neoplasms; Cyclin D1; Cyclins; Humans; Lymphatic Metastasis; Middle Aged; Neoplasm Invasiveness; Oncogene Proteins; Prognosis; Survival Rate

Cyclin D1 gene amplification and/or overexpression occurs in several human cancers. The level of expression of cyclin D1 protein during the multistage process of human colon carcinogenesis was determined.. Cyclin D1 protein abundance was determined by immunostaining samples of normal colonic mucosa(n=23), transitional normal mucosa adjacent to adenomas or adenocarcinomas (n=41), hyperplastic polyps (n=8), adenomatous polyps (=35), and adenocarcinomas (n=27), using a polyclonal anti-human cyclin D1 antibody.. Cyclin D1 nuclear staining occurred in 30% of adenocarcinomas and 34% of adenomatous polyps but not in hyperplastic polyps or normal or transitional mucosa. Nuclear staining did not correlate with sex, age, size, or dysplasia of the adenomatous polyps or with differentiation and Dukes' staging of the adenocarcinomas. Left-sided colon neoplasms showed nuclear staining more frequently than those right-sided lesions. Diffuse or supranuclear cytoplasmic staining occurred in about one third of hyperplastic polyps, adenomas, and adenocarcinomas and in transitional mucosa adjacent to adenocarcinoma.. Increased nuclear expression of cyclin D1 occurs in around one third of colonic tumors as an early event during multistage process of colon carcinogenesis. Increased expression of cyclin D1 may perturb cell-cycle control in benign adenomas and thereby enhance tumor progression.

Topics: Adenocarcinoma; Adenoma; Adolescent; Adult; Aged; Cell Nucleus; Chi-Square Distribution; Colon; Colonic Polyps; Colorectal Neoplasms; Cyclin D1; Cyclins; Cytoplasm; Female; Humans; Immunohistochemistry; Intestinal Mucosa; Logistic Models; Male; Middle Aged; Oncogene Proteins

Colorectal cancer is the second leading cause of death from cancer in the United States. Continuous use of aspirin and other nonsteroidal anti-inflammatory drugs (NSAIDs) has been shown to reduce the risk of colorectal cancer in humans by 40-50%. Patients with familial adenomatous polyposis who take NSAIDs, such as sulindac, undergo a regression of intestinal adenomas. Rodents exposed to carcinogens that cause colon cancer have a 50-60% reduction in the size and number of colonic tumors when treated continuously with NSAIDs. One common target for these drugs is prostaglandin endoperoxide synthase, also referred to as cyclooxygenase (COX). We and others have shown recently that COX-2 levels are increased dramatically in 85-90% of human colorectal adenocarcinomas and in 40-50% of colonic adenomas. We prepared intestinal epithelial cells that express the COX-2 gene permanently and found that they have altered adhesion properties and resist undergoing apoptosis. We report here that these cells also have a 3-fold increase in the duration of G1, lower levels of cyclin D1 protein, and a marked decrease in retinoblastoma kinase activity associated with cyclin-dependent kinase 4. The delay in G1 transit may relate to the resistance of these cells to undergo programmed cell death, which could affect their tumorigenic potential.

Topics: Adenoma; Animals; Anti-Inflammatory Agents, Non-Steroidal; Anticarcinogenic Agents; Aspirin; Cell Line; Colonic Neoplasms; Colorectal Neoplasms; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinases; Cyclins; DNA; Epithelium; G1 Phase; Humans; Intestines; Kinetics; Oncogene Proteins; Open Reading Frames; Prostaglandin-Endoperoxide Synthases; Proto-Oncogene Proteins; Rats; Recombinant Proteins; Retinoblastoma Protein; Time Factors; Transfection; United States

Activating mutations in the c-K-ras gene occur in about 40% of human colorectal carcinomas, yet the role of this oncogene in tumorigenesis is not known. We have developed a model cell culture system to study this problem, utilizing the immortalized but non-tumorigenic epithelial cell line IEC18, originally derived from normal rat intestine epithelium. These cells were cotransfected with the drug resistance selectable marker tk-neo and the plasmid pMIKcys, which encodes a mini human c-K-ras gene (15 kb) containing a cysteine mutation at codon 12. Drug resistant clones were isolated. Clones which also expressed the activated c-K-ras gene displayed a transformed morphology, decreased doubling time, increased level of diacylglycerol, anchorage independent growth in soft agar and an aneuploid karyotype and they were also tumorigenic when injected into nude mice. These clones also displayed increased expression, at both the mRNA and protein levels, of cyclin D1 and Rb. These findings may be of clinical relevance since human colorectal tumors also frequently display increased expression of both cyclin D1 and Rb. This model system may be useful for understanding the role and interrelationship between activation of the c-K-ras oncogene and increased expression of cyclin D1 and Rb in colorectal tumorigenesis.

Topics: Animals; Cell Line, Transformed; Colon; Colorectal Neoplasms; Cyclin D1; Cyclins; Genes, ras; Genes, Retinoblastoma; Humans; Karyotyping; Mice; Mice, Nude; Oncogene Proteins; Rats

The PRAD-1/cyclin D1 proto-oncogene is localized on chromosome 11q13 and it is overexpressed in several tumour types as a consequence of gene amplification or chromosomal rearrangements. In this study, the abundance and patterns of cyclin D1 protein expression in normal/non-involved colon (n = 44), primary (n = 48) and metastatic (n = 9) colorectal carcinomas, and in a series of 4 colon cancer cell lines were investigated by immunochemical methods using the DCS-6 monoclonal antibody specific for cyclin D1. While examination of all normal colorectal tissue samples and 56% of the primary tumours revealed only weak to undetectable immunostaining signals, 23% of the primary carcinomas showed moderate and 21% showed strong aberrant accumulation of this cell-cycle regulatory oncoprotein. The immunohistochemical patterns in the secondary lesions were concordant with the matched primary tumours in all cases. The staining was nuclear both in the clinical specimens and in the colon cancer cell lines, in which the antibody-mediated knock-out experiments demonstrated a positive regulatory role of the cyclin D1 protein whose function was required for progression through the G1 phase of the cell cycle. These results indicate that the PRAD-1/cyclin D1 protooncogene may be deregulated in a significant subset of colorectal tumours, and warrant further analyses of such aberrations of the cyclin D1/retinoblastoma protein pathway to elucidate its potential involvement in the multistep pathogenesis of human colorectal cancer.

Topics: Chromosomes, Human, Pair 11; Colorectal Neoplasms; Cyclin D1; Cyclins; Humans; Immunohistochemistry; Oncogene Proteins; Proto-Oncogene Mas; Proto-Oncogenes; Retinoblastoma Protein; Tumor Cells, Cultured