ABSTRACT
OBJECTIVES: FoxP3 expression is a marker for Tregs which are known to be involved in tumor immunity. We aimed to evaluate FoxP3 promoter demethylation in human colorectal cancer (CRC) and rat intrahepatic cholangiocarcinoma (ICC). DESIGN AND METHODS: Bisulfite-treated genomic DNA templates of shock frozen paired samples were studied from 13 anonymous CRC patients and from 10 male rats (n=6 ICC induced by thioacetamide and n=4 age-matched controls). Real-time PCR was carried out using a LightCycler 480 system. Human FoxP3 and CD3 promoter demethylations were estimated using previously described assays; and rat FoxP3 promoter demethylation using a newly developed assay. RESULTS: A significant 3.5-fold increase of the demethylation in FoxP3 promoter region was found in human CRC and rat ICC (P<0.05). The average frequency of cells with FoxP3 demethylation in patients suffering from CRC was 0.26% in normal tissue and 0.92% in tumor tissue (n=11 paired samples). Although, no significant difference was found between the mean frequency of CD3 demethylation in normal tissue (4.80%, n=6) and in tumor tissue (4.14%, n=6) from CRC patients, the ratio of demethylated CD3/FoxP3 promoter areas was significantly lower in tumor specimens (P<0.05). Using our novel assay, we found a significant increase in mean frequencies of cells with FoxP3 demethylation in rats with ICC (7.42%, n=6) in comparison to controls (2.14%, n=4). CONCLUSION: FoxP3 seems to be an interesting biomarker for immune response to epithelial tumors. Functional consequences from the increase of Tregs remain to be demonstrated. Further studies with outcome data are necessary.
Subject(s)
Biomarkers, Tumor/metabolism , Cholangiocarcinoma/metabolism , Colorectal Neoplasms/metabolism , DNA Methylation , DNA, Neoplasm/metabolism , Forkhead Transcription Factors/metabolism , Neoplasm Proteins/metabolism , Promoter Regions, Genetic , Adult , Aged , Animals , Biomarkers, Tumor/genetics , Biomarkers, Tumor/immunology , CD3 Complex/genetics , CD3 Complex/immunology , CD3 Complex/metabolism , Cholangiocarcinoma/genetics , Cholangiocarcinoma/immunology , Cholangiocarcinoma/pathology , Colorectal Neoplasms/genetics , Colorectal Neoplasms/immunology , Colorectal Neoplasms/pathology , DNA, Neoplasm/genetics , DNA, Neoplasm/immunology , Female , Forkhead Transcription Factors/genetics , Forkhead Transcription Factors/immunology , Humans , Male , Middle Aged , Neoplasm Proteins/genetics , Neoplasm Proteins/immunology , Rats , T-Lymphocytes, Regulatory/immunology , T-Lymphocytes, Regulatory/metabolism , T-Lymphocytes, Regulatory/pathologyABSTRACT
OBJECTIVES: Unequivocal biomarkers are needed to predict susceptibility and progression of colorectal cancer. DESIGN AND METHODS: Paired samples of tumor and normal tissue from six patients with colorectal cancer of different localization, pTNM stage and grade were employed in the present study. MS analysis was used to identify differentially regulated proteins after 2-DE separation and densitometric analysis. RESULTS: Densitometric analysis revealed differential abundance of 55 spots in tumor as compared to normal tissues. Thirty nine out of 55 spots were unambiguously identified by MS representing 32 different proteins. CLIC1, TPD52 and FABPL were consistently overexpressed (>3-fold, P<0.05) in all tumor tissue samples, while TPM1, TPM2, TPM3, TAGL and MLRN were consistently down-regulated (>3-fold, P<0.05) compared to normal tissue. CONCLUSIONS: CLIC1 and TPD52 were significantly (P<0.05) up-regulated in all cases of colorectal cancer investigated, irrespective of localization, pTNM stage and grade of colon cancer highlighting their potential to serve as new biomarkers.
Subject(s)
Biomarkers, Tumor/metabolism , Chloride Channels/metabolism , Colorectal Neoplasms/metabolism , Neoplasm Proteins/metabolism , Aged , Aged, 80 and over , Demography , Electrophoresis, Gel, Two-Dimensional , Female , Humans , Male , Mass Spectrometry , Middle Aged , Neoplasm Proteins/chemistry , Reproducibility of ResultsABSTRACT
PURPOSE: Variation in genetic factors together with xenobiotic exposure may result in increased risk of colorectal cancer. The P-glycoprotein (P-gp) is highly expressed in the apical membrane of enterocytes, where it pumps xenobiotics from the enterocytes back into the intestinal lumen. Thus, polymorphisms that reduce the activity of the MDR1 (ABCB1) efflux pump are potential risk factors for colorectal carcinogenesis. The aim of the present study is to genotype the MDR1 2677G>T (rs2032582) and 3435C>T (rs1045642) polymorphism in patients with colorectal cancer and controls and to identify a possible association between individual genetic variation and susceptibility to colorectal cancer. METHODS: In the present study, 146 Bulgarian patients with sporadic colorectal cancer and 160 healthy Bulgarian volunteers were evaluated for the two polymorphisms in MDR1. Polymorphisms were identified using rapid-cycle real-time amplification with allele-specific probes and subsequent melting curve analyses on a LightCyclertrade mark (Roche Diagnostics, Mannheim, Germany). RESULTS: No differences were found between the frequencies of the two mutant alleles in the tumor tissue from the cases and lymphocytes from the controls [frequencies of 2677T: 43.5% in patients and 44.1% in controls; frequencies of 3435T: 48.3% in patients and 50.9% in controls (both P > 0.05)]. The MDR1 polymorphic sequence of the tumor tissue always matched that of normal intestinal tissue from the same patient. Consequently, genotyping of DNA from archived tumor tissues is a valid alternative to the use of leukocyte DNA. CONCLUSIONS: The present study suggests that MDR1 2677G>T and 3435C>T polymorphism is not a risk factor for sporadic colon cancer among Bulgarians and that somatic mutation at these sites is not involved in the genesis of colon tumors. Further examination using larger number of samples must be necessary to reach to more reliable conclusions.
Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1/genetics , Colorectal Neoplasms/genetics , Genetic Predisposition to Disease , Polymorphism, Single Nucleotide , ATP Binding Cassette Transporter, Subfamily B , ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , Aged , Bulgaria , Female , Genetic Variation , Genotype , Haplotypes , Humans , Male , Mutation , Risk FactorsABSTRACT
BACKGROUND: The purpose of the study was to genotype four polymorphisms in CYP3A5 (CYP3A5*2, CYP3A5*3, CYP3A5*3B, and CYP3A5*6) for a possible association between individual genetic variations and susceptibility to colorectal cancer. Another point of interest was to conduct a comprehensive analysis in tumor and normal intestine tissue from the same patient, searching for somatic hotspots. MATERIAL AND METHODS: In our study, 146 Bulgarian patients with sporadic colorectal cancer and 160 healthy volunteers were enrolled. CYP3A5 polymorphisms were identified using rapid-cycle real-time amplification with allele-specific probes and subsequent melting curve analysis on a LightCycler. RESULTS: The allele frequencies were comparable in both groups: frequency of CYP3A5*2 = 0.3% in patients vs. 0.6% in controls; frequency of CYP3A5*3 = 90.8% in patients vs. 93.1% in controls; in both groups no CYP3A5*3B and CYP3A5*6 variants were detected (p > 0.05). No difference was observed between genotype frequencies in tumor and surrounding normal tissues of 80 patients. CONCLUSIONS: The CYP3A5 variants are unlikely to have an important functional significance in patients with colorectal cancer. The studied CYP3A5 loci do not seem to be hotspots for somatic mutation. DNA from archived tumor tissues is a valid alternative to the use of leukocyte DNA for genotyping of these polymorphisms.
