Influence of functional polymorphisms in TNF-α, IL-8, and IL-10 cytokine genes on mRNA expression levels and risk of gastric cancer.

Functional polymorphisms in promoter regions can produce changes in the affinity of transcription factors, thus altering the messenger ribonucleic acid (mRNA) expression levels of inflammatory cytokines associated with the risk of cancer development. The goal of this study was to evaluate the influence that polymorphisms in the cytokine genes known as TNF-α-308 G/A (rs1800629), TNF-α-857 C/T (rs1799724), IL-8-251 T/A (rs4073), IL-8-845 T/C (rs2227532), and IL-10-592 C/A (rs1800872) have on changes to mRNA expression levels and on the risks of chronic gastritis (CG) and gastric cancer (GC). A sample of 723 individuals was genotyped using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique. Relative mRNA expression levels were measured using quantitative real-time PCR (qPCR). Polymorphisms TNF-α-308 G/A and IL-8-251 A/T were not associated with risks of these gastric lesions. However, TNF-α-857 C/T, IL-8-845 T/C, and IL-10-592 C/A were found to be associated with a higher risk of GC, and IL-10-592 C/A was found to be associated with a higher risk of CG. The relative mRNA expression levels (RQ) of TNF-α, IL-8, and IL-10 were markedly downregulated in the CG group (median RQs = 0.128, 0.247, and 0.614, respectively), while the RQ levels of TNF-α in the GC group were upregulated (RQ = 2.749), but were basal for IL-8 (RQ = 1.053) and downregulated for IL-10 (RQ = 0.179). When the groups were stratified according to wild-type and polymorphic alleles, only for IL-8-845 T/C the polymorphic allele was found to influence the expression levels of this cytokine. IL-8-845 C allele carriers were significantly upregulated in both groups (GC and CG; RQ = 3.138 and 2.181, respectively) when compared to TT homozygotes (RQ = -0.407 and 0.165, respectively). In silico analysis in the IL-8 promoter region revealed that the presence of the variant C allele in position -845 is responsible for the presence of the binding sites for two transcription factors (REL and CREB1), which are involved in increased gene expression. Polymorphic alleles were not shown to have any effect on the expression levels of TNF-α and IL-10. Taken together, our findings provide evidence for an association of TNF-α-857 C/T, IL-8-845 T/C, and IL-10-592 C/A with a higher risk of gastric cancer and also demonstrate the influence that the polymorphic C allele of IL-8-845 has on changes to the gene expression levels of this cytokine.

GPC3 reduces cell proliferation in renal carcinoma cell lines.

BACKGROUND: Glypican 3 (GPC3) is a member of the family of glypican heparan sulfate proteoglycans (HSPGs). The GPC3 gene may play a role in controlling cell migration, negatively regulating cell growth and inducing apoptosis. GPC3 is downregulated in several cancers, which can result in uncontrolled cell growth and can also contribute to the malignant phenotype of some tumors. The purpose of this study was to analyze the mechanism of action of the GPC3 gene in clear cell renal cell carcinoma. METHODS: Five clear cell renal cell carcinoma cell lines and carcinoma samples were used to analyze GPC3 mRNA expression (qRT-PCR). Then, representative cell lines, one primary renal carcinoma (786-O) and one metastatic renal carcinoma (ACHN), were chosen to carry out functional studies. We constructed a GPC3 expression vector and transfected the renal carcinoma cell lines, 786-O and ACHN. GPC3 overexpression was analyzed using qRT-PCR and immunocytochemistry. We evaluated cell proliferation using MTT and colony formation assays. Flow cytometry was used to evaluate apoptosis and perform cell cycle analyses. RESULTS: We observed that GPC3 is downregulated in clear cell renal cell carcinoma samples and cell lines compared with normal renal samples. GPC3 mRNA expression and protein levels in 786-O and ACHN cell lines increased after transfection with the GPC3 expression construct, and the cell proliferation rate decreased in both cell lines following overexpression of GPC3. Further, apoptosis was not induced in the renal cell carcinoma cell lines overexpressing GPC3, and there was an increase in the cell population during the G1 phase in the cell cycle. CONCLUSION: We suggest that the GPC3 gene reduces the rate of cell proliferation through cell cycle arrest during the G1 phase in renal cell carcinoma.

Deregulation of annexin-A1 and galectin-1 expression in precancerous gastric lesions: intestinal metaplasia and gastric ulcer.

OBJECTIVE: Annexin-A1 (ANXA1/AnxA1) and galectin-1 (LGALS1/Gal-1) are mediators that play an important role in the inflammatory response and are also associated with carcinogenesis. We investigated mRNA and protein expression in precancerous gastric lesions that participate in the progression cascade to gastric cancer, such as intestinal metaplasia (IM) and gastric ulcer (GU). METHODS: Quantitative real-time PCR (qPCR) and immunohistochemical techniques were used to analyze the relative quantification levels (RQ) of ANXA1 and LGALS1 mRNA and protein expression, respectively. RESULTS: Increased relative expression levels of ANXA1 were found in 100% of cases, both in IM (mean RQ = 6.22 ± 0.06) and in GU (mean RQ = 6.69 ± 0.10). However, the LGALS1 presented basal expression in both groups (IM: mean RQ = 0.35 ± 0.07; GU: mean RQ = 0.69 ± 0.09). Immunohistochemistry revealed significant positive staining for both the AnxA1 and Gal-1 proteins in the epithelial nucleus and cytoplasm as well as in the stroma of the IM and GU groups (P < 0.05) but absence or low immunorectivity in normal mucosa. CONCLUSION: Our results bring an important contribution by evidencing that both the AnxA1 and Gal-1 anti-inflammatory proteins are deregulated in precancerous gastric lesions, suggesting their involvement in the early stages of gastric carcinogenesis, possibly due to an inflammatory process in the gastric mucosa.

Expression of annexin-A1 and galectin-1 anti-inflammatory proteins and mRNA in chronic gastritis and gastric cancer.

OBJECTIVE: The anti-inflammatory proteins annexin-A1 and galectin-1 have been associated with tumor progression. This scenario prompted us to investigate the relationship between the gene and protein expression of annexin-A1 (ANXA1/AnxA1) and galectin-1 (LGALS1/Gal-1) in an inflammatory gastric lesion as chronic gastritis (CG) and gastric adenocarcinoma (GA) and its association with H. pylori infection. METHODS: We analyzed 40 samples of CG, 20 of GA, and 10 of normal mucosa (C) by the quantitative real-time PCR (qPCR) technique and the immunohistochemistry assay. RESULTS: High ANXA1 mRNA expression levels were observed in 90% (36/40) of CG cases (mean relative quantification RQ = 4.26 ± 2.03) and in 80% (16/20) of GA cases (mean RQ = 4.38 ± 4.77). However, LGALS1 mRNA levels were high (mean RQ = 2.44 ± 3.26) in 60% (12/20) of the GA cases, while low expression was found in CG (mean RQ = 0.43 ± 3.13; P < 0.01). Normal mucosa showed modest immunoreactivity in stroma but not in epithelium, while stroma and epithelium displayed an intense immunostaining in CG and GA for both proteins. CONCLUSION: These results have provided evidence that galectin-1 and mainly annexin-A1 are overexpressed in both gastritis and gastric cancer, suggesting a strong association of these proteins with chronic gastric inflammation and carcinogenesis.

Downregulation of OCLN and GAS1 in clear cell renal cell carcinoma.

Clear cell renal cell carcinoma (ccRCC) is the most common histological subtype of kidney cancer. This carcinoma is histologically characterized by the presence of clear and abundant cytoplasm. In the present study, we sought to identify genes differentially expressed in ccRCC and build a molecular profile of this cancer. We selected genes described in the literature related to cellular differentiation and proliferation. We analyzed the gene and protein expression by quantitative PCR (qPCR) and immunohistochemistry, respectively, and examined possible epigenetic mechanisms that regulate their expression in ccRCC samples and cell lines. Occludin (OCLN) and growth arrest-specific 1 (GAS1) genes were underexpressed in ccRCC, and we report that miR-122 and miR-34a, respectively, may regulate their expression in this cancer. Furthermore, we showed by qPCR and immunohistochemistry that solute carrier family 2 member 1 (SLC2A1) was significantly overexpressed in ccRCC. The set of genes identified in the present study furthers our understanding of the molecular basis and development of ccRCC.

Differentially expressed genes in giant cell tumor of bone.

Giant cells tumors of bone (GCTB) are benign in nature but cause osteolytic destruction with a number of particular characteristics. These tumors can have uncertain biological behavior often contain a significant proportion of highly multinucleated cells, and may show aggressive behavior. We have studied differential gene expression in GCTB that may give a better understanding of their physiopathology, and might be helpful in prognosis and treatment. Rapid subtractive hybridization (RaSH) was used to identify and measure novel genes that appear to be differentially expressed, including KTN1, NEB, ROCK1, and ZAK using quantitative real-time polymerase chain reaction (qRT-PCR) and immunohistochemistry in the samples of GCTBs compared to normal bone tissue. Normal bone was used in the methodology RaSH for comparison with the GCTB in identification of differentially expressed genes. Functional annotation indicated that these genes are involved in cellular processes related to their tumor phenotype. The differential expression of KTN1, ROCK1, and ZAK was independently confirmed by qRT-PCR and immunohistochemistry. The expression of the KTN1 and ROCK1 genes were increased in samples by qRT-PCR and immunohistochemistry, and ZAK had reduced expression. Since ZAK have CpG islands in their promoter region and low expression in tumor tissue, their methylation pattern was analyzed by MSP-PCR. The genes identified KTN1, ROCK1, and ZAK may be responsible for loss of cellular homeostasis in GCTB since they are responsible for various functions related to tumorigenesis such as cell migration, cytoskeletal organization, apoptosis, and cell cycle control and thus may contribute at some stage in the process of formation and development of GCTB.