| IntroductionConnexin 26 (Cx26) is one of the connexin (Cx) family members, with the smallest molecular weight (26 KD). The Cx proteins are encoded by a multigene family that so far 20 different human Cx genes have been identified, and named by molecular weight, for example Cx26, Cx32 et al. These trans-membrane proteins in the human, comprise the main subunits of gap junctions (GJs)-the connexon which is composed of six Cxs. Two connexons from adjacent cells forms a complete gap junction channel that allow intercellular passage of ions and molecules of up to 1000 Da, including second messengers. Gap junctional intercellular communication (GJIC), meditated by GJs, is believed to be involved in the regulation of cell proliferation differentiation, and tissue development. GJIC also plays an important role in the maintenance of tissue structure and functions, especially homeostasis.The majority of tumors show aberrant expression of Cxs, which have been classified as tumor suppressors. Analogous to other Cxs, Cx26 has been considered to serve as a tumor suppressor gene, however, it is more controversial than other members in cancer progression. Luiza Kanczuga-Koda et al revealed that loss of normal intercellular Cx26 occurred during colorectal carcinogenesis and alteration of expression pattern from membrane to cytoplasm. On the contrary, accumulating evidence indicated that Cx26 is overexpressed in several carcinomas, for example, prostate and skin carcinoma. Cxs may be also induced by growth hormones, and involved in regulation of cell proliferation and apoptosis indirectly. Together, these strands of evidence appear to show the contradictory roles of Cx26 in carcinomas, which needs further investigation.Little is known about the expression pattern of Cx26 and its roles in human gastric carcinoma (GC) up to now. In this study, we focused on the expression pattern of Cx26 in large amount cases of gastric cancer by means of immunohistochemistry, to explore its clinical significance and prognostic analysis. The protein level and mRNA level of Cx26 in fresh tissues were also analysed by Western blot and Realtime PCR analysis, meanwhile, pcDNA3.1-CFP-Cx26 eukaryon expression plasmid were transfected into gastric cancer cell line MGC-803. The effects on cell proliferation and apoptosis were observed and the related mechenisms were investigated. And our findings possess not only obviously theoretical significance for understanding the biological characteristics of tumors, but also clinical and social effects.Part I Expression of Cx26 in human gastric cancer and its relation with clinicopathologic variables and prognostic analysisObjective To assess Cx26 expression in gastric carcinoma and its relationship with clinic characters, including survival analyses.Methods 213 Paraffin-embedded tissue samples and 20 fresh tissue samples were obtained from patients of gastric carcinoma. Cx26 expression was assessed in 205 tissue samples by immunohistochemistry. Realtime PCR and Western blot was used to detect protein and mRNA level of Cx26 in fresh tissues.Results For each specimen, the evaluation of Cx26 expression was analyzed over ten different visual fields at a power of x400, and mean percentage of membranous staining cells or cytoplasmic staining cells were calculated. It was determined as positive when at least 10% of cancer cells exhibit cytoplasmic or membranous stained positively.79 of 205 (38.5%) gastric carcinoma cases were positive for Cx26 with mainly cytoplasmic localization comparing to sporadically membranous staining in normal epithelium, and the expression levels were confirmed by Western blot and Realtime PCR. Negative associations were revealed between Cx26 expression and most clinicopathologic features (all P<0.05). Notably, high Cx26 expression was associated with histological intestinal-type (P=0.017). The multivariate regression analysis revealed that positive Cx26 expression was an independent prognostic predictor of intestinal-type GC (P= 0.023, HR=2.019).Conclusions Aberrant expression of Cx26 in cytoplasma plays a tumor-suppressor role in gastric carcinoma and is an independent biomarker for favorable prognosis in intestinal-type gastric carcinoma. Partâ…¡Overexpression of Cx26 in regulation of proliferation and apoptosis in MGC-803 cell line and its related mechanisms explorationObjectives To clarify the effect of Cx26 on regulating proliferation and apoptosis in human gastric cancer cell line MGC-803 and its related mechanisms.Method pcDNA3.1-CFP-Cx26 eukaryon expression plasmid were transfected into MGC-803 cell line. Cell growth curve (MTT) and flow cytometry analysis and were used to analyze cells growth, apoptosis and proliferation. Western blot was used to detect the expression of apoptosis-related protein Fas, Fas-L, DR5, caspase-3, Bax, Bcl-2, Bcl-xL, cyclin-dependent kinases inhibitory protein (CKI) p21, proliferating cell nuclear antigen (PCNA) and insulin-like growth factor receptor-I.Results Transfected with pcDNA3.1-CFP-Cx26 plasmid suppressed MGC-803 cells growth, increased the fraction of apoptotic cells and arrested cell cycle in G2/M phase.In Cx26 overexpressing MGC-803 cells, pro-apoptotic factors, Fas, Fas-L, DR5, Bax, were remarkably higher than control cells. Anti-apoptotic factors, Bcl-2 and Bcl-xL were significantly lower than control cells. Active caspase-3 was also upregulated. p21 and PCNA was unchanged. IGF-IR was gradually increased following the overexpression of Cx26.Conclusion Overexpression of Cx26 suppressed the growth of MGC-803 cells, induced cell apoptosis and promoted the expression of IGF-IR. The effects of Cx26 on cancer cells growth were various and multiple.
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