Effects Of MnSOD Transfection And Tanâ…¡A On The Growth Of Gastric Cancer Cell Lines And The Molecular Mechanisms

Effects of MnSOD Transfection and TanⅡA on the growth of gastric cancer cell lines and the molecular mechanisms Part 1. MnSOD expression is relevant with endogenous ROS differentiation degree and proliferation in gastric cancer cell linesObjective Try to measure maganese superoxide dismutase(MnSOD) expression in normal gastric mucosa and MKN-28 SGC7901 BGC823 HGC-27 gastric cancer cell lines. Furthermore,try to elucidate the relationships among MnSOD expression and endogenous reactive oxygen species(ROS), tumor differentiation degree and proliferate activities in these cell lines. Methods Enzyme activities of alkaline phosphatase (ALP) lactate dehydrogenase (LDH), which represent the differentiation degree of these cell lines, were measured by enzymatic catalysis assay. RT-PCR and DCFH-DA fluorescence assay were taken to identify the MnSOD transcription and ROS contents respectively. In addition, the proliferate activities were measured by MTT assay and direct cells count. Results Enzymatic activities of ALP and LDH are significantly different in SGC7901,BGC823,HGC-27 cell lines. Meanwhile,it indicates much less MnSOD expressions in these four gastric cancer cell lines compared with that in the normal gastric mucosa. With the decrease of MnSOD transcriptions, the endogenous ROS level rise synchronously as well as the proliferation activities in MKN-28 SGC7901 BGC823 HGC-27 gastric cancer cell lines. Part 2. Effects on the proliferation of SGC7901 via MnSOD transfectionObjective To observe the effects on proliferation of SGC7901 mediated by MnSOD gene transfection.Methods Transferring sense and antisense MnSOD cDNA carrier pHβA-SOD (-)/pHβA-SOD (+) or mock vector into SGC7901 gastric cancer cell by electroporation. Screening stable expression sublines by 400ug/ml G418solution and further confirmed by RT-PCR and western blot. Then the subtypes of SGC7901 were marked as MnSOD-7901 MnSOD-AS7901 and vector-7901 respectively. The Copper-Zinc superoxide dismutase (CuZnSOD) contents in the transfectants were also measured by radioimmunoassay. Evaluate the proliferation activities in these three subtypes by direct cells count, MTT assay, plate clone formation test and nude mice transplanting tumor model respectively. Results Compared with the vector-7901, MnSOD-7901 reveals such characteristics as followed: (a) Partial inhibition of proliferation activities reflected by growth curves；(b) Decreasing clone formation on plate; (d) Remarkable growth retardation of transplanting tumor in nude mice. On the contrary, the MnSOD-AS7901 subline reveals the promotion of tumor proliferation. Part 3. The molecular mechanisms involved in the proliferation modulated by MnSOD transfectionObjective Try to disclose the molecular mechanisms associated with MnSOD transfection, which are involved in modulating the proliferation activities in SGC7901 gastric cancer cell line.Methods DCFH-DA fluorescence assay were used to measure the ROS contents in MnSOD－7901 MnSOD－AS7901 and Vector－7901 sublines. Ultra structures of the transfectants were studied by transmission electron microscopy. Cell cycles were examined by flow cytometry. Immunohistochemistry assay were taken to identify P53 Bcl－2 Ki67 and C-erbB2 protein expression in the slices of nude mice transplanted tumor.P53 mRNA expression was further evaluated by RT-PCR.Results Compared with the vector-7901, MnSOD-7901 subline reveals such genomic or molecular changes: (a) Decreasing endogenous ROS level; (b) G1 phase arrest in cell cycle;(c) Down-regulated expression of Bcl－2 Ki67 and C-erbB2.(d) P53 mRNA expression was up-regulated. Meanwhile, the MnSOD-AS7901 subline show the opposite changes.Part4. Apoptosis induced by TashinoneⅡA in SGC7901 gastric cancer cell Objective To assess the anticancer effects of antioxidant TashinoneⅡA (TanⅡA) on SGC7901 gastric cancer cell in vitro.Methods SGC7901 gastric cancer cells were inoculated into the 96-well-plates. They were trea...