AIM: To determine the effect of cis -9, trans -11-conjugated linoleic acid (c9, t11-CLA) on the cell cycle of gastric cancer cells (SGC-7901) and its possible mechanism in inhibition cancer growth. METHODS: Using cell...AIM: To determine the effect of cis -9, trans -11-conjugated linoleic acid (c9, t11-CLA) on the cell cycle of gastric cancer cells (SGC-7901) and its possible mechanism in inhibition cancer growth. METHODS: Using cell culture and immunocytochemical techniques, we examined the cell growth, DNA synthesis, expression of PCNA, cyclin A, B(1), D(1), p16(ink4a) and p21(cip/waf1) of SGC-7901 cells which were treated with various c9, t11-CLA concentrations (25, 50, 100 and 200 micromol.L(-1))of c 9, t 11-CLA for 24 and 48h, with a negative control (0.1% ethane). RESULTS: The cell growth and DNA synthesis of SGC-7901 cells were inhibited by c9, t11-CLA.SGC-7901 cells. Eight day after treatment with various concentrations of c9, t11-CLA mentioned above, the inhibition rates were 5.92%, 20.15%, 75.61% and 82.44%, respectively and inhibitory effect of c9, t11-CLA on DNA synthesis (except for 25 micromol.L, 24h) showed significantly less (3)H-TdR incorporation than that in the negative controls (P【0.05 and P【0.01). Immunocytochemical staining demonstrated that SGC-7901 cells preincubated in media supplemented with different c9, t11-CLA concentrations at various times significantly decreased the expressions of PCNA (the expression rates were 7.2-3.0%, 24h and 9.1-0.9% at 48h, respectively), Cyclin A (11.0-2.3%, 24h and 8.5-0.5%,48h), B(1) (4.8-1.8% at 24h and 5.5-0.6% at 48h)and D(1) (3.6-1.4% at 24h and 3.7%-0 at 48h) as compared with those in the negative controls(the expressions of PCNA, Cyclin A, B(1) and D(1) were 6.5% at 24h and 9.0% at 48h, 4.2% at 24h and 5.1% at 48h, 9.5% at 24h and 6.0% at 48h,respectively)(P【0.01), whereas the expressions of P16(ink4a) and P21(cip/waf1), cyclin-dependent kinases inhibitors(CDKI), were increased. CONCLUSION: The cell growth and proliferation of SGC-7901 cell is inhibited by c9, t11-CLA via blocking the cell cycle, with reduced expressions of cyclin A,B(1) and D(1) and enhanced expressions of CDKI(P16(ink4a) and p21(cip/waf1)).展开更多
AIM To further investigate the effect of cyclin D1 on the biologic behavior of cancer cells and its potential role in gene therapy of tumor. METHODS A cyclin D1 subcloning plasmid termed BKSD1 was constructed by su...AIM To further investigate the effect of cyclin D1 on the biologic behavior of cancer cells and its potential role in gene therapy of tumor. METHODS A cyclin D1 subcloning plasmid termed BKSD1 was constructed by subcloning the human cyclin D1 cDNA into Bluescript KS, a plasmid vector with a pair of T7 and T3 promoters, with recombinant DNA technology of molecular biology. So, it is easy to generate digoxigenin (DIG) labeled RNA probes of antisense and sense to cyclin D1 using RKSD1 as a template vector. PDORD1AS, an eukaryotic expression vector containing the full length human cyclin D1 cDNA in its antisense orientation cloned into the retroviral vector pDOR neo, was successfully constructed with BKSD1 to change restriction sites. A gastric cancer cell line, SGC7901/VCR, was transfected with pDORD1AS by Lipofect Amine mediated introduction and a subline termed SGC7901/VCRD1AS, which had stable overexpression of antisense RNA to cyclin D1, was obtained by selection in G418. The subline, control subline transfected pDOR neo and SGC7901/VCR were evaluated by methods of immunohistochemistry, flow cytometry, molecular hybridization, morphology and cell biology. RESULTS Compared with control cell lines, SGC7901/VCRD1AS had a reduced expression of cyclin D1 (inhibition rate was about 36%), increased cell size and cytoplasm to nucleus ratio, increased doubling time (42 2h to 26 8h and 26 4h), decreased saturation density (18 9×10 4 to 4 8×10 5 and 4 8×10 5), increased percentage of cells in the G1/G0 phase (80 9%-64 6% and 63 8%), reacquired serum dependence, and a loss of tumorigenicity in nude mice (0/4 to 4/4 and 4/4). CONCLUSION Stable overexpression of antisense RNA to cyclin D1 can reverse the transformed phenotype of human gastric cancer cells and may provide an approach of gene therapy for gastric cancer.展开更多
基金the National Natural Science Foundation of China,No.39870661
文摘AIM: To determine the effect of cis -9, trans -11-conjugated linoleic acid (c9, t11-CLA) on the cell cycle of gastric cancer cells (SGC-7901) and its possible mechanism in inhibition cancer growth. METHODS: Using cell culture and immunocytochemical techniques, we examined the cell growth, DNA synthesis, expression of PCNA, cyclin A, B(1), D(1), p16(ink4a) and p21(cip/waf1) of SGC-7901 cells which were treated with various c9, t11-CLA concentrations (25, 50, 100 and 200 micromol.L(-1))of c 9, t 11-CLA for 24 and 48h, with a negative control (0.1% ethane). RESULTS: The cell growth and DNA synthesis of SGC-7901 cells were inhibited by c9, t11-CLA.SGC-7901 cells. Eight day after treatment with various concentrations of c9, t11-CLA mentioned above, the inhibition rates were 5.92%, 20.15%, 75.61% and 82.44%, respectively and inhibitory effect of c9, t11-CLA on DNA synthesis (except for 25 micromol.L, 24h) showed significantly less (3)H-TdR incorporation than that in the negative controls (P【0.05 and P【0.01). Immunocytochemical staining demonstrated that SGC-7901 cells preincubated in media supplemented with different c9, t11-CLA concentrations at various times significantly decreased the expressions of PCNA (the expression rates were 7.2-3.0%, 24h and 9.1-0.9% at 48h, respectively), Cyclin A (11.0-2.3%, 24h and 8.5-0.5%,48h), B(1) (4.8-1.8% at 24h and 5.5-0.6% at 48h)and D(1) (3.6-1.4% at 24h and 3.7%-0 at 48h) as compared with those in the negative controls(the expressions of PCNA, Cyclin A, B(1) and D(1) were 6.5% at 24h and 9.0% at 48h, 4.2% at 24h and 5.1% at 48h, 9.5% at 24h and 6.0% at 48h,respectively)(P【0.01), whereas the expressions of P16(ink4a) and P21(cip/waf1), cyclin-dependent kinases inhibitors(CDKI), were increased. CONCLUSION: The cell growth and proliferation of SGC-7901 cell is inhibited by c9, t11-CLA via blocking the cell cycle, with reduced expressions of cyclin A,B(1) and D(1) and enhanced expressions of CDKI(P16(ink4a) and p21(cip/waf1)).
文摘AIM To further investigate the effect of cyclin D1 on the biologic behavior of cancer cells and its potential role in gene therapy of tumor. METHODS A cyclin D1 subcloning plasmid termed BKSD1 was constructed by subcloning the human cyclin D1 cDNA into Bluescript KS, a plasmid vector with a pair of T7 and T3 promoters, with recombinant DNA technology of molecular biology. So, it is easy to generate digoxigenin (DIG) labeled RNA probes of antisense and sense to cyclin D1 using RKSD1 as a template vector. PDORD1AS, an eukaryotic expression vector containing the full length human cyclin D1 cDNA in its antisense orientation cloned into the retroviral vector pDOR neo, was successfully constructed with BKSD1 to change restriction sites. A gastric cancer cell line, SGC7901/VCR, was transfected with pDORD1AS by Lipofect Amine mediated introduction and a subline termed SGC7901/VCRD1AS, which had stable overexpression of antisense RNA to cyclin D1, was obtained by selection in G418. The subline, control subline transfected pDOR neo and SGC7901/VCR were evaluated by methods of immunohistochemistry, flow cytometry, molecular hybridization, morphology and cell biology. RESULTS Compared with control cell lines, SGC7901/VCRD1AS had a reduced expression of cyclin D1 (inhibition rate was about 36%), increased cell size and cytoplasm to nucleus ratio, increased doubling time (42 2h to 26 8h and 26 4h), decreased saturation density (18 9×10 4 to 4 8×10 5 and 4 8×10 5), increased percentage of cells in the G1/G0 phase (80 9%-64 6% and 63 8%), reacquired serum dependence, and a loss of tumorigenicity in nude mice (0/4 to 4/4 and 4/4). CONCLUSION Stable overexpression of antisense RNA to cyclin D1 can reverse the transformed phenotype of human gastric cancer cells and may provide an approach of gene therapy for gastric cancer.
