| Objective This study aimed to explore the effects of raltitrexed on proliferation and apoptosis of gastric cancer cells, and its underlying mechanisms which may provide theoretical foundation for the treatment of gastric cancer.Methods(1) CCK-8 assay was performed to examine the proliferation inhibition of different dose of raltitrexed for 24 h, 48 h and 72 h.(2) The morphological changes of apoptosis were observed by Hoechst 33258 staining.(3) The effects on apoptosis, cell cycle, mitochondrial transmembrane potential and reactive oxygen species were analyzed through flow cytometry.(4) The expression of Bax, Bcl-2, cyt-c and caspase-3 were measured by Western Blot.(5) Models of human gastric cancer in nude mice were established by subcutaneous transplantation; mental state, weight, and tumor growth were recorded; the expression of Ki67 and PCNA were detected by immunohistochemistry; TUNEL staining was measured to detect apoptosis; and the expression of Bax and caspase-3 were analyzed using Western Blot.Results(1) Raltitrexed effectively inhibited the growth of SGC7-7901 cells in a dose- and time-dependent manner.(2) Raltitrexed induced apoptosis, and morphological changes were observed using fluorescence microscope.(3) Raltitrexed significantly arrested cell cycle in G0/G1 phase with a time-dependent manner.(4) A compromised mitochondrialmembrane potential and overproduction of reactive oxygen species demonstrated the involvement of the mitochondrial signaling pathway.(5) The level of Bax, cyt-c and caspase-3 expression were increased by raltitrexed, while the expression of Bcl-2 was reduced.(6) Raltitrexed significantly inhibited the growth of gastric cancer bearing nude mice. Immunohistochemical analysis showed that raltitexed could suppress the expression of Ki67 and PCNA; TUNEL method validated that raltitrexed could induce apoptosis, which may be correlated with the enhanced expression of Bax and caspase-3.Conclusion(1) In vitro experiment, raltitrexed inhibited the proliferation and induced apoptosis of gastric cancer cells, at the same time, blocked the cell cycle at the G0/G1 phase, induced mitochondrial membrane potential decrease and reactive oxygen species generation. The mechanisms may be related to the up-regulation of Bax, cyt-c, caspase-3 and the down-regulation of Bcl-2 protein expression.(2) In vivo experiment, raltitrexed inhibited the growth of xenograft tumor in nude mice, decreased Ki67 and PCNA expression,and induced apoptosis by increasing the expression of Bax and caspase-3.(3) This study provides reliable theoretical data for the treatment of gastric cancer. |
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