Valepotriate-induced apoptosis of gastric cancer cell line MKN-45

AIM: To study the apoptosis of gastric cancer cell line MKN-45 induced by valepotriate and its relationship with the expression of Caspase, P53, and Survivin. METHODS: Gastric cancer cell line MKN-45 was divided into 4 groups, named group A (control), B (treated with Caspase-3, -8 and -9 inhibitors), C (treated with valepotriate) and D (treated with inhibitory agents plus valepotriate), respectively. The apoptosis rates of MKN-45 cells were tested by fluorescence activated cell sorter (FACS) at different time (24, 48 and 72 h) in each group. After exposure to different concentrations of valepotriate for different time (12, 24, 48 and 72 h), MKN-45 cells were collected and the RNA was extracted by tripure agent. The mRNA expression of Survivin was assayed by reverse transcription-polymerase chain reaction (RT-PCR), while the protein expression of P53 and Survivin were detected by immunohistochemical methods 24 hours after exposure to different concentrations of valepotriate (50 and 100 mg/L). RESULTS: The apoptosis rates of MKN-45 cells were not significantly different between group A and B at 24,48 and 72 h (P > 0.05). The apoptosis rates were significantly higher in MKN-45 cells exposed to valepotriate plus Caspase-3 inhibitor or Caspase-9 inhibitor for 24, 48 and 72 h than those in group A (24 h: 5.73%, 5.41% vs 4.38%, P < 0.01; 48 h: 6.88%, 6.32% vs 4.35%, P < 0.01; 72 h: 7.72%, 8.62% vs 4.54%, P < 0.01), but lower than those in group C (24 h: 5.73%, 5.41% vs 8.14%, P < 0.01; 48 h: 6.88%, 6.32% vs 12.31%, P < 0.01; 72 h: 7.72%, 8.62% vs 26.41%, P < 0.01). The apoptosis rates of MKN-45 cells exposed to valepotriate plus Caspase-8 inhibitor for 24, 48 and 72 h were notably increased in comparison with those in group A (8.02% vs 4.38%, P < 0.01; 11.05% vs 4.35%, P < 0.01; 24.86% vs 4.54%, P < 0.01), but was not significantly different from those in group C (P > 0.05). Valepotriate down-regulated the expression of Survivin mRNA in MKN-45 cells in both concentration- and time-dependent manner. Valepotriate also down-regulated the expression of Survivin protein but up-regulated the expression of P53 protein in MKN-45 cells in a concentration-dependent way. CONCLUSION: Valepotriate-induced apoptosis of MKN-45 cells is correlated with the high expression of P53 protein and low expression of Survivin mRNA and protein, and it can be inhibited by Caspase-3 inhibitor or Caspase-9 inhibitor, but not by Caspase-8 inhibitor.

Duke Scholars

Author Fengming Chen Pathology