The Study Of 3-methyladenine Enhances The Drug Sensitivity Of Cisplatin In Gastrical Cancer MGC-803 Cells

Objective: To investigate the cytotoxic effect of 3-Methylademne(3-MA) autophagy inhibitors combined with cisplatin on gastrical cancer cells MGC-803,and its possible mechanism.Methods:Human gastric cancer cell MGC-803 is cultured in vivro. Cell proliferation activity were determined by MTT assay.Four groups of gastrical cancer cells at logarithmic phase were designed in this experiment:Control, 3-Methylademne(3-MA),cisplatin,DDP) and 3-Methylademne+cisplatin(3-MA+DDP).MTT assay was used to observe cell viability.Plate clone formation assay was used to observe colony forming ability.The expression of autophagosomes and autolysosomes were observed by scanning electron microscopic.The aggregation of LC3Ⅱ,which as an autophagy-related protein were dectected by immunohistochemistry. Elisa asssy was used to observe the expression of nucleosomes. Western Blot were used to detect the expression of autophagy-related proteins(Beclin1、PI3KⅢ) and apoptosis-related proteins(caspase3、caspase9).The results:Csiplatin could depress the cell viability of gastric cancer cell MGC-803 in concentration-dependent manners. Compared with Control group and 3-MA group,There was no significant difference among these two groups in the cell viability,clone formation rate,the amount of nucleosome formation and the expression levels of apoptosis proteins(P>0.05). In the 3-Methylademne,the expression of autophagosomes and autolysosomes were less than the Control group. The IHC assay showed that the average optical density of LC3Ⅱin the the 3-MA group was significantly lower than the Control group(P<0.05). Western blot showed that the level of Beclinn1 and PI3KⅢ in the 3-MA group was significantly lower than the Control group(P<0.05). MTT assay showed that the cell viability of the DDP group was significantly lower than the Control group(P<0.05),as well as the clony formation rate(P<0.05). The the expression of nucleosomesand the level of apoptosid-related proteins in the DDP group was significantly higer than the Control group(P<0.05).While, compared with the Control group, the DDP group had a lot of autophagosomes autolysosome gathered and the expression of autophagy-related proteins were more than the Control group(P<0.05).Compared with the DDP group, the 3-MA+DDP group the expression of autophagosomes and autolysosomes were decreased siganificantly. The IHC assay showed that the average optical density of LC3Ⅱ in the 3-MA+DDP group was significantly lower than the DDP group(P<0.05). Western blot showed that the level of Beclin1 and PI3KⅢ in the 3-MA+DDP group were significantly lower than the DDP group(P<0.05), The apoptosis rate of the 3-MA+DDP group was significantly higher than the DDP group(P<0.05). Western Blot showed that the level of caspase3 and caspase9 in the 3-MA+DDP group was significantly higher than the DDP group(P<0.05).Conclusion:3-Methyladenine inhibits protective autophagy response of gastrical cancer cells to chemotherapy drugs,which can enhance the sensitivity of gastrical cancer cells to cisplatin.