Study On The Oridonin-induced DNA Damage And Autophagy In Gastric Cancer SGC-7901 Cells

Objects: To study the inhibitory effect of oridonin on the proliferation of gastric cancer SGC-7901 cells and the effect of oridonin-induced DNA damage.To explore whether oridonin can induce autophagy in gastric cancer SGC-7901 cells.Methods:(1)MTT assay and crystal violet were used to detect the cytotoxicity effect of oridonin on gastric cancer SGC-7901 cells.(2)Flow cytometry was used to detect the drug effect on the cell cycle of gastric cancer cells.(3)Acridine orange was used to detect autophagic acid vesicles.(4)The DNA damage related proteins of ?H2AX(S139),phospho-ATM(S1981),phospho-P53(S15),phospho-CHK2(T68),phospho-cdc2(Y15),phospho-cdc25c(S216)and autophagy-related proteins LC3-?,LC3-? and Beclin1 were detected by Western blot.(5)Immunofluorescence assay was used to detect the effect of oridonin on DNA damage-related protein phospho-ATM(S1981)and ?-H2AX(S139)in gastric cancer SGC-7901 cells.Results:(1)MTT results showed that oridonin could inhibit the growth of SGC-7901 cells in a dose-dependent manner.(2)Crystal violet showed that the numbers of the cells in each well of the six-well plates gradually decreased.The OD value measured on the microplate reader showed a gradual decrease.(3)Flow cytometry showed that the numbers of G0/G1 cells decreased gradually with the increase of the drug doses,while the numbers of G2/M phase cells gradually increased and reached the highest peak at 40?M,suggested G2/M arrest.(4)Acacrid orange showed that the numbers of acidic autophagic vesicles increased in a dose-dependent manner.(5)The protein levels of ?H2AX(S139),phospho-ATM(S1981),phospho-P53(S15),phospho-CHK2(T68),phospho-cdc2(Y15),phosp ho-cdc25c(S216)were increased in a dose-dependent manner.The autophagy-related proteins LC3-?decreased,while LC3-? and Beclin1 were increased.(6)The immunofluorescence showed that the signal intensity of phospho-ATM(S1981)and?H2AX(S139)increased with the increase of drug doses.Conclusion:(1)Oridonin can inhibit the proliferation of SGC-7901 cells and induce G2/M cell cycle arrest by “ATM-CHK2-Cdc2” pathway.The drug can induce DNA damage,the specific mechanism of Oridonin-induced DNA damage response need to be further studied.(2)In addition,Oridonin can induce the autophagy of gastric cancer SGC-7901 cells.The protein levels of autophagy-related LC3-?and Beclin1 increase with the increase of the drug doses.