Effect Of Altered Autophagy Level On Cisplatin Resistance In Gastric Cancer Cells And Its Molecular Mechanism

AimTo explore the difference of autophagic activity between gastric cancer cells SGC-7901 and its cisplatin-resistant cells SGC-7901/DDP;To explore the effect of altered autophagy level on cisplatin resistance of gastric cancer cells and possible molecular mechanisms.Methods1.The expression levels of autophagy related proteins in SGC-7901 and SGC-7901/DDP cells before and after cells were treated with everolimus and chloroquineThe expressions of autophagic related proteins Beclin-1,LC3?,P62,Atg5 and Atg7 in gastric cancer cells SGC-7901 and its cisplatin-resistant cells SGC-7901/DDP were detected by Western blot,and the expression changes of autophagic related proteins in the two cell lines were analyzed after cells were treated with the autophagyinducereverolimus?RAD001?andtheautophagyinhibitor chloroquine?CQ?,respectively.2.Changes of cell sensitivity to cisplatin after autophagy levels of SGC-7901and SGC-7901/DDP cells were changedAfter SGC-7901 and SGC-7901/DDP cells were treated with everolimus,chloroquine or shRNA-Beclin-1,respectively,for inducing or inhibiting cell autophagy,the two cell line cells were treated with cisplatin,and the colony forming ability,proliferation activity and apoptosis of SGC-7901 and SGC-7901/DDP cells were detected by colony formation assays,CCK-8 assays and flow cytometry,respectively.According to the above CCK-8 results,the IC₅₀0 values of cisplatinfor SGC-7901 and SGC-7901/DDP cells were calculated.And then drug resistance reversal fold was calculated by using the formula:drug resistance reversal fold?RF?=IC₅₀0 of cisplatin in drug combination group/IC₅₀0 of cisplatin in group treated with cisplatin alone.3.The molecular mechanism of changes of cell sensitivity to cisplatin after the autophagy level of SGC-7901 and SGC-7901/DDP cells were changedAfter SGC-7901and SGC-7901/DDP cells were treated with everolimus,chloroquine or shRNA-Beclin-1,respectively,for inducing or inhibiting cell autophagy,the two cell line cells were treated with cisplatin,and total proteins were extracted.The expressions of autophagy related proteins Beclin-1,LC3 II,Atg5 and Atg7,apoptosis related proteins Bcl-2 and Bax,mTOR signaling pathway related proteins p-m TOR?Ser2448?,Raptor,p-p70S6K,p-Akt?ser473?and Akt in both cells were detected by Western blot in order to analyze the molecular mechanism of the autophagy effect on cisplatin resistance of gastric cancer cells.Results1.ThedifferenceofautophagicactivitybetweenSGC-7901and SGC-7901/DDP cells?1?The expression levels of Atg5 and LC3 II in cisplatin-resistant SGC-7901/DDP cells were significantly lower than that in SGC-7901 cells,and the expression levels of P62 and Bcl-2 were significantly higher than that in SGC-7901cells?P<0.001?,but the expression levels of Beclin-1 and Atg7 had no significant difference in the two cell line cells?P>0.05?.?2?Compared with SGC-7901 and SGC-7901/DDP cells,respectively,the expression levels of autophagy related proteins P62,Beclin-1,LC3 II,Atg5 and Atg7in the two cell line cells treated with everolimus or chloroquine alone were increased in varying degrees?P<0.05?.2.The effect of altered autophagy level on cisplatin resistance in gastric cancer cells?1?Compared with SGC-7901 and SGC-7901/DDP cells untransfected or transfected with empty plasmid and then treated with cisplatin alone,respectively,colony forming ability of cells,cell proliferation activity and cell apoptosis had no obvious change in SGC-7901 and SGC-7901/DDP cells were pretreated with everolimus and then with cisplatin?P>0.05?;colony forming ability of cells and cell proliferation activity were significantly decreased,and cell apoptosis rate was significantly increased in SGC-7901 and SGC-7901/DDP cells were pretreated with chloroquine and transfected with shRNA-Beclin-1 plasmid and then with cisplatin?P<0.001?.It suggested that autophagy might promote the survival of SGC-7901 and SGC-7901/DDP cells.?2?SGC-7901 and SGC-7901/DDP cells were pretreated with chloroquine?10,20 and 30?mol/L?and then treated with different concentrations of cisplatin,the drug resistance reversal fold demonstrated that the sensitivity of SGC-7901 cells to cisplatin was increased by 1.73 times,3.12 times and 8.32 times,and the sensitivity of SGC-7901/DDP cells to cisplatin was increased by 2.36 times,5.01 times and13.87 times.It was suggested that chloroquine had better reversal effect on cells treated with cisplatin,and cisplatin-resistant cells were more sensitive to autophagy regulation.3.The molecular mechanism of autophagy affecting on resistance of gastric cancer cells to cisplatin?1?Compared with SGC-7901 and SGC-7901/DDP cells,the expression levels of mTORC1 related proteins p-mTOR?Ser2448?,p-p70S6K and Raptor were decreased slightly after the two cell line cells were treated with everolimus alone?P<0.05?;Compared with SGC-7901 and SGC-7901/DDP cells treated with cisplatin,in the two cell line cells pretreated with everolimus then cisplatin,the expression levels of autophagy related proteins were increased slightly?P<0.05?,mTORC1 related proteins p-mTOR?Ser2448?,p-p70S6K and Raptor were decreased slightly?P<0.05?,and the ratio of Bcl/Bax had no obvious change?P>0.05?.?2?Compared with SGC-7901 and SGC-7901/DDP cells,the expression levels of mTORC1 related proteins p-mTOR?Ser2448?,p-p70S6K and Raptor were significantly decreased after the two cell line cells were treated with chloroquine alone?P<0.001?;compared with SGC-7901 and SGC-7901/DDP cells treated with cisplatin,the two cell line cells pretreated with chloroquine then with cisplatin,the expression levels of autophagy related proteins were significantly increased?P<0.001?,mTORC1 related proteins p-mTOR?Ser2448?,p-p70S6K and Raptor and the ratio of Bcl/Bax were significantly decreased?P<0.001?.It was suggested that autophagy inhibition of chloroquine enhanced the cisplatin induced apoptosis effect in SGC-7901 and SGC-7901/DDP cells,and the mechanism was associated with the accumulation of autophagy related proteins induced by chloroquine,which significantly reduced the activity of mTORC1 related proteins.?3?Compared with SGC-7901 and SGC-7901/DDP cells transfected with shRNA-NC,respectively,the expression levels of Beclin-1 and p-Akt?ser473?were significantly decreased after SGC-7901and SGC-7901/DDP cells were transfected with shRNA-Beclin-1?P<0.001?;compared with SGC-7901 and SGC-7901/DDP cells transfected with shRNA-NC and then treated with cisplatin,the expression level of Beclin-1 and the ratios of p-Akt?ser473?/Akt and Bcl-2/Bax were significantly decreased after SGC-7901 and SGC-7901/DDP cells were transfected with shRNA-Beclin-1 and then treated with cisplatin?P<0.001?.It was suggested that autophagy inhibition of shRNA-Beclin-1 can enhance the cisplatin induced apoptosis effect in SGC-7901 and SGC-7901/DDP cells,and its mechanism was related to the significant down-regulation of p-Akt?ser473?activity by shRNA-Beclin-1.ConclusionsThere are significant differences of autophagy levels between SGC-7901 and SGC-7901/DDP cells.Chloroquine and shRNA-Beclin-1 can reverse the cisplatin resistance in gastric cancer cells.The mechanism may be related that autophagy inhibition of chloroquine caused the accumulation of autophagy related proteins then downregulating mTORC1 related proteins and knocking down the expression level of autophagy related protein with shRNA-Beclin-1downregulated the expression of p-Akt?ser473?.