The Function And Mechanism Of Cisplatin-induced Autophagy Of Retinoblastoma Y79 Cells

Objectives To investigate the role and mechanism of cisplatin-induced autophagy in retinoblastoma Y79 cells.Methods 1 The Y79 cells were cultured at 0,1,5,10,and 20μM concentrations in Cis,and the IC50 was measured by CCK-8.2 Y79 cells were divided into control group,Cis group,and cisplatin group.Cisplatin was used in combination with the autophagy blocker 3-methyladenine（Cis+3-MA）and the autophagosomes were observed by transmission electron microscope.3 The cells were stained with Dansylcadaverine（MDC）and the optical density（OD）of each group was measured by flow cytometry.4 Each group of cells was cultured at 0,5,10,25,50,and 100μM in Cis,and cell inhibition rate was measured by CCK-8 method.5 The apoptosis of each group was detected by Annexin V/PI double staining.6 The expression of apoptosis and survival（Bax,Bcl-2）and drug-resistance（MRP3,MRP7,P-gp）genes was detected by q-PCR in each group.7Fluo-4 AM calcium ion fluorescent probe was used to detect the intracellular calcium level in each group.8 The expression of CaMKK2,AMPK,mTORC1,and LC3 protein in each group was detected by Western Blot.Results 1 After treated with cisplatin,the number of autophagosomes in double-layered or multilamellar membranes was significantly increased by transmission electron microscopy.After autophagy blocking agent 3-MA was applied,the number of autophagosomes decreased.2 MDC staining flow cytometry OD₃₅₅,Cis group OD values were significantly higher than the Control group（P<0.01）,Cis+3-MA group OD values lower than the Cis group（P<0.05）.3 After pretreatment of cells with autophagy blocker3-MA,the inhibitory rate of cisplatin at each concentration point was greater than that of cells treated with cisplatin alone.4 After treated with cisplatin,the apoptotic rate was increased compared with the control group（P<0.05）.After 3-MA was used to downregulate autophagy,the apoptosis rate was further increased（P<0.05）.5 Cisplatin treated cells,compared with the Control group,the inhibition of apoptosis protein Bcl-2expression was down-regulated（P<0.01）,Bax expression was up-regulated（P<0.01）,and the resistance-related genes MRP3 in each group.The transcription level of MRP7 and P-glycoprotein decreased（P<0.01）;Bcl-2 expression continued to decrease after adding autophagy blocker 3-MA（P<0.01）,while Bax continued to rise（P<0.05）.The transcription levels of drug-related genes MRP3,MRP7,and P-glycoprotein continued to decrease（P<0.05）.6 Fluo-4 AM calcium ion probe staining laser confocal microscopy observation showed that after cisplatin treatment of tumor cells,intracellular calcium ion fluorescence was significantly higher than the control group.7 Western-blot results showed that compared with the control group,calmodulin-dependent protein kinase kinase 2（CAMKK2）,p-AMPK,and LC3 II were significantly up-regulated and mTORC1 was down-regulated in the Cis group（P<0.05）.Conclusions 1 Cisplatin can induce autophagy in retinoblastoma y79 cells.2 Inhibition of autophagy can increase the apoptosis rate of cisplatin-induced retinoblastoma y79 cells,and reduce the expression of cell resistance-related genes,suggesting that cisplatin-induced autophagy plays a protective role on cells and participates in the chemotherapy resistance of retinoblastoma y79 cells.3 Cisplatin may activate cam kk 2/AMPK/MTO rc1 pathway by increasing intracellular calcium concentration to induce autophagy in y79 cells.