| Background and Objective:Insulin resistance(IR)is a chronic pathological process that liver,skeletal muscle and fat tissues behave resistance to insulin or impaired glucose utilization,which is also closely related to the occurrence,development and prognosis of liver cancer.There are a large number of exploratory researches around the relationship between insulin resistance and tumors,but so far the relevance of insulin-resistance and drug-resistance in liver cancer is still unclear.In this study,the hepatoma HepG2 cells are induced with high concentration of insulin to establish a stable insulin-resistant cell model,named as HepG2/IR cells,and to study the sensitivity to conventional chemotherapy drugs,autophagy activity and endoplasmic reticulum stress(ER stress)response of HepG2/IR cells,and the ultimate aim is to explore the relationship between insulin resistance and drug sensitivityin hepatocellular carcinoma,and then to reveal the roles of autophagy activity and ER stress in drug resistance of insulin-resistant hepatocarcinoma cells.Methods:HepG2 cells underwent long induction with high concentration of insulin to establish an insulin-resistant HepG2/IR cell model with well stability.Glucose oxidase-peroxidase method was used to detect glucose consumption,and Periodic acid Schiff stain was for testing the glycogen synthesis.Flow cytometry and qRT-PCR were employed to determine the expression of insulin receptor and glucose transporters-2,and the cell adhesion,migration and invasive abilities were observed with Transwell assay.MTT colorimetric assay and Annexin V/PI double staining were used to examinthe cell proliferation andapoptosis,respectively.The ultrastructure of the cells and ER and autophagosomes were anlysized by tansmission electron microscopy(TEM),and Monodansylcadaverin(MDC)staining was used to observe the formation of autophagic vacuole with fluorescent microscope.The expressions of autophagy-related proteins Beclin-1,LC3,P62,HSC70 and LAMP2a,ER stress-related proteins GRP78,XBP-1,PERK and p-PERK,apoptosis-related proteins Bcl-2 and Caspase-3 and P-glycoprotein(P-gp)were detected by western-blotting.Flow cytometry is used to assess reactive oxygen,mitochondrial membrane potential and the efflux function of P-glycoprotein.Results:1.In stable HepG2/IR model cells,established with inducetion of 0.5-1?mol/Linsulin for 72 h,the glucose consumption and glycogen synthesis were significantly reduced,meanwhile the expression of insulin receptor and glucose transporter-2 was obviously down-regulated compared with the parent HepG2 cells,which maintained steadily insulin-resistance more than 72 h and could be reversed by insulin sensitizer pioglitazone hydrochloride(PH)of 10mmol/L.These data meaned that the insulin-induced HepG2/IR cells obtained stable insulin resistance.Compared with the parental cells,the sensitivity of HepG2/IR cells to chemotherapy drugs,such as cisplatin,5-fluorouracil,vincristine and mitomycin,significantly decreased,the value of IC50 increased by 0.59 to 2.80 folds and apoptosis rate decreased by 13.21-24.64%,and the drug-sensitivity of HepG2/IR cells could be reversed by PH and restored to near parental HepG2 cells.Compared with HepG2 cells,the expression of Bcl-2 increased by 23.89%and that of Caspase-3 decreased by 33.16%in cisplatin-treated HepG2/IR cells.In addition,the ablities of adhesion,migration and invasion of HepG2/IR cells obviously enhanced and could be reversed by PH.These data demonstrate that insulin-resistant HepG2/IR cells possess enhanced drug-reistance and abilities of adhesion,migration and invasion.2.Electron microscopy and MDC staining observation showed that HepG2/IR cells have more autophagic vacuoles than HepG2 cells.The autophagy activity was significantly increased during the process of insulin-resistance induction.The macroautophagy increased during the early phase and fell during the late,while CMA increased much later and lasted long steadily,and the reversal of insulin-resistance by insulin sensitizer PH led to the decrease of autophagy activity.Cisplatin induced both HepG2/IR cells and HepG2 cells to apoptosis,accompanied by enhancement of autophagy activity.3-MA,an autophagy inhibitor,could improve the sensitivity of HepG2/IR and HepG2 cellsto cisplatin,the apoptosis rate increased by 59.5%(HepG2)and 114.9%(HepG2/IR),respectively;The expression of Bcl-2 down-regulated by 39.2%(HepG2)and 53.2%(HepG2/IR),and the activation of Caspase-3 up-regulated by 52.4%(HepG2)and 67.7%(HepG2/IR),compared to the cisplatin treatment alone.The autophagy inducer Rapamycin played the opposite effects by enhancing autophagy.These results confirm that the elevated autophagy is one of the main mechanism of drug resistance in HepG2/IR cells.3.The decrease of mitochondrial membrane potential accompanied with the increase of ROS existed during the process of insulin resistance induction,and ultrastructure observation showed that ER expansion and degranulation,accompanied with mitochondria swelling,in HepG2/IR cells.Compared with parental HepG2 cells,the expression of ER-related proteins GRP78,p-PERK and XBP-1 in HepG2/IR cells up-regulated by 0.66 to 1.72 folds,and reversal of IR bring about the reduce of the increased expression of the above three proteins.Enhanced autophagy activity by rapamycin could reduce the expression of ER stress-related proteins,decreased by 29.9%(GRP78),24.6%(XBP-1)and 38.1%(pPERK/PERK)in HepG2 cells,and by 35.6%(GRP78),33.0%(XBP-1)and 46.0%(pPERK/PERK)in HepG2/IR cells,respectively.On the contrary,inhibited autophagy activity by 3-MA promoted the ER stress-related proteins,for HepG2 cells increase by 48.1%(GRP78),134.4%(XBP-1)and 42.9%(pPERK/PERK),and for HepG2/IR cells by 43.2%(GRP78),113.4%(XBP-1)and 22.4%(pPERK/PERK),respectively.During the induction of IR in HepG2 cells,P-glycoprotein expression was gradully increased by 0.34 to 1.37 times and its efflux function also enhanced,and which could be reversed by inhibition of IR.These data demonstrate that the insulin-resistant HepG2 cells are provided with elevated autophagy activity and ER stress response,which coordinated with each other to protect the cells from being damaged.Conclusion:1.The insulin-resistantHepG2/IR model cells induced with high concentration of insulin possess much reduced sensitivity to converntional chemotherapy drugs and enhaced abilities of cell adhesion,migration and invasion.2.The enhanced activitiesof macroautophagy and chaperone-mediated autophagy exists in HepG2/IR cells and can be inhibited by reversion of insulin resistance.The resistance to DDP of HepG2/IR cells can be reinforced by promoting autophagy and weakened by inhibiting autophagy.3.Insulin-resistant HepG2/IR cells have higher ER stress responsewhich can be reversed by insulin sensibilization.The ER stress response in HepG2/IR cells is able to be strengthened by inhabiting autophagy or be lowered by enforcing autophagy.These data prompt that in insulin-resistant hepatoma cells,ER stress response coordinates with autophagy activity to play the effects of protecting the cells against injury.4.The expression and efflux function of P-gp dynamically exhibited significant increase during the formation of insulin resistance in HepG2 cells,which is quite consistent with drug resistance and ER stress response and can be reduced via increasing the insulin sensitivity.These findings suggest that P-gp may be a powerful factor mediating ER stress response and drug resistance in HepG2/IR cells. |
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