Rosiglitazone Enhances The Anti-tumor Effects Of Chemotherapy Drugs In Insulin-resistant Hepatoma HepG2/IR Cells

Research background and purpose:Liver cancer is the fifth most common malignant tumor in the world.Although the clinical diagnosis and treatment of liver cancer have made great progress in recent years,liver cancer still has high recurrence rate and very low survival rate.Liver cancer also has a low sensitivity to most anticancer drugs,so there is an urgent need to find novel ways to increase the sensitivity of chemotherapy.In addition,insulin resistance,one of the causes of liver cancer,promotes the occurrence of liver cancer.Rosiglitazone belongs to the class of thiazolidinedione insulin sensitizers.Recent studies have shown that rosiglitazone has a certein anticancer effect on human malignant cells and can reduce the side effects of chemotherapy.In this paper,we observed the effects of rosiglitazone combined with adriamycin on proliferation,autophagy,apoptosis,cell cycle,migration and growth of hepatocellular carcinoma HepG2/IR cells with insulin resistance from both cellular and animal aspects,and subsequently elucidated the molecular mechanism of the synergistic anti-insulin resistance tumor effects of the combination treatment.Experimental methods:1.Establishment of insulin-resistant cell modelGlucose oxidase-peroxidase method was used to detect the glucose consumption of HepG2 cells induced by insulin and the expression of insulin receptor on HepG2 cells after insulin induction was detected by Western blotting assay.According to the level of glucose consumption and insulin receptor,we determined the optimal induced concentration of insulin to induce insulin resistance in HepG2 cells.It was further determined whether the insulin-resistant HepG2 cells(HepG2/IR cells)model was successfully established by detecting the triglyceride(TG)level of HepG2 cells.2.Cytotoxicity of ROSI and chemotherapy drugs against HepG2 and HepG2/IR cellsMTT assay was used to determine the inhibitory effect of DDP,ADM,5FU,SOR,VCR and TAX used alone or in combination with ROSI on the cell proliferation of HepG2 and HepG2/IR.The resistant fold of HepG2/IR cells to chemotherapeutic drugs was calculated by determining the IC50 of these drugs on sensitive Hep(G2 cells and insulin-resistant HepG2/IR cells.Moreover,the drug concentrations of ROSI and chemotherapeutic drugs to induce optimal synergistic inhibition were determined.At the same time,the cytotoxicity of ROSI and ADM on human normal cells HUVEC and HL-7702 were examined.3.Determination of whether the anti-tumor effect of the combination of ROSI and ADM on HepG2/IR cells depended on PPARy pathwayWhether the synergistic anti-tumor effect of ROSI and ADM on HepG2/IR cells depends on PPARy pathway was determined by using PPARy antagonist GW9662 by MTT assay.4.Detection of changes of P-gp function and expression between HepG2 and HepG2/IR cellsThe changes of P-gp efflux function and P-gp protein expression between HepG2 and HepG2/IR cells were assessed by detecting the intracellular accumulation of Rhodamine 123 and Western blotting method.5.Detection of the effects of ROSI and ADM on autophagy of HepG2/IR cellsAcidine orange staining was used to detect acidic autophagic vesicles in HepG2 cells and HepG2/IR cells.Western blotting was used to detect the effect of ROSI and ADM on the expression of autophagy-associated protein MAP1LC3B2,EGFR,p-EGFR,ERK and p-ERK in HepG2 and HepG2/IR cells.6.Detection of the apoptosis induction of ROSI and ADM on HepG2/IR cellsAfter treatment with ROSI and ADM for 48 hours,the apoptosis morphology of HepG2/IR cells was observed by Hoechst 33342 staining.Annexin V-FITC/PI double staining(flow cytometry)was used to quantitatively detect the apoptosis rate of HepG2/IR cells treated by ROSI and ADM.At the same time,the expression levels of apoptosis-related proteins Bcl-2 and Bax in HepG2/IR cells were detected by Western blotting method.7.Investigation of the effect of the combination of ROSI and ADM on cell cycle distribution of HepG2/IR cellsThe effect of the combination of ROSI and ADM on the cell cycle distribution of HepG2/IR was detected using PI single staining(flow cytometry).8.Detection of the effects of the combination of ROSI and ADM on migration of HepG2/IR cellsWound scratch assay was used to detect the effect of ROSI and ADM on the migration of HepG2/IR cells.The effect of ROSI and ADM on the expression of MMP-2 protein was determined by Western blotting method.9.Detecting the effects of the combination of ROSI and ADM on the migration and tube formation of HUVEC cellsWound scratch assay and tube formation assay were used to detect the effect of the combination of ROSI and ADM on HUVEC cell angiogenesis.10.Determination of the effect the combination of ROSI and ADM on Akt/mTOR pathwayThe effect of ROSI and ADM on the expression of Akt,p-Akt,mTOR,p-mTOR in HepG2/IR cells was detected by Western blotting method.11.Establishment of insulin-resistant mice modelThe insulin-resistant mice model was induced by DXMS and STZ methods.The body weight,fasting blood glucose level(FBG),fasting insulin level(FINS),oral glucose tolerance(OGTT),and insulin resistance index(HOMO-IR)were measured to determine the optimal induction conditions for the insulin-resistant mice model.12.Detection of the inhibitory effects of ROSI and ADM on the growth of allografts of H22 tumor cells in insulin-resistant miceThe insulin-resistant mice H22 allografts model was used to observe the effects of ROSI and ADM by continuous administration for 6 days[ROSI(intragastric administration,4mg/kg),ADM(intraperitoneal injection,2mg/kg)]on the tumor growth by measuring the tumor volume and tumor weight.The body weight of mice was detected,and liver and heart were weighed to calculate the liver index and heart index.Experimental results:1.Insulin-resistant HepG2/IR cell model was successfully induced by insulin.After induction of HepG2 cells with insulin at 0.1 μmol/L,0.5 μmol/L and 1μmol/L for 48 h,the cellular glucose consumption of HepG2 cells was decreased by 10.2%,22.8%and 47.7%,respectively.1μmol/L insulin significantly inhibited the expression of insulin receptor(IR)in HepG2 cells.In addition,1 μmol/L insulin treated for 48 hours significantly decreased triglyceride(TG)levels in HepG2 cells.The results suggested that the insulin-resistance HepG2/IR cell model was successfully induced by 1 μmol/L insulin treated HepG2 cells for 48 h.2.ADM combined with ROSI produced a synergistic effect against the proliferation of HepG2/IR cells in vitro.Compared with HepG2 cells,the IC50 of ADM,5FU,SOR,VCR,and TAX were significantly increased in HepG2/IR cells.HepG2/IR cells showed highest resistance to ADM,and the resistant fold reached to 7.03.When combined with different concentrations of ROSI,ADM showed higher inhibition on the proliferation of HepG2/IR cells in comparison to ADM treatment alone.Among them,the resistance reversal index of 10 μmol/L ROSI combined with ADM was the largest,by reducing the IC50(μmol/L)of ADM to HepG2/IR cells from 2.43 ± 0.39 to 0.95± 0.08.In the subsequent experiments,10 μmol/L ROSI was used in the combination treatment.The cytoxicity test on normal cells showed that the combination of ROSI and ADM had little toxicity on human normal cells HUVEC and HL-7702,indicating that the cytotoxicity of ROSI and ADM has selectivity on tumor cells.3.Synergistic effect of ROSI and ADM on the proliferation of HepG2/IR cells was independent of PPARγ pathway.PPARγ antagonist GW9662 did not inhibit the synergistic anti-proliferation effect of ROSI and ADM on HepG2/IR cells.4.The P-gp function and expression in HepG2/IR cells did not change significantly in comparison with HepG2 cells.Compared with HepG2 cells,the function and expression of P-gp in HepG2/IR cells were not significantly changed by Rhodamine 123 accumulation test and Western blotting test,indicating that the resistance of HepG2/IR cells to chemotherapeutics has no concern with P-gp.5.HepG2/IR cells showed higher autophagy level than HepG2 cells and the combination of ROSI and ADM inhibited the autophagy of HepG2/IR cells.The acridine orange staining results showed that the number of acidic autophagic vesicles in HepG2/IR cells was significantly higher than HepG2 cells.However,after treatment with ROSI and ADM,the number of acidic autophagic vesicles in HepG2/IR cells decreased significantly than that in the cells treated with ADM alone.Western blotting results showed that the autophagy-related protein MAP1LC3B2,EGFR,p-EGFR and ERK were overexpressed in HepG2/IR cells than that in HepG2/cells.However,the combination of ROSI and ADM significantly inhibited the expression of MAP1LC3B2,EGFR,p-EGFR,ERK,p-ERK in HepG2/IR cells.6.The combination of ROSI and ADM induced the apoptosis of HepG2/IR cells.Hoechst staining results showed that the cell nucleus of control HepG2/IR cells was full ellipse and the chromatin was uniformly colored,while the irregular nuclear morphology,apoptotic bodies and apoptotic morphology were observed in the ADM treated cells.Moreover,the number of apoptotic cells in combined ROSI and ADM cells was significantly higher than that in the cells treated with ADM alone.Annexin V-FITC/PI double staining results showed that the apoptotic rate of the combination group was significantly higher than control HepG2/IR cells.The apoptotic rate was higher in combination group than ADM signal group,by increasing from 14.69%to 23.51%.In addition,the combination of ROSI and ADM increased the Bax/Bcl-2 ratio in HepG2/IR cells,indicating that they triggered a synergistic effect on promoting apoptosis of HepG2/IR cells.7.The combination of ROSI and ADM arrested HepG2/IR cells at S phase in cell cycle distribution as the same as ADM effect.The combination of ROSI and ADM arrested the cell cycle distribution of HepG2/IR cells at S phase,as the same as ADM-treated alone group.8.The combination of ROSI and ADM inhibited the migration of HepG2/IR cellsIn wound scratch assay,the combination of ROSI and ADM inhibited the migration of HepG2/IR cells more significantly than ADM signal group.The migration rate decreased from 30.42%to 16.92%for 24 h treatment.Moreover,the expression level of MMP-2 was significantly decreased in the combination of ROSI and ADM compared with the blank group and the ADM signal group.These results indicated that the combination of ROSI and ADM might inhibit the migration of HepG2/IR cells by decreasing the expression of MMP-2 protein.9.Detecting the effects of the combination of ROSI and ADM on the migration and tube formation of HUVEC cellsIn wound scratch assay and tube formation assay,ROSI could not increase the inhibitory effect of ADM on the migration and tube formation of HUVEC cells.10.The combination of ROSI and ADM inhibited AKT/mTOR signaling pathway in HepG2/IR cells.The combination of ROSI and ADM could significantly inhibit the protein expression levels of Akt,p-Akt and p-mTOR in HepG2/IR cells.11.Insulin-resistant mice model was successfully established by STZ method.Compared with DXMS induction group,the KM mice fed with high-sugar and high-fat diet for 4 weeks and intraperitoneal injected with 80mg/kg STZ induced insulin-resistant mice model by increasing body weight,and the level of FBG,FINS,OGTT and HOMO-IR.12.The combination of ROSI and ADM inhibited the growth of H22 allografts in insulin-resistant mice modelThe inhibition rates of ROSI(4 mg/kg)or ADM(2 mg/kg)on tumor growth were 23.18%or 58.54%,respectively,and the inhibition rate of combination of the two drugs increased to 76.14%.The combination of ROSI and ADM could also significantly reduce the FBG level in insulin-resistant mice.These results showed that the combination of ROSI and ADM synergistically inhibited the growth of H22 allografts in insulin-resistant mice and reduced the blood glucose level.In addition,the body weight,heart and liver index of mice in each experimental group showed no significant change,indicating that the combination treatment showed synergistic effects against tumor growth in insulin-resistant mice without obvious toxicity and side effects on mice.Conclusion:1.Insulin-resistant HepG2/IR cell model was successfully induced by 1 μmol/L insulin treated HepG2 cells for 48 h.2.Compared with HepG2 cells,HepG2/IR cells were less sensitive to chemotherapeutic drugs,with lowest sensitivity to ADM.The combination of ROSI and ADM synergistically inhibited the proliferation of HepG2/IR cells with weak cytotoxicity on human normal cells HUVEC and HL-7702.The synergistic effects of ROSI and ADM on the proliferation of HepG2/IR cells were related to downregualtion of Akt/mTOR pathway.3.Compared with HepG2 cells,the acidic vesicle organelles in HepG2/IR cells were significantly increased.The combination of ROSI and ADM inhibited the autophagy level of HepG2/IR cells,reduced the expression of autophagy-related protein MAP1LC3B2 and downregulated EGFR/ERK pathway in HepG2/IR cells.4.ROSI enhanced the effect of ADM on apoptosis of HepG2/IR cells,which might be related to the down-regulation of Bcl-2 protein and the up-regulation of Bax protein in the mitochondrial apoptosis pathway.Combination of ROSI and ADM inhibits the migration of HepG2/IR cells by inhibiting MMP-2 expression.5.Insulin-resistance animal model was established by feeding Kunming mice with high-sugar and high-fat diet for 4 weeks and intIn vivo experiments,the combination of ROSI and ADM induced significant raperitoneal injection of 80mg/kg STZ.synergistic growth inhibitory effects on H22 allografts,and reduced FBG level in insulin-resistant mice as the same as ROSI alone.