The Mechanism Of ER Stress And Autophagy Induced By Celastrol In Hepatocellular Carcinoma Cells

Hepatocellular carcinoma(HCC)is the most common malignant tumor worldwide.China has a large population suffering from the disease.The incidence of HCC is in the position immediately after lung cancer and gastric cancer,and its mortality rate is very high.At present,surgical resection and liver transplantation are the most preferred method to cure HCC.Studies have shown that endoplasmic reticulum stress exists in malignant tumors,including HCC.Endoplasmic reticulum stress and unfolded protein response with stress signal transduction mission can determine cell fate through precise molecular regulatory network.Extracted from the root of celastrus with a triterpenes chemical structure,celastrol has a developing potential of treating inflammatory as an inhibitor of NF-kB.In recent years,research data indicate this natural compound has antitumor activities.The purpose of this study is to investigate the molecular mechanism of its anti tumor activity in HCC cells and observe its anti tumor effect of celastrol in HCC cells in vivo.The dose dependent inhibitory effect of celastrol on two human HCC cell line Hep G2 and Bel7402 was observed in vitro.CCK-8,plate colony formation,flow cytometry and western blot were used to investigate the cell proliferation of HCC cells exposed to celastrol.PI/Annexin V double labeling,TUNEL staining and western blot was used to observe the apoptosis induced by celastrol in HCC cells.To observe celastrol induced endoplasmic reticulum stress in HCC cells Flow cytometry used to detect intracellular Ca2+ concentration.Ubiquitinated protein and protein synthesis control associated molecular chaperones on endoplasmic reticulum detected by western blot.Autophagy induced by celastrol in HCC cells was observed by biological electron microscopy and western blot.Using CCK-8 and western blot to obseve autophagy in HCC cells.Using the BALB/c tumor bearing mice model to observe the therapeutic effect of celastrol by intragastric administration.The results showed that celastrol inhibited cell viability and colony formattmg in Hep G2 and Bel7402.Celastrol caused Hep G2 and Bel7402 cells arrested in G2/M phase with doses dependent.The cell cycle arrest in HCC cells is associated with higher expression of p21 and lower level expression of cyclin B1 in the cells.Celastrol can induce apoptosis in HCC cells.Celastrol activatied apoptosis related protein caspase-3 and PARP in both Hep G2 and Bel7402 cells was significantly increased.Expression of endoplasmic reticulum molecular chaperones,PDI and Ero1-La decreased suggested that celastrol induced endoplasmic reticulum stress in HCC cells.The Ca2+ concentration and ubiquitin protein expression increased in HCC cells enhanced endoplasmic reticulum stress.The key protein of unfolded protein response p-el F2a and p-IRE1a expression increased.Bip and the transcription factor,ATF4,CHOP and XBP1 s m RNA expression were upregulated by celastrol in HCC cells.Combined use of endoplasmic reticulum stress reliever TUDCA,attenuated ER-stress induced by celastrol in both Hep G2 and Bel7402 cells.The protein expression level of p-el F2a,p-IRE1a and the m RNA expression level of ATF4 and CHOP decreased in Hep G2.The aopoptosis key protein cleaved caspase-3 and cleaved PARP expression level also decreased.Under electron microscope,the phagocytic bubble containing substance are visible HCC cells treated with celastrol.Celastrol also enhanced expression of LC3 in HCC cells.The use of autophagy inhibitor 3-MA compared to use celastrol alone,decreased LC3 protein expression.Opposited to Hep G2,the cell viability of Bel7402 increased by co-treated with celastrol and 3-MA.Use celastrol intragastric injection to mice hepatocarcinoma cells H22 bearing BALB/c mice everyday.Tumor volume was significantly smaller than the solvent control group using by 5mg/kg dose.There was no significant difference in body weight between the control group,2.5mg/kg dose,group and 5mg/kg dose group.The indexes of liver and kidney function in the blood has not show significant difference among three groups.Immunohistochemical staining showed the expression of cleaved caspase-3 in tumor tissue was higher in the treated group than in the solvent control group.Conclusions: Celastrol can inhibit the proliferation of HCC cells by induced cell cycle arrest.calcium homeostasis and ubiquitinated protein accumulation induced by celastrol and TUDCA reduced apoptosis protein expression suggesting that celastrol can induce ER stress-related apoptosis.Celastrol can induce autophagy in two HCC cells.And the results that 3-MA co-treated with celastrol in HCC cells suggest that celastrol induced autophagy may playing different roles in two HCC cells in.In BALB/c mice bearing mice HCC cells H22 model,celastrol inhibited the growth of HCC.