Effects Of Celastrol On Hepatocellular Carcinoma And Its Mechanisms

Heppatocellular carcinoma (HCC), is one of the primary tumor which lead to cancer deaths in the world. It is highly malignant and poor prognosis. These patients often died within4-6months without treatment. The incidence of HCC increased continuously in the recent years, there are0.5to1million new patients increased every year. Liver transplantation、liver surgical resection、radiotherapy and chemotherapy are common therapic methods of hepatocarcinoma. At present, liver surgical resection offers the best chance for cure of these patients. Unfortunately, it is always metaphase or afternoon when the hepatocarcinoma is diagnosed. These patients who had resectable tumors are less than20%. Now, the chemotherapy is used to those patients who do not adapt the surgery, however the clinical effect of chemotherapy is not very significant. So, lowly-toxicity and highly-effect drugs have to be exploredCelastrol (Cel), a quinone methide triterpene, is an active component of Tripterygium Wilfordii. Cel has been used in the treatment of autoimmune and neurodegenerative diseases for its antioxidative and anti-inflammatory effects. Recently, Cel was found to exhibit significant anticancer activities in vitro and in vivo, including the induction of apoptosis in different cancer cells, and inhibiting the growth of glioma, melanoma and prostate cancer in nude mice. However, anticancer activity of Cel in hepatocellular carcinoma and its mechanism are not completely clear.ObjectiveThe objective of the study was to evaluate the anticancer effects of Cel in hepatocellular carcinoma and to explore its possible mechanisms.Methods1. Liver cancer was induced in rats with DEN. The rats were randomly divided into6groups, namely normal control group, liver cancer model group, Cel (2,4,8mg·kg-1) treated groups and XAP (800mg·kg-1)treated group. The activities of ALT, AST, ALP in serum were evaluated by spectrophotometry using commercially kits. The levels of AFP in serum were determined by using rat enzymelinked immunoadsordent assay (ELISA) kit. The protein expressions of MDM2、P53、Bax and Bcl-2were measured by western blot. The samples were stained with HE for histopathological examination. 2. Effect of Cel on the proliferation of Bel-7402cells was measured by MTT assay. The apoptosis rate was detected by flow cytometry and the morphological change of apoptosis was observed by Hoechst33342fluorescent staining and TUNEL. The expression of MDM2、P53、Bax、Bcl-2、Caspase-3、Caspase-8、Caspase-9、Cytochrome c (Cyt c)、Fas and FasL were assayed by western blot.Results1. Therapeutic effects of Cel on DEN-induced HCC rats1.1Effect of Cel on hepatic index、nodules and nodules volume in DEN-induced HCC ratsAfter20-weeks Cel treatment, all rats were sacrificed and nodules number and volume of tumor were examined. Cel treatment prevented the increase of liver index. Macroscopically, in normal group no nodules or additional abnormalities were found in liver, in model group, multiple whitish nodules distributed on the liver surface were noted. While the other rats in Cel treatments groups were found to have fewer tumors and the tumor size was reduced compared with the model group1.2Effect of Cel on histopathology change in DEN-induced HCC ratsMicroscopically, the hepatic sections from the normal rats revealed normal liver parenchyma characterized by typical hepatic lobules and small uniform nuclei. Liver tissue samples from the model group exhibited disordered architecture with a large number of pseudolobules, infiltration of inflammatory cells and abnormal cells with irregular-shaped cytoplasm and enlarged hyperchromatic nuclei, and these HCC cells generally tended to be poorly differentiated. In Cel treatment groups, the hepatic pathological lesions with pseudolobules, fibrosis, inflammatory cell infiltration and cellular atypia were markedly improved compared with model group, and the tumors in Cel treatment groups were histologically well-differentiated1.3Effect of Cel on the levels of serum AFP, ALT, AST and ALP in DEN-induced HCC ratsDEN induction resulted in a marked increase in AFP levels in liver and serum as compared with that of the control group. Treatment with Cel significantly decreased the AFP level. The levels of alanine aminotransferase(ALT), aspartate aminotransferase(AST), alkaline phosphatase(ALP) in serum were signifieantly increased in model group in compared with those in control group, while the treatment with Cel markedly reduced all the above criteria compared with model group.1.3Effect of Cel on the expression of MDM2、P53、Bax and Bcl-2in liver tissues in DEN-induced HCC ratsP53and Bax proteins were more obviously upregulated in Cel-treated rats than those in model rats, while Bcl-2and MDM2proteins were statistically decreased.2Effect of Cel on human hepatoma cell lines Bel-74022.1Effect of Cel on the proliferation of human hepatoma cell lines Bel-7402The results suggested that Cel at concentrations of0.78～12.5μg·ml-1had the inhibitory effect on the proliferation of Bel-7402cells. Cel at concentrations of0.39μg·ml-1do not has obvious growth inhibition.2.2The apoptotic effect of Cel on human hepatoma cell lines Bel-7402The typically apoptotic changes include chromatin condensation and deformed and fragmented nuclei. The results showed that Bel-7402cells cultured in Cel for24h exhibited typical morphological changes of apoptotic cells under fluorescent microscope, including shrinkage of cell condensation of chromatin, breakage of nuclear, formation of apoptotic bodies and obviously increased number of apoptotic cells. A Flow Cytometry (FCM) assay was performed to analyze apoptotic rate. The apoptotic rate of Cel at concentrations of0.78～3.12μg·ml-1on Bel-7402is (18.6±3.7)%、(34.6±2.8)%、(40.3±1.7)%respectively.2.3Effect of Cel on the expression of apoptotic related proteins in human hepatoma cell lines Bel-7402Celastrol increased the expression of P53, inhibited the activity of MDM2, elevated the ratio of Bax/Bcl-2in Bel-7402cells and in DEN induced rat liver. Furthermore, Celastrol induced the release of Cyt c, increased the expression of Fas, FasL, cleavage of caspase-3, caspase-8and caspase-9in Bel-7402cells.ConclusionCel has obvious anti-HCC effect, its mechanism may be related to the induction of apoptosis of liver cancer cells.