Functional Role And Regulatory Mechanisms Of Xanthatin In Hepatocellular Carcinoma

Among primary liver cancers,hepatocellular carcinoma(HCC)accounts for about 85%~90% of the total,and is the second highest cause of cancer death worldwide,with more than one million new cases each year nowadays.Hepatocellular carcinoma almost originates from chronic liver injuries,such as hepatitis B or hepatitis C virus infection,alcoholic fatty liver,nonalcoholic fatty liver,smoking and aflatoxin exposure.Alcoholic fatty liver and nonalcoholic fatty liver in Europe and the United States are the main causes of hepatocellular carcinoma.In China,due to the spread of viral hepatitis,HCC accounts for nearly half of the new cases of carcinomas in the world every year.Although China is currently vigorously promoting the vaccination of viral liver cancer vaccines,the incidence of liver cancer has not been expected to decrease drastically.Many patients with HCC are in the absence of the above risk factors.It is obvious that there must be other risk factors or molecular mechanisms to be further clarified for HCC.Currently,liver transplantation and partial hepatectomy are considered to be the main treatments for HCC.Meanwhile,due to the high recurrence rate of HCC,the lack of liver donors,the difficulty of early HCC diagnosing,and the poor systemic chemotherapy effect of patients with advanced HCC,the options of treatment for patients with HCC is very limited.The multi-kinase inhibitor Sorafenib is the only effective drug for HCC chemotherapy.Therefore,it is necessary to thoroughly explore the molecular mechanism of HCC and find more effective clinical therapeutic drugs to improve the overall survival rate of patients,including advanced HCC patients.In recent years,it has been found that xanthatin,the main pharmacological active ingredient of sesquiterpene lactones extracted from xanthium sibiricum,has obvious anti-tumor effects in breast cancer,non-small cell lung cancer,gastric cancer and melanoma.Through in vitro molecular biology experiments and in vivo nude mice tumor formation experiments,we found the effects of xanthatin on the malignant biological characteristics of hepatoma cells and analyzed its potential molecular mechanisms.We firstly observed the effect of xanthatin on the morphology of human hepatoma cells.Light microscopy showed that the human hepatoma cell lines gradually shrunk and rounded after stimulation with xanthium,accompanied by floating dead cells.Subsequent MTT assay showed that xanthatin significantly inhibited the viability of human hepatoma cells in time-and concentration-dependent manner.Cell growth is usually affected by cell cycle arrest and the cell apoptosis.In this study,the cell cycle and cell apoptosis of human hepatoma cells treated by xanthatin were detected by flow cytometry.It was found that the medium concentration of xanthatin had obvious cell cycle arrest and apoptosis.The apoptotic cells induced by xanthatin were visually displayed by TUNEL staining and Annexin-V/PI double staining experiments.Western blot analysis showed that xanthatin increased the level of cleaved-caspase-3 and Bax protein,and suppressed the expression of anti-apoptotic protein Bcl-2,indicating xanthatin triggers the apoptotic signaling cascade of human hepatoma cells.Unfolded protein response during endoplasmic reticulum stress is an important mechanism for inducing cell apoptosis.It mainly functions through three signaling pathways: PERK-e IF2α-ATF4,IRE-XBP1 and ATF6.In this study,cytoplasmic condensation,nuclear rupture,endoplasmic reticulum expansion and apoptotic bodies were observed by transmission electron microscopy in xanthatin treated cells,suggesting that endoplasmic reticulum stress might be involved in xanthatin-induced apoptosis.Western blot analysis showed that the levels of endoplasmic reticulum stress-related proteins GRP78/Bip,PERK,p-PERK,IRE1 and ATF6 in human hepatoma cells were increased after treatment with xanthatin.In order to clarify the specific pathway involved in the regulation of xanthatin in apoptosis,the downstream proteins of the above signaling pathways were detected.The level of e IF2α,phosphoryled e IF2α,ATF4,and XBP1 were significantly increased after xanthatin treatment.However,the levels of ERDJ4 and EDEM1,the downstream targets of the IRE-XBP1 pathway,were not significantly changed.However,the level of CHOP,downstream of the PERK-e IF2α-ATF4 pathway,was up-regulated in xanthatin-treated human hepatoma cells,suggesting that xanthatin may trigger the endoplasmic reticulum stress-induced apoptosis mainly through PERK-e IF2α-ATF4 signaling pathway.Moreover,CHOP-si RNA blocked these effects of xanthatin on apoptosis and the molecules in PERK-e IF2α-ATF4 pathway.In this study,we also found that the cytoplasmic ATF4 was transfered into the nuclei after treatment with xanthatin.At the same time,luciferase reporter gene assay showed that xanthatin significantly increased the transcriptional activity of ATF4 in Hep G2 cells.Therefore,xanthatin may promote the endoplasmic reticulum stress-indcuced apoptosis by activating PERK-e IF2α-ATF4 signaling pathway and its downstream targets.In order to demonstrate this finding,we also carried out in vivo experimental study to establish a liver cancer tumor model in male BALB/c nude mice.The tumor volume and growth rate in xanthatin group were lower than those in control group.The mouse tumor tissues were collected and immunohistochemical experiments were performed.We observed that the positive cells of cleaved-caspase3,GRP78/Bip,PERK,ATF-4,p-e IF-2α,and CHOP protein in the xanthatin group were more than than in the control group,strongly suggesting that the PERK-e IF2α-ATF4 signaling pathway and its downstream target CHOP participate in the biological function of xanthatin in apoptosis of hepatoma cells.More importantly,in vitro experiments showed that xanthatin nearly has no obvious effect on inhibiting the growth of human L02 cell line,indicating that xanthatin specifically inhibits cell viablity.In summary,xanthatin has a significant suppressor effect on hepatoma cells by activating PERK-e IF2α-ATF4 signaling pathway and its downstream target CHOP.This finding provides an experimental basis for the potential treatment of human HCC,which will shed a light on the anti-hepatocarcinoma drugs.