Celastrol Induces Cell Death Via TFEB-Lysosomal Pathway In Hepatocellular Carcinoma

Objective:Primary liver cancer is currently one of the most common malignant tumors and the second leading cause of tumor-related deaths in China,which seriously threatens people’s lives and health.China has rich traditional Chinese medicine resources,but majority of them are not well studied.So it is urgent to research and develop for better clinical application service.Tripterygium wilfordii Hook is one of the traditional Chinese medicine in China.And Celastrol,which is one of main active ingredients in Tripterygium wilfordii Hook,is a triterpenoid compound isolated from Tripterygium wilfordii Hook root of.Although increasing evidence has revealed the effects of Celastrol on significant antitumor activity,the exact molecular mechanism for the anti-HCC role of Celastrol has not been fully unveiled to date.This study was designed to explore the anti-cancer effect of Celastrol through multi-aspect based on,hepatocellular carcinoma cell lines and nude mouse xenograft model to provide a theoretical basis for targeted treatment of HCC with Chinese herbal medicine.Method:(1)The effects of Celastrol on proliferation,morphology,colony formation ability and survival rate of HCC cells were detected by MTT assay,optical microscopy,clonogenesis assay and flow cytometry.(2)The effects of Celastrol on ROS,iron content,MDA,SOD,GSH-Px,lipid peroxidation,y-H2AX,Apoptotic bodies,caspase 3,PARP,ferritin(FTH,FTL)and transferrin receptor TFRC in hepatocellular carcinoma cells were detected by CM-H2DCFDA assay,colorimetry,ELISA,BODIPYTM 581/591 C11,immunofluorescence,Hoechst staining,WB and other techniques respectively.(3)The effects of Celastrol on lysosome of hepatoma cells were detected by flow cytometry and laser confocal microscopy.Subsequently,The expression of MDA,SOD,GSH-Px,ACSL4,GPX4,FTH,FTL and TFRC were observed by WB and ELISA methods in hepatoma carcinoma cell which were treated with lysosome inhibitors.(4)Expression of TFEB and lysosomal membrane protein(Cathepsin D,Cathepsin B,ATP6V1B2,LAMP-1)in hepatocellular carcinoma cells treated with Celastrol were detected by WB.TFEB was silenced to observe the effects of Celastrol on the expression of lysosomal membrane proteins and cell survival rate.(5)Hepatocellular carcinoma cells was treated by Celastrol combined with Bid inhibitor,caspase inhibitor zVAD and ferroptosis inhibitor respectively.The expressions of Bid,tBid,Caspase 9 and PARP were detected by Western blot.Mitochondrial membrane potential was measured by TMRM.The cells survival rate were measured by propidium iodiede(PI)staining.(6)Establish nude mice Subcutaneous transplantation tumor model and treated with Celastrol through intragastric administration.H&E staining and TUNEL staining were used to detect the effects of Celastrol on histomorphology and apoptosis of transplanted tumor.The expression of ACSL4,GPX4,FTH,TFL,TFRC,Cathepsin D,ATP6V1B2,LAMP-1 and other proteins in tumor tissues were detected by Western blot.Result:(1)To validate whether Celastrol exhibited anti-proliferation effect against HCC,MTT assay was performed to assess cell viability after Celastrol treatment in four human HCC cell lines.We found that Celastrol has significant inhibited effect on HCC cells and this effect on hepatocellular carcinoma cells show a time-and dose-dependent sensitivity towards Celastrol,and screened the cell line HepG2 and the dose of Celastrol for the later experiment.PI straining the HepG2 colonies which were under Celastrol treatment showed Celastrol had significantly inhibite effect.In addition,the result showed that Celastrol induced HepG2 cell death with the help of using many death inhibitor,which was associated with elevated ROS,apoptosis and ferroptosis.(2)The molecular mechanism of Celastrol induced hepatoma carcinoma cell death were explored by ROS,apoptosis and iron death.We used the fluorescent probe CM-H2DCFDA to monitor intracellular ROS levels,finding as a result that treatment with Celastrol caused an ROS level increasement as compared with non-treatment cells,and that this increase could be rescued by pre-treatment with NAC(an antioxidant).Next,we tested whether Celastrol was involved in activation of lipid peroxidation.The results revealed that Celastrol significantly promoted the occurrence of lipid peroxidation,while the changes could be mitigated using the ROS inhibitor NAC and ferroptosis inhibitor(Liproxstatin-1,Lip or Deferoxamine,DFO).Also,the antioxidant indexes,SOD,GSH-Px,GSH and MDA levels,were assayed.We showed that the intracellular SOD,GSH-Px,GSH activities were reduced and the expression level of MDA was significantly increased by Celastrol treatment,while the combination of Celastrol with NAC or ferroptosis inhibitor(Lip,DFO)could reverse this change.Furthermore,we observed that Celastrol treatment induced accumulation of Fe2+,And Western blot results showed that the expression of ACSL4,ferritin(FTH,FTL)and TFRC markedly decreased while GPX4 was significant increased after Celastrol-treatment.These results suggested that Celastrol induced ferroptosis in HCC cells.Meanwhile,the results of Hoechst staining and western blot revealed that Celastrol induced apoptosis of HCC cells that caused the PARP cleavage and caspase-3 activation.This change could be reversed by NAC and Celastrol treatment.It verified that Celastrol could cause apoptosis in HCC cells.In conclusion,these results demonstrate that Celastrol induced not only ferroptosis but also apoptosis in hepatocellular carcinoma.Celastrol can enhance lysosome activity in hepatoma cells.(3)We explored the mechanism of ferroptosis and apoptosis induced by Celastrol to verify the effect of Celastrol on lysosome.The study showed that Celastrol increase of lipid peroxidation and iron content when ROS increased significantly.Therefore,it is supposed to whether the effect of Celastrol on lipid peroxidation and iron level depends on transferrin lysosome degradation?By flow cytometry and confocal microscopy confirmed Celastrol could activate lysosome after Lyso-Tracker Green/Red staining.When using Baf-Al and CQ,the effect of lipid peroxidation,iron levels,and induction of cell death through Celastrol can be reversed,so as to prove that Celastrol could activate lysosomes,which plays a key role in the process of anti-hepatoma carcinoma cell.(4)Find the lysosome and further explore how Celastrol activates the lysosome.RT-PCR results showed that Celastrol can promote the expression of TFEB mRNA level;The results of suborganelle isolation and Western Blot showed that Celastrol could promote the nuclear translocation of TFEB.The expression levels of lysosomal membrane proteins Cathepsin D,Cathepsin B,ATP6V1B2 and LAMP-1 were increased.TFEB gene silenced by siRNA was used for reverse validation.After TFEB gene silenced,TFEB induced by Celastrol was effectively reversed,confirming the effect of Celastrol induced TFEB transcriptional activity on lysosomal activation,iron release,increased lipid peroxidation levels,and induced hepatocellular carcinoma cell death.(5)Based on the results of the study,we attempted to elucidate the possible interaction or causal relationship between the two death forms of hepatoma carcinoma cell induced by Celastrol,including ferroptosis and apoptosis.Western Blot results showed that Celastrol group caused tBid accumulation and a decrease in Bid length,as well as a decrease in caspase-9 and PARP cleavage.However,Ferrostatin-1,ZVAD,and Bid inhibitor BI-6C9 all reversed these results.The mitochondrial membrane potential(δψM)was significantly decreased by Celastrol,and the mitochondrial membrane potential increased by Celastrol combined with baF-Al,BI-6C9 and zVAD,respectively.These results fully reveal the interaction of Bid in mediating Celastrol induced ferroptosis and apoptosis.(6)Tumor inhibition of Celastrol was verified in nude mice xenograft tumor model.The results showed that higher concentration of Celastrol inhibited tumor growth more significantly and tumor volume decreased significantly.H&E staining results showed that the tumor tissues in the control group had clear cell structure,dense arrangement,basically complete cell morphology,clear nucleus and almost no necrosis.In low concentration Celastrol group,most of the tissue structure was destroyed,while in high concentration Celastrol group the tissue structure was destroyed more seriously,and sparse cell arrangement and disappearance of cell morphology,including inflammatory cell infiltration and vacuolar change.TUNEL assay showed that apoptotic cells were rarely seen in the xenograft tissues of nude mice in the blank control group,while the number of apoptotic cells was significantly increased in the xenograft tissues treated with Celastrol.Western Blot results showed that the protein expressions of ACSL4,Cathepsin D,ATP6V1B2 and LAMP-1 were increased in Celastrol treatment group,while the protein expressions of GPX4,FTH,FTL and TFRC were significantly decreased.Conclusion:(1)Celastrol inhibits the growth and proliferation of the four kinds of hepatoma carcinoma cells.The accumulation of ROS in the dead hepatoma carcinoma cells induced by Celastrol is positive related;Hepatoma carcinoma cells treated with Celastrol showed increased iron accompanied by increased lipid peroxidation level,which induced ferroptosis and apoptosis;(2)Celastrol activates lysosome function through nuclear translocation of TFEB,promoting degradation of ferritin heavy chain,light chain and transferrin receptor,resulting in lysosomal dependent ferroptosis;(3)Celastrol induces lipid peroxidation in hepatocellular carcinoma cells and mediates the interaction between ferroptosis and apoptosis through Bid cleavage;(4)Celastrol has good tumor inhibition in nude mice xenograft tumor model.