Anticancer Mechanism Of Trichosanthin

Trichosanthin(TCS) is a protein extracted from root tubers of Trichosanthes kirilowii Maxim.It is a functional component of traditional Chinese medicine "Tian Hua Fen",which has abortifacient,anti-tumor and anti-HIV activities.TCS is a member of single chain ribosome-inactivating protein family.In the past, investigators paid more attention on the structures and pharmacology of TCS.The mechanism of apoptosis and signal transduction pathways induced by TCS have been little exploited.The further study is necessary for better understanding anticancer mechanism of TCS and directing its clinical applications.Objective To investigate the molecular mechanism of apotosis and signal transduction caused by TCS using new theory and approach of cell biology including ER stress and autophagy.Methods Part I.Apoptosis and cycle arrest of cancer cells caused by TCS. The cell cycle arrest of L929 murine fibrosarcoma cells was analyzed by flow cytometry.The MTT method was used to evaluate the cytotoxicity of TCS on cancer cells such as Hela,L929,HepG2,HCT116,and Colo205 cells.In order to confirm the apoptosis caused by TCS in different cancer cells,Western blot was used to detect the changes of Caspase 3,PARP and JNK.Partâ…¡.ER stress induced by TCS in cancer cells.To study ER stress induced by TCS,the expression levels of protein markers of CHOP and BIP,and DR5 receptor of TRAIL upregulated by CHOP were analyzed by Western blot.The small RNA interference was employed to knock-down the CHOP gene so that the role of CHOP in the apoptosis caused by TCS can be investigated.L929 cells transfected with CHOP siRNA and scrambled siRNA were subjected to the cytotoxicity test to the combined treatment of TCS and TRAIL using flow cytometry.Partâ…¢.The involvement of autophagy in TCS-induced apoptosis.In this part,the autophagy induced by TCS was studied.First,the autophagic hallmarks including LC3-conversion and an increased number of autophagosomes were observed by Western blot and confocal microscopy.In order to study on investigate the importance, autophagy-related genes of Atg 5,Atg 7 and beclin were knocked down by siRNA interference.Finally,the knock-down cells were treated by TCS and TRAIL,to compare the cytotoxicity analyzed by flow cytometry.Results:Partâ… .Apoptosis and cycle arrest of cancer cells caused by TCS.After treated with TCS for 24 h and 48 h,L929 cells showed different -phase cycle arrests:G0/G1 and S phase arrest,respectively.We found that TCS had cytotoxicity to almost all cancer and normal cells tested and the cytotoxicities onto different cancer cells were different.The apoptotic death caused by TCS was confirmed by Hoechst 33342 and Sytox Green staining.The dose-dependent cleavage of PARP,a substrate of caspase 3,was observed by Western blotting analysis,indicating that caspase 3 was activated during TCS treatment.The c-jun N-terminus kinase(JNK) and caspase 3 were also activated during theTCS treatment.Partâ…¡.ER stress induced by TCS in cancer cells.The expression of chaperone BIP,a sensor of ER stress,was increased with the treatment of TCS in time and dose-dependent manners.After treatment with TCS for 24 h,another marker protein,CHOP was increased dramatically.In the meantime,caspase 12 was also activated.The upregulation of DR5,a receptor of TRAIL was verified by Western blot,and the TCS-treated cells were sensitized to TRAIL.The sensitization can be abolished while the CHOP gene was knocked down by siRNA interference in L929 cells.Partâ…¢.The involvement of autophagy in TCS-induced apoptosis.We observed the autophagic hallmark of LC3 was present in Hela and L929 cells treated with TCS. The conversion of LC3-I to LC3-â…¡was observed during TCS treatment for 3-6 hrs in Hela and L929 cells.The autophagosomes in Hela,HepG2 and L929 were visualized followed the transfection with mRFP-GFP tagged LC3 construct examined by a confocal microscopy.These results provided evidence that autophagy involed the TCS- induced apoptosis.Cloroquine,known as inhibitor of cathepsin in lysosomes was able to increase the cytotoxicity of TCS,whileas the activator of autophagy,rapamycin,can decrease the cytotoxicity.Finally,the autophage-related genes,Atg 5,Atg 7,and beclin were knocked down by small RNA interference.The cytotoxicities of TCS to the Atg 5 and Atg 7 knock-down cells were increased to 4.16 and 1.87- fold.The above results suggest the autophagy plays a protective role during TCS-induced apoptotic cell death.Conclusion:1.TCS can inhibit cancer cells growth through inducing cell cycle arrest and apoptosis.2.ER stress can be markedly induced by TCS in L929 cells,indicated by the increasing expression of hallmarks of BIP and CHOP.3.DR5,a receptor of TRAIL can be upregulated in the process of TCS-induced ER stress,which increases the susceptibility of cancer cells to TRAIL treatment.4.Autophagy is involved in the apoptosis caused by TCS,and chloroquine,an inhibitor of cathepasin enzyme in autophagosomes can enhance the cytotoxicity of TCS,suggesting autophagy plays a protective role in TCS-induced apoptosis in L929 cells.5.Chloroquine and TRAIL can enhance the cytotoxicity of TCS,which may have clinical significance. Taken together,these results provide insights for further study on anticancermechanisms of TCS and other related ribosome-inactivating proteins.