The Mechanism Of Apoptosis Induced By Ivalin In Hepatocarcinoma Cells

Hepatoma is one of the most common cancers in the world.Clinically,surgical resection,radiotherapy,chemotherapy and other treatments are used to treat hepatoma While,because of its high degree of deterioration,strong ability of migration and drug resistance,research and development of new drugs anti-hepatoma is urgent.Ivalin is a natural compound separated from Carpesium Macrocephalum(C.Macrocephalum)by professor Weidong Xie.Carpesium Macrocephalum is widely distributed in Sichuan,Guizhou,Hunan,Fujian province and northeast China,which can be used to treat cold,headache and acute conjunctivitis.In the early research work of our research group,we found that Ivalin had the anti-migration effects in breast cancer.In this subject,we found that can induce apoptosis of hepatocarcinoma.Therefore,in the follow-up study,we used the most effective SMMC-7721 cells as research objects,and discussed its mechanism of action.Firstly,we used MTT assay to analyze the cytotoxicity ocf Ivalin against human liver cells and hepatoma cell lines.We found that Ivalin had lower cytotoxicity against human liver cells LO2(IC50=25.86±0.87 ?mol/L)and much higher cytotoxicity against hepatoma,especially for hepatoma cell lines SMMC-7721(IC50=4.34±0.10 ?mol/L),which was chosen for further molecular mechanism researches.We investigated that Ivalin could promote the depolymerization of microtubes in hepatoma,which cause the G2/M arrest and following the apoptosis.Using the immunofluorescence staining assay and western blot assay,we found that Ivalin could promote the depolymerization of microtubes in hepatoma like Vincristine.Microtubes play a part in the cytoskeletal support and intracellular transportation as well as adjusting the cell cycles.Thus,we detected the cell cycle distribution after treating with Ivalin.The results showed that Ivalin could lead to the G2/M cell cycle arrest in hepatoma.Furthermore,we performed the western blot assay to detect the expression level of cell cycle-related proteins.We found that both Cdc25A and Cyclin B1 were up-regulated after Ivalin treating.Additionally,we found the G2/M arrest causing by Ivalin reached the highest point at 24 h and then went down.So we speculated that cells under long-term cycle arrest promoted the programmed death,which was further verified by Annexin V and PI staining.Meanwhile,we found that Ivalin could induce the mitochondrial apoptosis in SMMC-7721 cells.Then we found that Ivalin could induce the accumulation of reactive oxygen in hepatoma cell lines.Then we performed RT-PCR and western blot assay and found that Ivalin could regulate the transcription and protein expressions of NF-?B,p53 and Bax,which suggested that the Ivalin-induced apoptosis was associated with the activation of NF-?B and p53.The results of JC-1 staining assay showed that Ivalin could reduce mitochondrial potential and increase the membrane permeability.In this case,cytochrome C was released into cytoplasm and further to trigger Caspase 3 activity and induce apoptosis.In conclusion,Ivalin perhaps was a compound with multiple targets.Firstly,Ivalin could induce the G2/M cycle arrest and apoptosis in hepatoma by promoting the depolymerization of microtubes.Secondly,Ivalin increased the expression of NF-?B and p53 through the accumulation of reactive oxygen,which lead to the mitochondrial apoptosis.Ivalin,a natural anti-cancer compound,is worthy of study and develop deeply.And our studies also laid a solid foundation for its structure modified and drug development.