Study On The Mechanism Of Hepatocarcinoma HepG2 Cells Apoptosis Induced By Costunolide

Objective:Hepatocellular carcinoma is a very common malignant tumor with a high morbidity and lethality among the most advanced tumors,which seriously threatens human health.Currently,surgical resection,liver transplantation,radiotherapy,chemotherapy and others methods are mainly used for the clinical treatment of hepatocellular carcinoma.Due to the poor prognosis resistance is easy to occur.However,some studies have shown that the active ingredients of certain Chinese herbal medicines have anti-tumor effects,and have mild and toxic effects on the body,and some of the active ingredients of Chinese herbal medicines have been used clinically to treat tumors.As the active extract of radix aucklamdias,costunolide has been reported to inhibit the growth of certain tumor cells,but the specific molecular mechanism of how to inhibit the growth of Hep G2 cells remains to be elucidated.In this paper,Human hepatocellular carcinoma Hep G2 cells were taken as the main body of study,and the mechanism of apoptosis induced by costunolide in Hep G2 cells was preliminarily discussed,which provides theoretical for the use of costunolide as a potential drug in the treatment of hepatocellular carcinoma.Methods:Hep G2 cells were stimulated with different concentrations of costunolide,the effect of costunolide on Hep G2 cells proliferation was determined by MTT assay.Flow cytometry were used to detect cell cycle,apoptosis,and intracellular ROS levels in Hep G2 cells.WB was used to detect changes of costunolide on Hep G2 cell cycle,apoptosis,autophagy,and ERS protein expression levels.In addition,WB was used to detect the effect of inhibitors(NAC,3-MA)on ERS and autophagy protein induced by costunoilde in Hep G2 cells.MTT assay was used to detected the effect of ROS and autophagy on Hep G2 cells activity.Results:(1)The results of MTT experiments showed that costunolide could significantly inhibit the proliferation of Hep G2 cells,and the inhibitory effect showed a concentration-dependent relationship.(2)The results of Flow cytometry showed that costunolide significantly increased the proportion of S-phase of Hep G2 cell cycle.WB results shown that costunolide can decrease the expression level of cyclin A2 protein in Hep G2 cells,increase the expression level of p21 protein.(3)The results of Flow cytometry showed that costunolide could induce apoptosis and intracellular ROS accumulation in Hep G2 cells,in a concentration-dependent manner.WB results showed the increase of the expression level of Cleaved PARP,p-e IF2α and CHOP protein in Hep G2 cells treated with costunolide.And NAC can reduce the expression of p-e IF2α and CHOP.After pretreatment with NAC and stimulation with costunolide,MTT results showed that NAC could attenuate the effect of costunolide on the proliferation of Hep G2 cells.(4)The results of WB show that costunolide can increase the expression levels of LC3B-II and Beclin-1 proteins in Hep G2 cells in a time-dependent manner.And NAC can significantly reduce the expression levels of LC3B-II and Beclin-1 proteins.When cells were pretreated with 3-MA and then stimulated with costunolide,MTT results showed that autophagy could attenuate the effect of costunolide on Hep G2 cell proliferation.Conclusions:Through the above results,it can be shown that the costunollide can significantly inhibit the proliferation of Hep G2 cells by inducing S-phase arrest,and promote the apoptosis of Hep G2 cells through ROS-mediated endoplasmic reticulum stress and apoptotic autophagy.By studying the mechanism of costunolide promoting cell apoptosis,it provides a new idea for the treatment of hepatocellular carcinoma,which may make costunolide a potential drug for the treatment of hepatocellular carcinoma.