The Anti-tumor Effect Of Parthenolide By Inducing Apoptosis And Autophagy Of Human Hepatoma Cells And Its Mechanism

Objective The incidence and mortality of malignant tumors are increasing year by year around the world,showing a trend of getting younger.Especially for liver cancer,the cure rate is low and the mortality rate is high.However,the pathogenesis is still unclear.Many natural sesquiterpene lactones have been found to have excellent antitumor activity and have no side effect on normal cells.The antitumor effect of Parthenolide was mainly verified by cell experiments,and its mechanism in human hepatocellular carcinoma HepG2 cells were mainly studied in this paper.Methods The WST-8 method was used to detect the effect of parthenolide on the toxicity of HepG2 at different time points and concentrations;flow cytometry(FCM)was adopted to detect the effect of parthenolide on mitochondrial membrane potential(?Ψm),intracellular calcium ions,cell cycle and apoptosis ratio;Western blot was used to detect the effect on HepG2 cell death(apoptosis and autophagy)-related protein and cycle-related protein;FCM was adopted to detect the effect of parthenolide on the reactive oxygen specise(ROS)in Hep G2 cell;The effect of parthenolide on reduced glutathione(rGSH)in cells was determined by colorimetricmethod;the effect of PN combined with ROS scavenger N-acetyl-L-cysteine(NAC)or BSO on the above-mentioned indicators of HepG2.Results With the increase of the action time and dosing concentration,HepG2 cells are rounded and wrinkled,the nuclear chromatin is dense and deeply stained and the cell have a poor cell adherence;the proliferation of HepG2 cell was inhibited and its vitality gradually decreased;with the increase of parthenolide action time,the MMP gradually decreased,When parthenolide acted on HepG2 cells for 12 h,the cell mitochondrial membrane potential(?Ψm)almost disappeared,and when parthenolide acted on HepG2 cells for 24 h,the cell mitochondrial membrane potential completely disappeared.with the increase of parthenolide action time,calcium ions flowed inwards,the cell cycle was arrested in phase G1,And the cell apoptosis rate,especially late apoptosis and necrotic cells,increased.The shear expression of apoptosis-related proteins caspase-3 and caspase-9 was up-regulated;the shear expression of AIF,MIF and PARP1 proteins associated with Caspase-independent apoptosis,i.e.Parthanatos apoptosis,showed time-dependent up-regulation;the long and short expressions of anti-apoptosis protein FLIP showed different degrees of decrease;the expression of autophagy-related proteins LC3Ⅱ and becin-1 was up-regulated;the expression of P62 protein was down-regulated;the expression of cycle-related proteins P53,P27 and P21 increased significantly;theexpression of CyclinD1 and CyclinE1 decreased.FCM was used to detect the increase of ROS with the action time of PN;an its generation level showed an increasing trend(P<0.001);Chemiluminescence detection of intracellular rGSH level found that with the increase of parthenolide action time,the intracellular rGSH level showed a decreasing trend(P < 0.001).However,when combined with the ROS scavenger NAC,the intracellular ROS production level was significantly reduced and cell death was almost completely blocked.When parthenolide combined with BSO,The cell apoptosis,especially the proportion of necrosis,was significantly increased compared with parthenolide or BSO alone(P < 0.001),and the intracellular ROS production level was also significantly increased(P < 0.001).Conclusion Parthenolide may increase intracellular ROS levels and induce to cell cycle arrest,apoptosis and autophagy through eliminating intracellular GSH levels and leading to cellular redox equilibrium broken,thus playing an anti-tumor effect.However,The BSO enhanced the sensitivity of HepG2 to parthenolide.