Molecular Mechanism Of Autophagy And Apoptosis Of Human Hepatocellular Carcinoma Cells Induced By Juglone From Juglans Mandshurica Maxim

Research Purpose:Juglans,belonging to Juglans Mandshurica Maxim,are widely distributed in China,and have a variety of pharmacological activities.In China and South Korea,the immature exocarp of Juglans mandshurica has been widely used in the treatment of diseases such as tumors.JG,a kind of naphthoquinone,is widely distributed in the fresh root bark,branch bark and green peel of Juglans mandshurica.According to prevours studies,JG could inhibit proliferation of various tumors in vitro,but the molecular mechanism of JG-induced liver cancer cell apoptosis and autophagy is still unclear.Therefore,our study will take human hepatoma HepG2 cell line as a model,using molecular biology,cell biology and nude mice xenograft experiments and other technical means to explore the effects of JG on autophagy and apoptosis.Furthermore,the molecular mechanism of autophagy on the cell death by JG was investigated.Thus,it provides a scientific basis for the development of a new anti-tumor drug based on the JGResearch Methods:In this study,we examined the inhibitory effects of JG on the proliferation in HepG2 cells by MTT,CCK-8 and colony formation assays.Then,JG-induced cell cycle arrest was explored by flow cytometry and western blotting.The configuration and nuclei changes of HepG2 cells after JG treatment were observed by fluorescence microscope.Next,DNA damage,apoptosis,MMP and apoptosis-related protein were detected.Moreover,AO staining,MDC staining,Ad-mCherry-GFP-LC3B transfection,immunofluorescence double staining and western blotting were applied to determine the effect of JG on autophagy and autophagy.At the same time,the relationship between autophagy and apoptosis was investaged by treatment with the autophagy inhibitor 3-MA or wortmannin.The possible pathway of JG-induced autophagy activation and changes in ROS were then examined.Finally,the anti-tumor effect of JG in vivo was observed by establishing a nude mouse xenograft.Research Methods:Results in vitro1.JG can inhibit HepG2 cell proliferation and induce apoptosis.Compared with the control group,JG treatment significantly decreased the viability of HepG2 cells in a time-and dose-dependent manner,while JG had a low toxicity to normal human hepatocytes.Meanwhile,treatment with JG effectively inhibited the number of cell clones and induced the cell cycle arrest at G2/M phase.It was found that after JG treatment,cells were turned to round shape,loosely arranged,and the number of adherent cells was reduced along with shrinkage and condensation.Annexin-V-FITC/PI double staining showed that the percentage of apoptosis increased from 4.08%(0 ?M)to 20.90%(30 p,M).The results of immunoblotting further showed that the expression of cleaved PARP and cleaved caspase-3 increased with increasing JG concentration.At the same time,JG treatment caused rapid accumulation of p53 in cells.However,after the knockdown of p53 gene,apoptosis was significantly reduced.2.JG can induce autophagy in HepG2 cells.JG treatment led to a significant the accumulation of autophagic vacuoles labeled by AO and MDC.Then,western blotting results confirmed that an increasing level of LC3II,Beclin-1 and Atg5 proteins,while the expression of p62 protein was gradually decreased.Next,the laser confocal microscopy revealed that JG markedly induced the increase of red and yellow fluorescent puncta in HepG2 cells transfected with adenovirus mCherry-GFP-LC3.However,after the combination of inhibitors CQ and Baf A1,almost all of cells were yellow puncta.In addition,both MTT and Annexin-V-FITC/PI results confirmed that JG-induced apoptotic effects were blocked in the presence of 3-MA and wortmannin compared to JG treatment alone.3.JG can induce autophagy-related pathway activation in HepG2 cells.Western blotting revealed that an increase in phosphorylation of p38 MAPK and JNK was found after JG treatment,but not p-ERK 1/2.Further research confirmed that JG-induced autophagy was associated with p3 8 and JNK through the utilization of SP60012(JNK inhibitor)and SB203580(p38MAPK inhibitor).Subsequently,phosphorylation levels of AMPK? and ACC were significantly increased,but the protein expression of p-mTOR was inhibited.Next,suppression of AMPKa significantly reduced autophagy caused by JG.In addition,JG-induced autophagy was inhibited by silencing p53.4.JG can induce mitochondria-mediated ROS production in cells.Flow cytometry revealed that JG decreased MMP in a time-and concentration-dependent manner.However,treatment with NAC effectively suppressed JG-induced reduction of MMP.Meanwhile,elevating the ratio of Bax/Bcl-2 was observed with the addition of JG,and treatment with NAC reversed this phenomenon.Further studies have found that JG obviously elevated the levels of O2·-and H2O2.After combined CAT(H2O2 scavenger)or SOD(O2·-scavenger)with JG,the survival rate of the cell measured by MTT was risen to varying degrees,which was consistent with the apoptosis obtained by flow cytometry.Moreover,western blotting analysis also showed that both CAT and SOD could inhibit JG-induced p53 accumulation and activation of apoptotic proteins PARP and caspase-3.In addition,the laser confocal microscopy revealed that CAT treatment could significantly inhibit the production of fluorescent puncta in adenovirus mCherry-GFP-LC3 transfected cells compared with SOD does.Results in vivoJG can inhibit the growth of xenografted tumors in nude mice.Compared with the control group,treatment JG significantly inhibited the growth of the volume and weight of solid tumors.At the same time,western blotting and immunohistochemistry analysis revealed that JG-treated tumor tissues caused a higher expression in mean areas for cleaved caspase-3,LC3II and p53.Finally,a slight loss in body weight was observed after JG injection,but it was no obvious toxicity to major organs by hematoxylin and eosin(H&E)staining,which indicated that the side effects were relatively low.Research Conclusion:In summary,our study demonstrated that JG had significant anti-tumor activity.Subsequently,JG could effectively inhibit the proliferation of HepG2 cells,induce cell cycle arrest and apoptosis.Moreover,JG also caused autophagy in HepG2 cells,and inhibition of autophagy attenuated JG-induced apoptosis.Additionally,both autophagy and apoptosis induced by JG were associated with levels of oxidative stress in the cells.Finally,JG significantly inhibited the growth of tumor xenografts compared to vehicle-treated without obvious side effects.In summary,this study provides a scientific basis for the clinical application of JG.