| ［Background］ Hepatic carcinoma is the second leading cause of cancer-related deaths with an incidence of more than 1 million cases per year worldwide,and the development is closely related to cellular molecular mechanisms.Currently,the main treatment of hepatic carcinoma are surgery,radiotherapy and chemotherapy.However,most of hepatic carcinoma can be detected in the progress stage,which only medical can be adopted with further treatment.With the deepening of research on hepatic carcinoma,an increasing treatment of chemical and novel target drugs focus on hepatic carcinoma.However,the prognosis of hepatic carcinoma was still poor because of drug resistence in some patients.Thus,the research of novel drugs will be a promising strategy for the treatment of hepatic carcinoma.With the discovery of antioxidative,antitumor and other functional activities in natural plant monomer compounds,active extracts of various natural medicinal plants have been used in cancer therapy and shown favorable antitumor effects.Alantolactone(Ala)and tanshinone Ⅰ(TanI)have been shown to significant anti-cancer effects and low toxicity,whereas the role of Ala and TanI in anti-hepatic carcinoma has also not been studied and reported.Thus,the effect and molecular mechanism of Ala and TanI in anti-hepatic carcinoma remain to be further elucidated.［Aims］ This study intends to systematically investigate the associated mechanisms of anti-hepatic carcinoma effects in Ala and TanI natural compounds through the analysis of both in vitro and in vivo experiments,which provived a theoretical and experimental basis for the clinical application of Ala and TanI as novel anti-cancer drugs.［Methods］ 1.Inverted microscope was used to observe the morphological changes in HepG2 cells.To explore the effects of Ala and TanI on the proliferation of hepatic carcinoma and normal human hepatocytes,the cell viability was determined using CCK-8 and RTCA system assay.2.Cell cycle arrest and apoptosis were detected by PI,DAPI and V-FITC/PI staining,respectively.The expression levels of apoptic and cycle proteins were also exhibited by western blot(WB)in order to preliminary study the molecular mechanism of Ala and TanI-mediated HepG2 apoptosis.3.After staining HepG2 cells with DCFH-DA,the level of intercellular ROS was analyzed by flow cytometry.ROS scavenger NAC was pre-treated HepG2 cells,and then determine the apoptosis and proliferation in Ala and TanI-treated HepG2 cells.Meanwhile,the expression levels of p-AKT,p-ERK,and apoptosis-related proteins were detected to clarify the effect and mechanism of ROS on HepG2 cell apoptosis.4.Under the Olympus Confocal Laser Scanning microscopy,JC-1 experiment was performed to observe mitochondrial membrane potential,while Mito Tracker combined Lyso Tracker were used to observe mitophagy.The expression of mitophagy-related proteins was detected by WB,and then mitophagy inhibitor Mdivi-1 was used to determine the relationship between mitophagy and apoptosis in Ala-or TanI-stimulated HepG2 cells.5.The overexpression vector of PINK1 and Parkin were also constructed to validate Alaor TanI-mediated mitophagy inhibition was crucial for the proliferation in HepG2 cells.6.To screen differentially expressed miRNAs,both treating and controlling cells were collected for miRNA-seq sequencing.Bioinformatics software miRDB was applied to identify the binding domain between PINK1 and miRNAs.Transient transfection of hsa-miR-2467-5p inhibitor was used to validate the interaction between PINK1 and hsa-miR-2467-5p.7.BALB-c nude mice model was constructed to clarify effect of Ala and TanI on proliferation of hepatic carcinoma cells,and further confirmed the molecular mechanism of Ala-and TanI-induced apoptosis in vivo by HepG2 cells subcutaneous injection in BALB-c nude mice.［Results］ 1.Both Ala and TanI significantly suppressed the proliferation of human HepG2 cells in a dose and time-dependent manners,whereas Ala and TanI showed no significant inhibition in HL-7702 normal liver cells with the same treatment conditions.2.Ala and TanI could induce G2/M and G0/G1 cell cycle arrest in HepG2 cells respectively,and the percentage of cells accumulated in G2/M and G0/G1 phase was obviously enhanced to 22.7% and 69.83%.The expression levels of corresponding cycle proteins(including cyclin A1,cyclin B1 and cyclin D1)were decreased,whereas p21 expression was significantly increased in a dose-dependent manner.3.Both Ala-and TanI-treated HepG2 cells exhibited the characteristic of apoptosis,including roundness,nucleus pyknosis and decreased in cell number.Furthermore,Ala and TanI could induce apoptosis through increasing the expression of apoptotic related proteins,such as cleaved-PARP,cleaved-caspase-3 and cleaved-caspase-8,whereas decreasing the protein expression of Bcl-2,and activating apoptosis.4.Ala and TanI resulted in a significantly increase of intracellular ROS in HepG2 cells.After pretreatment with NAC,cell viability significantly enhanced,while apoptosis significantly attenuated.Meanwhile,Ala induced the decrease of p-AKT protein expression,whereas TanI inhibited the expression of p-ERK,and then resulted in the expression level of cleaved-caspase-3,cleaved-caspase-8 and cleaved-PARP increased.5.Both Ala and TanI could induce mitochondrial membrane potential decreased,then inhibited mitophagy and the expression of related proteins in HepG2 cells.The influence of Ala or TanI on cell viability with Mdivi-1 strikingly decreased and consequently promoted apoptosis.6.After transient transfection with PINK1 or Parkin overexpression vector,the suppression effect of Ala or TanI on HepG2 cell proliferation was significantly attenuated.7.It was found that the expression of hsa-miR-2467-5p was related to mitophagy by miRNA-seq.Combined with bioinformatics,we found that hsa-miR-2467-5p binded to PINK1 gene in the 278-284 3’-UTR region.After transfecting the hsa-miR-2467-5p inhibitor into HepG2 cells,both the expression of PINK1 mRNA and protein were significantly increased compared with the untransfected cells.8.In the nude mice model,both Ala and TanI could effectively reduce tumor size and promote apoptosis in tumor cells.Ala decreased the expression of p-AKT protein,and TanI inhibited the expression level of p-ERK protein.In addition,in vivo experiments suggested that the expression level of PINK1 significantly decreased by Ala-or TanI-stimulating,and then mitophagy-inhibited.［Conclusion］ 1.In this study,we demonstrated that Ala and TanI could inhibit the growth of hepatic carcinoma cells by in vivo and in vitro experiments.2.Ala induced G2/M cell cycle arrest by inhibiting the expression of cyclin A1 and cyclin B1,whereas TanI mediated cell cycle arrest in G0/G1 phase through inhibiting the expression of cyclin D1.3.Both Ala and TanI could promote the increase of intracellular ROS level.Meanwhile,Ala or TanI induced HepG2 cell apoptosis by ROS/AKT or ROS/ERK pathway,respectively.4.hsa-miR-2467-5p could supressed the expression of PINK1 protein by targeting the 3’-UTR of PINK1 gene,which resulted in the inhibition of PINK1-mediated mitophagy.5.It was confirmed that Ala or TanI promoted intracellular ROS level,apoptosis and the growth inhibition of hepatic carcinoma cells by preventing PINK1/Parkin-mediated mitophagy. |
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