| Objective: Hepatocellular carcinoma is one of the most common malignant tumors and the third leading cause of death among tumor patients.At present,although the commonly used chemotherapeutic drugs in clinical practice have achieved certain efficacy,they can only prolong the survival of patients for a few months and cause toxic effects.Natural products are widely available and have low side effects,which is one of the main sources for discovering new anti-tumor drugs.As a natural product,Lycium ruthenicum is rich in anthocyanins,flavonoids,polysaccharides and other components,and has biological activities such as antitumor,anti-inflammatory and anti-aging.Studies have shown that anthocyanins,as one of the main active components of Lycium ruthenicum,have good anti-tumor activity.However,the anti-tumor effect and mechanism of anthocyanins from Lycium ruthenicum(abbreviated as ALR)in hepatocellular carcinoma are still unclear.To this end,this project investigated the anti-tumor activity and mechanism of ALR in hepatocellular carcinoma using Hep G2 cells and mouse subcutaneous hepatocellular carcinoma transplantation tumors,to provide new reference for the development and clinical application of anti-hepatocellular carcinoma drugs.Methods: 1.In vitro anti-hepatocellular carcinoma effect and mechanism study of ALR:Hep G2 cells were used as the study target.Firstly,the action time and dose of ALR were determined by CCK-8 assay.Secondly,the effects of ALR on Hep G2 cell morphology,clone formation ability,function,apoptosis and cycle were observed by plate cloning assay,scratch assay,transwell assay,AO/EB staining,V-FITC/PI staining and flow cytometry assay to evaluate the anti-tumor effect of ALR.Further,the effects of ALR on the autophagic morphology,autophagy-related proteins(Beclin-1,LC3-Ⅰ,LC3-Ⅱ)and autophagic pathwayrelated proteins(p-AMPK,AMPK,p-m TOR,m TOR)expression levels of Hep G2 cells were observed by AO,MDC,m GFP-LC3 adenovirus staining and Western blot assay,the mechanism of its anti-hepatocellular carcinoma action was investigated in vitro.2.In vivo anti-hepatocellular carcinoma effect and mechanism study of ALR: A mouse subcutaneous hepatocellular carcinoma transplantation tumor model was used as the study target.Firstly,the anti-tumor effect of ALR was evaluated by measuring tumor volume and tumor weight in mice using ALR alone or combined with autophagy inhibitor CQ.Secondly,the effects of ALR on autophagy-related proteins(Beclin-1,LC3-II),apoptosis levels,apoptosis-related proteins(Bcl-2,Bax,cleaved caspase-3,cleaved caspase-9)and autophagic pathway-related proteins(p-AMPK,p-m TOR)expression levels in vivo to investigate the antitumor mechanism of ALR.Results: 1.In vitro anti-hepatocellular carcinoma effect and mechanism of ALR: ALR inhibited Hep G2 human hepatocellular carcinoma cell viability in a time-and dose-dependent manner,with significant inhibition on Hep G2 cell viability at concentrations of 500 μg/m L,750μg/m L,1000 μg/m L and 1500 μg/m L for 24 h(P < 0.01),while concentrations of 500 μg/m L,750 μg/m L and 1000 μg/m L had no significant inhibition on LO2 human normal hepatocyte viability,so 500 μg/m L and 1000 μg/m L for 24 h were selected for the follow-up experiments.Further study revealed that ALR could significantly inhibit Hep G2 cell clone formation ability,migration and invasion,promote apoptosis and G2/M phase block(P < 0.05).Mechanistic studies showed that ALR was able to increase the number of autophagic vesicles and autophagosome,upregulate the expression of autophagy-related proteins Beclin-1 and LC3-II/LC3-I,and induce autophagy in Hep G2 cells(P < 0.05).The effects of ALR on Hep G2 cell viability,migration and invasion capacity,apoptosis and G2/M phase block were reversed by inhibiting cell autophagy with the autophagy inhibitor 3-MA(P < 0.05),indicating that ALR inhibited cell viability,migration and invasion capacity,induced apoptosis and G2/M phase block by inducing Hep G2 cell autophagy.Further research showed that it was shown that ALR was able to induce autophagy in Hep G2 cells by activating the AMPK/m TOR signaling pathway.2.In vivo anti-hepatocellular carcinoma effect and mechanism study of ALR: ALR could significantly inhibit the growth of subcutaneous hepatocellular carcinoma transplantation tumors in mice(P < 0.05).Mechanistic studies showed that ALR could significantly up-regulate the expression of Beclin-1 and LC3-II in tumor tissues(P < 0.001),thus inducing autophagy in tumor tissues.In addition,ALR was able to significantly upregulate the expression of apoptosisrelated proteins Bax,cleaved caspase-3 and cleaved caspase-9 and downregulate the expression of Bcl-2 in tumor tissues by inducing autophagy(P < 0.001),thus promoting apoptosis in tumor tissues.Further studies showed that ALR could activate AMPK/m TOR signaling pathway,thus inducing autophagy in tumor tissues.Conclusion: ALR was able to inhibit Hep G2 cell viability,migration and invasion ability,promote apoptosis and G2/M phase block by activating AMPK/m TOR autophagy pathway.In addition,ALR was able to activate AMPK/m TOR autophagy-related pathway and induce autophagy-dependent apoptosis in tumor tissues,thereby significantly inhibiting subcutaneous hepatocellular carcinoma transplant tumor growth in mice. |
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