| Objective:This study was to investigate the inhibitory effects of two phenanthrenes compounds isolated from Spiranthes sinensis on human hepatocellular carcinoma cell line HepG-2,human gastric cancer cell line AGS,and mouse melanoma cell line B16-F10,and the underlying mechanisms.Methods:1.The relative cell viability was detected by an MTT assay after the treatment of HepG-2 cells with different concentrations of orchinol for 48 h.A cell clone formation assay was carried out to analyze the effect of different concentrations of orchinol on HepG-2 cell proliferation.Flow cytometry was applied to detect changes of the apoptosis rate and mitochondrial membrane potential in HepG-2 cells caused by orchinol.Immunofluorescence staining was performed to detect the effects of orchinol on autophagy vesicles quantity and autophagy-related protein LC3Ⅱ expression.Western blot was carried out to observe the effect of orchinol on the expression of apoptosis-related proteins(Cleaved Caspase-3,Cleaved Caspase-9,Bax,Cytochrome C,and Bcl-2),autophagy-related proteins(LC3Ⅱ,Beclin-1,Atg5,and Atgl2)and PI3K/AKT/mTOR pathway key proteins(p-AKT,p-mTOR and p-PI3K).In addition,LY294002,an inhibitor of PI3K/AKT/mTOR signal pathway,was co-treated with orchinol and their effects on the apoptosis,autophagy and PI3K/AKT/mTOR pathway were then examined.The autophagy agonist Rapamycin(RAPA)was co-treated with orchinol and their effects on the relative cell viability,apoptosis and autophagy of HepG-2 cells were also investigated.2.The effect of spiranthesphenanthrene A on the relative cell viability after treating AGS cells with different concentrations of spiranthesphenanthrene A for 24,48 and 72 h was determined by the MTT assay.The apoptosis of AGS cells was examined by the flow cytometry after the cells were treated by spiranthesphenanthrene A for 48 h.Western blot analysis was used to observe the effects of spiranthesphenanthrene A on the expression of apoptosis-related proteins(Cleaved Caspase-3,Cleaved Caspase-9,Bax,Cytochrome C and Bcl-2).3.The effect of spiranthesphenanthrene A on relative cell viability of mouse melanoma B16-F10 cells was evaluated by the MTT assay after treating B16-F10 cells with different concentrations of spiranthesphenanthrene A for 48 h.A wound healing assay was performed to reveal the influence of spiranthesphenanthrene A on the migration ability of AGS cells.Western blot assay was applied to evaluate the effects of spiranthesphenanthrene A on the expression of EMT-related proteins N-cadherin,E-cadherin Vimentin and Snail.Results:1.After HepG-2 cells were treated with orchinol for 48 h,the relative cell viability decreased and the inhibitory ability increased with the increase of the concentration of orchinol,indicating that orchinol could dose-dependently inhibit the relative viability of HepG-2 cells.The half-inhibitory concentration(IC50)of orchinol was 16.20 ± 0.24 μmol/L.Meanwhile,orchinol could inhibit the ability to form clones of HepG-2 cells in a dose-dependent manner.Flow cytometry results showed that orchinol could increase the apoptosis rate and decrease the mitochondrial membrane potential in a dose-dependent manner after treating for 48h.The results of immunofluorescence assay demonstrated that orchinol could increase the numbers of autophagy vesicles and up-regulate LC3Ⅱ protein expression.The results of Western blot showed that orchinol could increase the protein expression levels of Cleaved Caspase-3,Cleaved Caspase-9,Bax,Cytochrome C,LC3Ⅱ,Beclin-1,Atg5 and Atgl2,and decrease those of Bcl-2,p-AKT,p-mTOR and p-PI3K.In addition,compared with the treatment with orchinol or LY294002 alone,the co-treatment with LY294002 and orchinol further increased the apoptosis rate;up-regulated the protein expression levels of Cleaved Caspase-3,Cleaved Caspase-9,LC3Ⅱ and Beclin-1;and decreased those of p-AKT,p-mTOR and p-PI3K in HepG-2 cells.A further decrease in the relative cell viability and increases in the apoptosis rate and protein expression levels of Cleaved Caspase-3,Cleaved Caspase-9,LC3Ⅱ and Beclin-1 were found in the RAPA and orchinol co-treatment group compared with the orchinol and RAPA groups.2.AGS cells were incubated with different concentrations of spiranthesphenanthrene A for 24,48,and 72 h,respectively.With the increase of the time and concentration,the relative cell viability decreased,together with increased inhibitory rate,indicating that spiranthesphenanthrene A could dose-and time-dependently inhibit the relative viability of AGS cells.The IC50 values of spiranthesphenanthrene A after 24,48 and 72 h were 40.55 ±5.85,24.61 ± 2.84 and 13.41±1.16 μmol/L,respectively.The total apoptosis rates of AGS cells at the concentrations of 0,2.5,5,and 10 μmol/L were 3.90± 0.81%,23.42 ± 5.56%,44.92 ± 8.43%,and 51.19 ± 5.59%,respectively.The above results displayed that spiranthesphenanthrene A significantly induced apoptosis in AGS cells in a dose-dependent manner.Compared with the control group,different concentrations of spiranthesphenanthrene A could apparently increase the expression levels of Cleaved Caspase-3,Cleaved Caspase-9,Bax,and Cyt-C proteins,and decrease that of Bcl-2 protein in a dose-dependent manner.3.Spiranthesphenanthrene A had a significant killing ability on B16-F10 cells after 48 h treatment,and the IC50 value was 19.0 ± 7.3 μmol/L.Spiranthesphenanthrene A could significantly inhibit the migration of B16-F10 cells,up-regulate the expression level of E-cadherin protein and down-regulate those of Vimentin,N-cadherin and Snail proteins.Conclusion:Orchinol can inhibit the proliferation of HepG-2 cells,which may be achieved by inducing apoptosis and autophagy through inhibition of the PI3K/AKT/mTOR pathway;meanwhile,orchinol-mediated autophagy can promote apoptosis in HepG-2 cells.Spiranthesphenanthrene A can inhibit the relative viability of AGS cells and promote apoptosis probably through the mitochondria-mediated intrinsic pathway.Spiranthesphenanthrene A can inhibit the relative viability and migration of B16-F10 cells,which may be related to the inhibition of EMT. |
PDF Full Text Request