| OBJECTIVE Selecting human hepatocellular carcinoma cell line SMMC-7721 in vitro as the research object,we aimed to study the inhibitory effect of JS-K on hepatoma cell proliferation and its influence on cell cycle and apoptosis by detecting changes in mitochondrial apoptosis pathway,PI3K/Akt pathway and cycle-related proteins.METHODS(1)Inhibitory effect of NO donor JS-K on SMMC-7721 cell proliferation: SMMC-7721 cells were treated with different concentrations of JS-K(0.75,1.50,3.13,6.25,12.50,25 μM)and different concentrations of 5-FU(50,100,200 μM)for 24 h,48 h,72 h,respectively.The inhibitory effect of them on cell proliferation was detected by MTT assay.The morphological changes of cells were detected by inverted microscope after 24 h treatment with JS-K.The proliferation of cells was observed by crystal violet staining after 24 h treatment with JS-K.(2)Induction of apoptosis of SMMC-7721 cells by NO donor JS-K: cells were treated with JS-K(0,2.5,5,10 μM)and 5-FU(100 μM)for 24 h,respectively.The morphological changes of apoptosis cells were observed by DAPI staining.The apoptosis rate was detected by Annexin V-FITC/PI double staining.The changes of mitochondrial membrane potential(MMP)were detected by Rhodamine staining and JC-I staining respectively.Western blotting was used to detect the expression of mitochondrial apoptosis-related protein and caspase inhibitor was added to verify the pathway.Fluorescence microscopy and flow cytometry were used to detect the NO level in cells.The level of NO,mitochondrial membrane potential,apoptosis rate and the expression of apoptosis-related protein were detected by adding NO scavenger Carboxy-PTIO.(3)Effect of NO donor JS-K on cell cycle arrest: the cell cycle was analyzed by PI staining.Western blot was used to detect the expression of cycle related proteins and PI3K/Akt pathway proteins after JS-K treatment.NO scavengers was added to detect the expression of these proteins.RESULTS(1)Effect of NO donor JS-K on proliferation of SMMC-7721 cells:MTT results showed that different concentrations of JS-K and 5-FU significantly inhibited cell proliferation,and the IC50 values of JS-K group at 24 h,48 h,and 72 h were(5.12 ± 0.02),(4.01 ± 0.01)and(3.10 ± 0.03)μM.The cell morphology of JS-Kgroup was found to be more rounded,wrinkled and smaller under inverted microscope.The results of crystal violet staining showed that different concentrations of JS-K could inhibit cell proliferation.(2)Effects of NO donor JS-K on apoptosis of SMMC-7721 cells: The typical apoptotic morphological changes such as cell chromatin condensation and nuclear division were observed under fluorescence microscopy after JS-K treatment.The results of double staining showed that the apoptotic rate of JS-K group were(19.23 ±0.08)%,(40.70 ± 0.12)% and(80.51 ± 0.05)%,higher than the control group(2.73 ±0.02)%(P<0.01).The results of JC-1 staining showed that JS-K caused an obvious collapse of MMP in a dose-dependent manner.Compared with the control group,the proportion of cells with depolarized MMP increased from(10.30 ± 1.75)% of the control cells to(23.42 ± 2.85)%,(50.74 ± 3.75)% and(71.62 ± 2.96)%.Rhodamine staining showed that the fluorescence intensity of Rh123 in the cells decreased from(660 ± 24.72)of the control cells to(584 ± 22.30),(384 ± 18.51)and(281 ± 11.50).Western blot showed that JS-K could induce the decrease of Bcl-2 expression,while the expression of Bax and activated caspase-3,caspase-9 increased significantly,and the ratio of Bax/Bcl-2 increased.After activating the caspase inhibitors Z-LEHD-FMK and Z-DEVD-FMK,the apoptotic rate was reduced from(77.72 ± 0.43)% in the JS-K group to(50.76 ± 0.31)%,(66.53 ± 0.44)%,respectively.DAF-FM DA fluorescence staining showed that the control group showed low-density fluorescence,while the fluorescence of the JS-K group gradually increased.Flow cytometry showed that intracellular NO levels in the JS-K group were(151.20 ± 1.16)%,(248.62 ± 1.61)%and(347.60 ± 2.42)%,compared with the control group(100%).After adding NO scavenger Carboxy-PTIO treatment,the NO level in JS-K group decreased from(323.16 ± 1.71)% to(210.32 ± 0.80)%.The fluorescence intensity of Rh123 in cells increased from the original(311.52 ± 18.57)to(534.50 ± 30.52),reversing the loss of mitochondrial membrane potential induced by JS-K.At the same time,Carboxy-PTIO significantly decreased the apoptosis rate,ranging from(80.50 ± 3.42)% to(23.42 ±8.25)%,and reduced the down-regulation of Bcl-2,up-regulation of Bax and activation of caspase-9/3 induced by JS-K.At the same time,the positive drug 5-FU also caused significant changes in the above targets.(3)Effect of NO donor JS-K on cell cycle arrest: PI staining showed that the percentage of cells in G2/M phase increased from(21.53 ± 5.13)% of the control cells to(35.67 ± 1.83)%,(46.93 ± 7.22)% and(59.73 ± 1.86)%,suggesting that JS-Kinduced cell cycle arrest in G2/M phase in a concentration-dependent manner.Western blot results showed that JS-K induced down-regulation of cyclin B1 expression and up-regulation of p-cdc2 and p21 protein expression.Adding NO scavenger partially reversed the changes of JS-K induced protein expression.In addition,JS-K blocked the PI3K/Akt pathway and decreased the expression of p-PI3 K and p-Akt.After adding NO scavengerr,the changes of JS-K induced protein expression were reversed.CONCLUSIONS(1)JS-K could significantly inhibit the proliferation of SMMC-7721 cells in a time and dose dependent manner.(2)JS-K could induce apoptosis of SMMC-7721 cells,which may be related to the activation of mitochondrial apoptosis pathway.(3)JS-K could induce cell G2/M phase arrest in SMMC-7721 cells,which may be related to blocking the PI3K/Akt pathway,upregulating p-cdc2,p21 protein expression,and down-regulating cyclin B1 protein expression.In summary,NO released by JS-K could induce cell G2/M cycle arrest by inhibiting the PI3K/Akt pathway and activate mitochondrial pathway to induce apoptosis. 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