Research In Mechanism Of Effects Of Salinomycin On Proliferative, Invasive And Metastatic Abilities Of Hepatoma Cell Lines

Objective:To investigate effects of salinomycin on the proliferative, invisive and metastatic abilities of hepatoma cell line in vitro and analyze the possible mechanisms. To construct lentivirus-mediated shRNA expression vector targeting c-Src and to establish stable hepatoma cell line HepG2. To explore the effect RNAi-mediated inhibition of c-Src gene on apoptosis and chemosensitivity to Salinomycin in hepatoma HepG2cells and its underlying mechanism.Methods:1、MTT was used for determining the proliferative abilities of hepatoma cells and human normal liver L02cells. Morphological changes were observed under the inverted phase-contrast microscope and photographed. Cell apoptosis was detected by Hoechst33258staining and flowcytometry analysis. The morphological alteration was observed by Hoechst33258staining. The cytoskeleton was observed using confocal laser scanning microscopy after F-actin staining by FITC-labeled phalloidin. The invasive and migratory abilities were detected by Transwell assay. Cell-cycle analysis was detected by flowcytometry. The relative protein expressions of Src/PI3K/Akt signal pathway were evaluated by Western Blotting.2、Lentiviral shRNA targeting c-Src was applied to construct stable knock-down hepatoma cell line HepG2, Western Blotting techniques were used to detect the alteration of protein level. HepG2cells were conventionally cultured and divided into6groups:control group (CON), negative control group (NC), RNAi c-Src group (KD), control combined with Salinomycin group (CON+Sal), negative control combined with Salinomycin group (NC+Sal), RNAi Src combined with Salinomycin group (KD+Sal). The proliferation of above mentioned HepG2cells was examined by MTT assay. Flow cytometry (FCM) was employed to detect the cell apoptosis and cell cycle. The protein expressions of MDRland Src/PI3K/Akt signal pathway were evaluated by Western Blotting. Results:1、The treatment of L02cells with salinomycin (0～8μmol/L) for24h and48h had no significant effects on cell viability. However, treatment of similar doses of salinomycin suppressed the growth of hepatoma cells in a time and concentration dependent manner. Salinomycin could lead to characteristic morphologieal changes of apoptosis in human hepatoma cells after24h and48h in a concentration dependent manner. Hepatoma cells was treated by salinomycin (1μmol/L,4μmol/L) for24h to detect the invasive and migratory abilities. Compared to that in the control group, the number of invasive and migratory cells was remarkably decreased (P<0.01), the structure of F-actin microfilament cytoskeleton was significantly changed. Salinomycin could also induce a cell cycle arrest of hepatoma cells at the phase of G0/G1. Marked changes in apoptotic morphology and apoptosis rates were clearly observed after salinomycin treatment. In addition, Immunoblot showed that salinomycin inhibited the activation of Src/PI3K/Akt pathway in a dose-dependent manner in hepatoma cells. Activation of GSK-3β, caspase-9, caspase-3, cleavage of PARP, upregulation of Bad, Bax, E-cadherin, downregulation of c-Src, Akt, Bcl-2, c-myc, cyclin Dl, survivin, MMP-2and MMP-9were also found in salinomycin-treated hepatoma cells.2、KD group had a significantly lower protein level of c-Src Src and MTT value than CON group and NC group (P<0.05), and had a higher apoptotic rate and larger percentage of G0/G1arrested cells and lower MDR1, Akt, cyclinDl expression than NC group (P<0.05). In KD+Sal group, MTT value was more lower and stronger caspase-9expression and lower MDR1, Akt, cyclinDl expression and larger percentage of G0/G1arrested cells than in KD group and NC+Sal group (P<0.05).Conclusion:1、The results suggested that the proliferative, invasive and migratory abilities of hepatoma cells can be inhibited by salinomycin, which may be related to blocking the Src/PI3K/AKT kinase cascade signaling pathway.2、RNAi could specifically supress c-Src gene expression, effectively inhibit the proliferation and increase G0/G1cell population and accelerate the apoptosis of HepG2cells, and enhance its chemosensitivity to Salinomycin. The mechanism may be related to the suppression of the activation of MDRland Src/PI3K/Akt pathway and the activation of Caspase signaling pathways. Objective:Cancer chemopreventive agent curcumin have been shown to possess inhibition cell growtlh and induction apoptosis properties in several types of cancer. However, the detailed molecular mechanisms of the compound remain far from clear in EJ bladder cancer cells. In order to explore the possible mechanism and offer experimental evidence of anti-cancer effcet of curcumin combined with LY294002, we investigated the anti-proliferation and apoptosis induction effcet of curcumin combined with LY294002on human bladder cancer cells.Methods:1. The effect of curcumin on EJ cells growth inhibition and apoptosis was detected by MTT assays and flow cytometry. The phosphorylation levels of PTEN, PDK1, Akt, GSK-3β, c-Raf, Bad and the expression levels of c-myc, Bax, Bcl-2, caspase-9, caspase-7, caspase-3, PARP by curcumin administration were examined by Western Blotting.2. After EJ cells were pretreated with the PI3k inhibitor LY294002(20μM) in complete medium for1h, and then various doses of Curcumin (0,12.5、25and50μM) was added to co-culture for the indicated time periods. The growth inhibition of curcumin and LY294002alone or in combination on bladder cancer EJ cells by MTT assay. Morphological changes were observed under the inverted phase-contrast microscope and photographed. Apoptotic rates of human EJ bladder carcinoma cells were determined by flow cytometry (FCM). Curcumin and LY294002alone or in combination regulated caspase-9、caspase-7、caspase-3and PARP protein expression in EJ cells by Western Blotting analysis.Results:1. Curcumin suppressed the growth of EJ cells in a time-and concentration-dependent manners. Immunoblot showed that curcumin increased expression levels of c-myc and inhibited the activation of PI3K/Akt pathway in a time-dependent manner in EJ cells. Activation of PTEN, GSK-3(3, c-Raf, caspase-9, caspase-7, caspase-3, cleavage of PARP, upregulation of Bad, Bax, downregulation of Akt, Bcl-2were also found in curcumin-treated EJ cells.2. EJ cells viability showed remarkable decrease in a concentration-and time-dependented manner in the curcumin (12.5,25and50μM)+LY294002(20μM) group as compared with curcumin (12.5,25and50μM)-treated control group or with LY294002(20μM) alone. Curcumin and LY294002could lead to characteristic morphologieal changes of apoptosis in human bladder carcinoma cells after24h including cellular rounding, shrinkage, chromatin condensation, and membrane blebbing, the appearance of membrane-associated apoptotic bodies and separation from neighboring cells. Combined treatment with LY294002and curcumin increased the percentage of apoptotic cell death in EJ cells as compared with either compound alone. The presenting time of the cleavage of caspase-9,-7,-3and PARP following the LY294002(20μM)/curcumin (25or50μM) combination administration is earlier than that of curcumin (25or50μM) alone treatment in EJ cells by Western Blotting analysis.Conclusion:1.These findings esteblish a mechanistic linkup or interaction between c-myc, Bax, Bad, Bcl-2, caspase cascades, PI3K/Akt pathway and curcumin-induced apoptosis of EJ cells, suggest that c-myc and PI3K/Akt signaling pathway plays an important roles in curcumin-induced apoptosis of EJ bladder cancer cells.2. Curcumin or LY294002could significantly inhibit the growth of human EJ bladder carcinoma cells, LY294002could effectively increase the growth inhibition of curcumin in EJ cells in vitro. The curcumin and LY294002can all induce human EJ carcinoma cells apoptosis. When the two drugs combined, the inductive effect for cell apoptosis became more obvious, which suggest a possible underlying molecular mechanism Where by curcumin and LY294002could induce the apoptosis signaling pathway in human EJ bladder carcinoma cells by caspase family protein activation and cleavage of PARP protein through mitochondrion-dependent pathway. LY294002could enhance the anti-tumor effect of curcumin, which provide the new thread and methods for the clinical thrapy of bladder cancer.