Cepharanthine induces autophagy via PI3K/AKT/mTOR signaling pathway in ovarian cancer SKOV3 cells

AIM: To investigate the autophagy of human ovarian cancer SKOV3 cells induced by cepharanthine and to explore its mechanism. METHODS: The effect of cepharanthine on the viability of ovarian cancer SKOV3 cells was measured by CCK-8 assay. The SKOV3 cells were treated with cepharanthine, and then the formation of autophagosome was observed with acridine orange staining under fluorescence microscope. The protein levels of LC3, AKT, p-AKT, mTOR, p-mTOR and GAPDH in the SKOV3 cells treated with cepharanthine were determined by Western blot.RESULTS: Cepharanthine significantly inhibited the viability of ovarian cancer SKOV3 cells in a dose-dependent manner (P<0.05). The number of the intracellular acidic autophagosomes with bright red fluorescence was significantly increased after cepharanthine treatment in the SKOV3 cells. The expression of LC3-Ⅱ in SKOV3 cells was significantly enhanced after cepharanthine treatment. Furthermore, treatment with cepharanthine in the SKOV3 cells also resulted in a significant down-regulation of phosphorylated form of AKT and mTOR (P<0.01), while the total protein level was not changed. Combination of cepharanthine and 3-methyladenine resulted in a substantial decrease in the cell viability compared with using cepharanthine alone.CONCLUSION: Cepharanthine significantly inhibits the growth of human ovarian cancer SKOV3 cells and induces the autophagy, which may be correlated with down-regulation of PI3K/AKT/mTOR signaling pathway.     

Cepharanthine promotes A549 cell apoptosis by regulating miR-150 and miR-182

AIM: To investigate the effect of cepharanthine on the growth of human lung carcinoma A549 cells and the underlying mechanism. METHODS: After A549 cells were treated with cepharanthine, the growth inhibitory rate was detected by MTT assay. The cell morphological changes were observed under light microscope. The apoptosis of the A549 cells was analyzed by flow cytometry. The expression of microRNA (miR)-150, miR-182, p53 mRNA and FOXO1 mRNA were detected by real-time PCR. The downstream target genes were predicted by software, and the expression of p53 and FOXO1 was determined by Western blot. RESULTS: After cepharanthine treatment, the growth of A549 cells was inhibited, the apoptosis rate was significantly increased, and the expression levels of miR-150 and miR-182 were significantly decreased. With cepharanthine treatment at 10 μmol/L, the expression levels of p53 and FOXO1 were elevated; however, with cepharanthine at 30 μmol/L, the expression levels of p53 and FOXO1 were decreased. After transfection with miR-150, the expression of p53 was significantly decreased, while the expression of FOXO1 was significantly decreased after transfection with miR-182. CONCLUSION: Cepharanthine inhibits the growth of A549 cells and promotes the apoptosis of A549 cells by inhibiting the expression of miR-150 and miR-182. miR-150 and miR-182 may down-regulate the expression of p53 and FOXO1, respectively.     

Cepharanthine induces miRNA expression in human lung adenocarcinoma LTEP-a-2 cells

AIM: To observe the effect of cepharanthine on human lung adenocarcinoma LTEP-a-2 cell growth, and to explore the changes of related microRNA (miRNA) expression in the cells. METHODS: LTEP-a-2 cells were treated with cepharanthine at concentrations of 0 μmol/L, 10 μmol/L, 20 μmol/L and 40 μmol/L. The growth inhibition rate was detected by MTT assay, and the cell morphological changes were observed under light microscope. The cell apoptosis was analyzed by flow cytometry. The expression of let-7c, miR-34a and miR-34b was measured by real-time PCR. RESULTS: Cepharanthine inhibited the cell activity of LTEP-a-2 cells in a dose-dependent manner. With the increase in cepharanthine concentration, the pyknosis of the cells was visible under the inverted microscope. Flow cytometry analysis found that different concentrations of cepharanthine induced the increase in the apoptotic rates of LTEP-a-2 cells. The results of real-time PCR showed that the cepharanthine also increased the expression of let-7c, miR-34a and miR-34b. CONCLUSION: Cepharanthine inhibits the growth of LTEP-a-2 cells, and induces apoptosis. Cepharanthine increases the expression of let-7c, miR-34a and miR-34b, indicating that these miRNAs in LTEP-a-2 cells has the function as tumor suppressor genes.     

p38 MAPK involves in cepharanthine-induced apoptosis in cardiomyocytes

AIM: To investigate the apoptotic effect of cepharanthine (CEP) on neonatal rat cardiomyocytes(NRCMs) and the underlying mechanisms. METHODS: MTT assay was used to detect the viability of the cells. CEP-induced apoptosis in NRCMs was evaluated by Hoechst 33342 staining and the expression of activated caspase-3. The phosphorylation levels of mitogen-activated protein kinases (MAPKs),such as extracellular signal-regulated kinase (ERK), c-jun N-terminal kinase (JNK) and p38 MAPK,were examined by Western blotting. The specific inhibitors of ERK and p38 MAPK were applied for identifying the roles of the corresponding signal pathways in CEP-induced apoptosis of cardiomyocytes. RESULTS: CEP inhibited the viability of NRCMs in a dose-and time-dependent manners. Positive nuclear fragmentation and activated caspase-3 were found in CEP-treated NRCMs. The phosphorylation levels of ERK and p38 MAPK were significantly elevated in CEP-treated NRCMs, but the change of JNK was not obvious. SB203580, an inhibitor of p38 MAPK, significantly alleviated the apoptotic effect induced by CEP. However, PD98059, an inhibitor of ERK1/2, did not significantly reduce the apoptotic effect.CONCLUSION: p38 MAPK is involved in CEP-induced apoptosis in NRCMs.     

Cepharanthine promotes apoptosis of RL-952 cells by regulating eIF4E-related miR-215

AIM:To investigate the effect of cepharanthine (CEP) on the viability and apoptosis of human endometrial carcinoma RL-952 cells and its mechanisms. METHODS:RL-952 cells were treated with cepharanthine at different concentrations. The cell viability and apoptosis were measured by MTT assay and flow cytometry, respectively. The optimal drug concentration was also screened. The miR-215 expression induced by cepharanthine was detected by real-time PCR. The effects of cepharanthine on the protein le-vels of eukaryotic initiation factor 4E (eIF4E) and p-eIF4E were determined by Western blot. The downstream target genes were predicted by bioinformatic software. Finally, after miR-215 was transfected into RL-952 cells, the change of GFP expression was evaluated by flow cytometry and the protein levels of eIF4E and p-eIF4E were determined by Western blot. RESULTS:With the increasing concentrations of cepharanthine, the inhibitory rate of the viability in RL-952 cells was increased gradually and the rate of apoptosis was increased. The most effective concentration was 30 μmol/L. After treatment with cepharanthine at this concentration, the expression level of miR-215 was significantly higher than that in control group (P<0.01). The bioinformatic software prediction indicated that there were binding sites of miR-215 in the 3'UTR of eIF4E. The protein levels of eIF4E and p-eIF4E in the cepharanthine group were lower than those in control group (P<0.05). After the miR-215 and pcDNA-GFP-eIF4E-3'UTR were cotransfected into the RL-952 cells, the expression of GFP declined (P<0.05). After transfection with miR-215, the protein le-vels of eIF4E and p-eIF4E were decreased (P<0.05). CONCLUSION:Cepharanthine effectively inhibits the viability and promotes the apoptosis of RL-952 cells. The mechanism may be related to up-regulating the expression of eIF4E-related miR-215 and then suppressing the protein levels of eIF4E and its active form p-eIF4E.