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BIM-mediated AKT phosphorylation is a key modulator of arsenic trioxide-induced apoptosis in cisplatin-sensitive and -resistant ovarian cancer cells.

Yuan Z, Wang F, Zhao Z, Zhao X, Qiu J, Nie C, Wei Y - PLoS ONE (2011)

Bottom Line: However, surprisingly, gene silencing of AKT or FOXO3A had little effect on BIM expression and phosphorylation.Moreover, the activation of caspase-3 by ATO treatment improved AKT dephosphorylation, not only by cleaving the regulatory A subunit of protein phosphatase 2A (PP2A), but also by increasing its activation.Furthermore, our data indicated that the c-Jun N-terminal kinases (JNK) pathway is involved in the regulation of BIM expression.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University, Chengdu, China.

ABSTRACT

Background: Chemo-resistance to cisplatin-centered cancer therapy is a major obstacle to the effective treatment of human ovarian cancer. Previous reports indicated that arsenic trioxide (ATO) induces cell apoptosis in both drug-sensitive and -resistant ovarian cancer cells.

Principal findings: In this study, we determined the molecular mechanism of ATO-induced apoptosis in ovarian cancer cells. Our data demonstrated that ATO induced cell apoptosis by decreasing levels of phosphorylated AKT (p-AKT) and activating caspase-3 and caspase-9. Importantly, BIM played a critical role in ATO-induced apoptosis. The inhibition of BIM expression prevented AKT dephosphorylation and inhibited caspase-3 activation during cell apoptosis. However, surprisingly, gene silencing of AKT or FOXO3A had little effect on BIM expression and phosphorylation. Moreover, the activation of caspase-3 by ATO treatment improved AKT dephosphorylation, not only by cleaving the regulatory A subunit of protein phosphatase 2A (PP2A), but also by increasing its activation. Furthermore, our data indicated that the c-Jun N-terminal kinases (JNK) pathway is involved in the regulation of BIM expression.

Conclusions: We demonstrated the roles of BIM in ATO-induced apoptosis and the molecular mechanisms of BIM expression regulated by ATO during ovarian cancer cell apoptosis. Our findings suggest that BIM plays an important role in regulating p-AKT by activating caspase-3 and that BIM mediates the level of AKT phosphorylation to determine the threshold for overcoming cisplatin resistance in ovarian cancer cells.

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ATO induces apoptosis in cisplatin-sensitive and -resistant human ovarian cells.A. Dose-dependent effects of cisplatin in ovarian cell lines. Cells were exposed to cisplatin at the indicated concentrations in DMEM or RPMI-1640 with 10% FBS in 96 well plate for 72 h and cell viability was assessed by MTT assay. Graphs showing results of MTT assay. All data are depicted graphically as the means ± standard errors of the means for at least three independent experiments. B. Analysis of cell apoptosis. Cells were treated with cisplatin (5 µM) for different periods of time. Cell apoptosis was quantitatively detected by a cell death ELISA kit. Graphs showing results of quantitative analyses. All data are depicted graphically as the means ± standard errors of the means for at least three independent experiments. *, P<0.05, **, P<0.01. C. Effect of ATO on apoptotic death in cisplatin-sensitive and -resistant cells. Cells were cultured in the presence or absence of ATO (2 µM) for different periods of time, then cell apoptosis was measured as described in Materials and Methods. All data are depicted graphically as the means ± standard errors of the means for at least three independent experiments. *, P<0.05, **, P<0.01. D. Detection of cell apoptosis with flow cytometry analysis. COC1 and COC1/CP cells were treated with ATO (2 µM) for 48 h, then stained with Annexin V and examined by flow cytometry. E and F. Analyses of cyt c release. After treatment with ATO for different periods of time, cells were subjected to subcellular fractionation. The cytosolic or mitochondrial fractions were immunoblotted (30 µg of protein/lane) with antibody specific for cyt c. β-Actin and Cox IV were used as a protein loading control. Densitometric analysis of the Western blots was performed and the amount of cyt c was compared to the protein loading control. For cyto cyt c, relative amount of cyt c from treated cells (72 h) was set as 1, while relative amount from untreated cells (0 h) was set at 1 for mito cyt c. Data are representative of at least three independent experiments.
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pone-0020586-g001: ATO induces apoptosis in cisplatin-sensitive and -resistant human ovarian cells.A. Dose-dependent effects of cisplatin in ovarian cell lines. Cells were exposed to cisplatin at the indicated concentrations in DMEM or RPMI-1640 with 10% FBS in 96 well plate for 72 h and cell viability was assessed by MTT assay. Graphs showing results of MTT assay. All data are depicted graphically as the means ± standard errors of the means for at least three independent experiments. B. Analysis of cell apoptosis. Cells were treated with cisplatin (5 µM) for different periods of time. Cell apoptosis was quantitatively detected by a cell death ELISA kit. Graphs showing results of quantitative analyses. All data are depicted graphically as the means ± standard errors of the means for at least three independent experiments. *, P<0.05, **, P<0.01. C. Effect of ATO on apoptotic death in cisplatin-sensitive and -resistant cells. Cells were cultured in the presence or absence of ATO (2 µM) for different periods of time, then cell apoptosis was measured as described in Materials and Methods. All data are depicted graphically as the means ± standard errors of the means for at least three independent experiments. *, P<0.05, **, P<0.01. D. Detection of cell apoptosis with flow cytometry analysis. COC1 and COC1/CP cells were treated with ATO (2 µM) for 48 h, then stained with Annexin V and examined by flow cytometry. E and F. Analyses of cyt c release. After treatment with ATO for different periods of time, cells were subjected to subcellular fractionation. The cytosolic or mitochondrial fractions were immunoblotted (30 µg of protein/lane) with antibody specific for cyt c. β-Actin and Cox IV were used as a protein loading control. Densitometric analysis of the Western blots was performed and the amount of cyt c was compared to the protein loading control. For cyto cyt c, relative amount of cyt c from treated cells (72 h) was set as 1, while relative amount from untreated cells (0 h) was set at 1 for mito cyt c. Data are representative of at least three independent experiments.

Mentions: We first determined the growth-inhibitory effect of cisplatin in various cisplatin-sensitive (COC1 and A2780) and -resistant (COC1/CP, A2780/CP and OVCAR-3) human ovarian cell lines, as described previously [31]. Cell viability was detected by the MTT assay after 72 hours of treatment. As depicted in Figure 1A, cisplatin caused a dose-dependent reduction of cell viability in COC1 and A2780 cells, while COC1/CP, A2780/CP and OVCAR-3 cells demonstrably exhibited resistance to cisplatin. To examine cisplatin-induced apoptosis, the cells were treated with 5 µM cisplatin. Next, apoptosis was confirmed by a DNA fragmentation ELISA assay at various time points. These results demonstrated that cisplatin effectively induced apoptosis in cisplatin-sensitive ovarian cells, but not in cisplatin-resistant cells (Figure 1B). To investigate the effects of ATO on apoptosis in all ovarian cancer cells, we treated ovarian cancer cells with ATO and analyzed the cell apoptosis process by a DNA fragmentation ELISA assay. Our data suggested that ATO induced cell apoptosis in all ovarian cancer cells, regardless of their differences in chemo-sensitivity (Figure 1C). Flow-cytometry analysis with Annexin V staining further revealed that there was no difference in ATO-induced apoptosis between cisplatin-sensitive and -resistant cells (Figure 1D).


BIM-mediated AKT phosphorylation is a key modulator of arsenic trioxide-induced apoptosis in cisplatin-sensitive and -resistant ovarian cancer cells.

Yuan Z, Wang F, Zhao Z, Zhao X, Qiu J, Nie C, Wei Y - PLoS ONE (2011)

ATO induces apoptosis in cisplatin-sensitive and -resistant human ovarian cells.A. Dose-dependent effects of cisplatin in ovarian cell lines. Cells were exposed to cisplatin at the indicated concentrations in DMEM or RPMI-1640 with 10% FBS in 96 well plate for 72 h and cell viability was assessed by MTT assay. Graphs showing results of MTT assay. All data are depicted graphically as the means ± standard errors of the means for at least three independent experiments. B. Analysis of cell apoptosis. Cells were treated with cisplatin (5 µM) for different periods of time. Cell apoptosis was quantitatively detected by a cell death ELISA kit. Graphs showing results of quantitative analyses. All data are depicted graphically as the means ± standard errors of the means for at least three independent experiments. *, P<0.05, **, P<0.01. C. Effect of ATO on apoptotic death in cisplatin-sensitive and -resistant cells. Cells were cultured in the presence or absence of ATO (2 µM) for different periods of time, then cell apoptosis was measured as described in Materials and Methods. All data are depicted graphically as the means ± standard errors of the means for at least three independent experiments. *, P<0.05, **, P<0.01. D. Detection of cell apoptosis with flow cytometry analysis. COC1 and COC1/CP cells were treated with ATO (2 µM) for 48 h, then stained with Annexin V and examined by flow cytometry. E and F. Analyses of cyt c release. After treatment with ATO for different periods of time, cells were subjected to subcellular fractionation. The cytosolic or mitochondrial fractions were immunoblotted (30 µg of protein/lane) with antibody specific for cyt c. β-Actin and Cox IV were used as a protein loading control. Densitometric analysis of the Western blots was performed and the amount of cyt c was compared to the protein loading control. For cyto cyt c, relative amount of cyt c from treated cells (72 h) was set as 1, while relative amount from untreated cells (0 h) was set at 1 for mito cyt c. Data are representative of at least three independent experiments.
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pone-0020586-g001: ATO induces apoptosis in cisplatin-sensitive and -resistant human ovarian cells.A. Dose-dependent effects of cisplatin in ovarian cell lines. Cells were exposed to cisplatin at the indicated concentrations in DMEM or RPMI-1640 with 10% FBS in 96 well plate for 72 h and cell viability was assessed by MTT assay. Graphs showing results of MTT assay. All data are depicted graphically as the means ± standard errors of the means for at least three independent experiments. B. Analysis of cell apoptosis. Cells were treated with cisplatin (5 µM) for different periods of time. Cell apoptosis was quantitatively detected by a cell death ELISA kit. Graphs showing results of quantitative analyses. All data are depicted graphically as the means ± standard errors of the means for at least three independent experiments. *, P<0.05, **, P<0.01. C. Effect of ATO on apoptotic death in cisplatin-sensitive and -resistant cells. Cells were cultured in the presence or absence of ATO (2 µM) for different periods of time, then cell apoptosis was measured as described in Materials and Methods. All data are depicted graphically as the means ± standard errors of the means for at least three independent experiments. *, P<0.05, **, P<0.01. D. Detection of cell apoptosis with flow cytometry analysis. COC1 and COC1/CP cells were treated with ATO (2 µM) for 48 h, then stained with Annexin V and examined by flow cytometry. E and F. Analyses of cyt c release. After treatment with ATO for different periods of time, cells were subjected to subcellular fractionation. The cytosolic or mitochondrial fractions were immunoblotted (30 µg of protein/lane) with antibody specific for cyt c. β-Actin and Cox IV were used as a protein loading control. Densitometric analysis of the Western blots was performed and the amount of cyt c was compared to the protein loading control. For cyto cyt c, relative amount of cyt c from treated cells (72 h) was set as 1, while relative amount from untreated cells (0 h) was set at 1 for mito cyt c. Data are representative of at least three independent experiments.
Mentions: We first determined the growth-inhibitory effect of cisplatin in various cisplatin-sensitive (COC1 and A2780) and -resistant (COC1/CP, A2780/CP and OVCAR-3) human ovarian cell lines, as described previously [31]. Cell viability was detected by the MTT assay after 72 hours of treatment. As depicted in Figure 1A, cisplatin caused a dose-dependent reduction of cell viability in COC1 and A2780 cells, while COC1/CP, A2780/CP and OVCAR-3 cells demonstrably exhibited resistance to cisplatin. To examine cisplatin-induced apoptosis, the cells were treated with 5 µM cisplatin. Next, apoptosis was confirmed by a DNA fragmentation ELISA assay at various time points. These results demonstrated that cisplatin effectively induced apoptosis in cisplatin-sensitive ovarian cells, but not in cisplatin-resistant cells (Figure 1B). To investigate the effects of ATO on apoptosis in all ovarian cancer cells, we treated ovarian cancer cells with ATO and analyzed the cell apoptosis process by a DNA fragmentation ELISA assay. Our data suggested that ATO induced cell apoptosis in all ovarian cancer cells, regardless of their differences in chemo-sensitivity (Figure 1C). Flow-cytometry analysis with Annexin V staining further revealed that there was no difference in ATO-induced apoptosis between cisplatin-sensitive and -resistant cells (Figure 1D).

Bottom Line: However, surprisingly, gene silencing of AKT or FOXO3A had little effect on BIM expression and phosphorylation.Moreover, the activation of caspase-3 by ATO treatment improved AKT dephosphorylation, not only by cleaving the regulatory A subunit of protein phosphatase 2A (PP2A), but also by increasing its activation.Furthermore, our data indicated that the c-Jun N-terminal kinases (JNK) pathway is involved in the regulation of BIM expression.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University, Chengdu, China.

ABSTRACT

Background: Chemo-resistance to cisplatin-centered cancer therapy is a major obstacle to the effective treatment of human ovarian cancer. Previous reports indicated that arsenic trioxide (ATO) induces cell apoptosis in both drug-sensitive and -resistant ovarian cancer cells.

Principal findings: In this study, we determined the molecular mechanism of ATO-induced apoptosis in ovarian cancer cells. Our data demonstrated that ATO induced cell apoptosis by decreasing levels of phosphorylated AKT (p-AKT) and activating caspase-3 and caspase-9. Importantly, BIM played a critical role in ATO-induced apoptosis. The inhibition of BIM expression prevented AKT dephosphorylation and inhibited caspase-3 activation during cell apoptosis. However, surprisingly, gene silencing of AKT or FOXO3A had little effect on BIM expression and phosphorylation. Moreover, the activation of caspase-3 by ATO treatment improved AKT dephosphorylation, not only by cleaving the regulatory A subunit of protein phosphatase 2A (PP2A), but also by increasing its activation. Furthermore, our data indicated that the c-Jun N-terminal kinases (JNK) pathway is involved in the regulation of BIM expression.

Conclusions: We demonstrated the roles of BIM in ATO-induced apoptosis and the molecular mechanisms of BIM expression regulated by ATO during ovarian cancer cell apoptosis. Our findings suggest that BIM plays an important role in regulating p-AKT by activating caspase-3 and that BIM mediates the level of AKT phosphorylation to determine the threshold for overcoming cisplatin resistance in ovarian cancer cells.

Show MeSH
Related in: MedlinePlus