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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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BIM mediated-AKT dephosphorylation regulates apoptosis induced by ATO.A. Effect of AKT siRNA on the BIM expression. Cells were transfected with either control siRNA or AKT1 siRNA for 48 h and then were exposed to ATO for 72 h. Cells were lysed and assayed for individual protein levels by Western blot. CF is referred to cleaved PARP. β-Actin was used as a protein loading control. Up, protein levels of AKT, BIM, cleaved caspase-3, and PARP were detected by Western blot. Relative amount of individual protein level was set as described in Figure 3B. p-AKT and AKT levels of Ctrl siRNA transfected and treated A2780 cells were considered as 1. Down, analysis of apoptotic cells. as described in Material and Methods. All data are depicted graphically as the means ± standard errors of the means for at least three independent experiments. P>0.05 (not significant). B. Effect of FOXO3A siRNA on the BIM expression and cell apoptosis. Cells were transfected with either control siRNA or FOXO3A siRNA for 48 h and then were exposed to ATO for 72 h. Up, Following transfection for 48 h, Immunoblot for BIM and FOXO3A protein expression was performed on whole cell lysates. Relative amount of individual protein level was set as described in Figure 3B. FOXO3A and BIM levels of Ctrl siRNA transfected COC1 cells were considered as 1. Down, protein levels of BIM, cytosol cyt c and PARP were assayed by Western blot in FOXO3A siRNA OVCAR-3 cells. For cyt c detection, cells were treated following Figure 1E. As described in Figure 3B, relative amount of BIM and cyt c in ATO treated cells (72 h) were regarded as 1. C. Effect of AKT on BIM phosphorylation. Cells were metabolically labeled with [32P] orthophosphoric acid and treated with ATO (2 µM) for 48 h, and BIM was immunoprecipitated by using an agarose-conjugated BIM antibody, then detection of phosphorylation of BIM. Phosphorylation of BIM was determined by autoradiography (upper panel). Western blot analysis was performed to confirm and quantify BIM protein (lower panel). Left, the effect of LY294002 on BIM phosphorylation. Cells were treated with ATO and/or LY294002 (25 µM), then detection of BIM expression and phosphorylation. As described in Figure 3B, relative amount of 32p-BIM and BIM in ATO treated cells were regarded as 1. Right, the effect of AKT siRNA on BIM phosphorylation. Cells were transfected with either control siRNA or AKT1 siRNA for 48 h and then were exposed to ATO for 48 h. Detection of BIM expression and phosphorylation as described. Relative amount of 32p-BIM and BIM in AKT transfected and ATO treated cells were set as 1. D. Effect of BIM shRNA on AKT phosphorylation. Cells were transfected with either control shRNA or BIM shRNA for 48 h and then were exposed to ATO for 48 h. Western blot examined BIM expression, caspase-3 cleavage, protein levels of p-AKT (Ser473) and AKT in cells. Relative amount of p-AKT, AKT and BIM in Ctrl shRNA transfected and ATO treated cells were set as 1. All data are representative of three independent experiments.
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pone-0020586-g004: BIM mediated-AKT dephosphorylation regulates apoptosis induced by ATO.A. Effect of AKT siRNA on the BIM expression. Cells were transfected with either control siRNA or AKT1 siRNA for 48 h and then were exposed to ATO for 72 h. Cells were lysed and assayed for individual protein levels by Western blot. CF is referred to cleaved PARP. β-Actin was used as a protein loading control. Up, protein levels of AKT, BIM, cleaved caspase-3, and PARP were detected by Western blot. Relative amount of individual protein level was set as described in Figure 3B. p-AKT and AKT levels of Ctrl siRNA transfected and treated A2780 cells were considered as 1. Down, analysis of apoptotic cells. as described in Material and Methods. All data are depicted graphically as the means ± standard errors of the means for at least three independent experiments. P>0.05 (not significant). B. Effect of FOXO3A siRNA on the BIM expression and cell apoptosis. Cells were transfected with either control siRNA or FOXO3A siRNA for 48 h and then were exposed to ATO for 72 h. Up, Following transfection for 48 h, Immunoblot for BIM and FOXO3A protein expression was performed on whole cell lysates. Relative amount of individual protein level was set as described in Figure 3B. FOXO3A and BIM levels of Ctrl siRNA transfected COC1 cells were considered as 1. Down, protein levels of BIM, cytosol cyt c and PARP were assayed by Western blot in FOXO3A siRNA OVCAR-3 cells. For cyt c detection, cells were treated following Figure 1E. As described in Figure 3B, relative amount of BIM and cyt c in ATO treated cells (72 h) were regarded as 1. C. Effect of AKT on BIM phosphorylation. Cells were metabolically labeled with [32P] orthophosphoric acid and treated with ATO (2 µM) for 48 h, and BIM was immunoprecipitated by using an agarose-conjugated BIM antibody, then detection of phosphorylation of BIM. Phosphorylation of BIM was determined by autoradiography (upper panel). Western blot analysis was performed to confirm and quantify BIM protein (lower panel). Left, the effect of LY294002 on BIM phosphorylation. Cells were treated with ATO and/or LY294002 (25 µM), then detection of BIM expression and phosphorylation. As described in Figure 3B, relative amount of 32p-BIM and BIM in ATO treated cells were regarded as 1. Right, the effect of AKT siRNA on BIM phosphorylation. Cells were transfected with either control siRNA or AKT1 siRNA for 48 h and then were exposed to ATO for 48 h. Detection of BIM expression and phosphorylation as described. Relative amount of 32p-BIM and BIM in AKT transfected and ATO treated cells were set as 1. D. Effect of BIM shRNA on AKT phosphorylation. Cells were transfected with either control shRNA or BIM shRNA for 48 h and then were exposed to ATO for 48 h. Western blot examined BIM expression, caspase-3 cleavage, protein levels of p-AKT (Ser473) and AKT in cells. Relative amount of p-AKT, AKT and BIM in Ctrl shRNA transfected and ATO treated cells were set as 1. All data are representative of three independent experiments.

Mentions: AKT mediates BIM activation through two primary pathways: (1) AKT phosphorylates BIM directly and inhibits BIM activation [19], [33], or (2) AKT phosphorylates FOXO3A, leading to its cytoplasmic retention by 14-3-3 proteins. Thereby, AKT could not translocate into the nucleus to induce BIM transcription, meaning that AKT regulates BIM activation indirectly. On the basis of these observations, we decided to examine whether AKT affects BIM activation in ovarian cancer cells. We first downregulated the expression of AKT1 by siRNA in ATO-treated A2780 and OVCAR-3 cells and found that the downregulation of AKT increased caspase-3 and PARP cleavage during ATO treatment. However, AKT downregulation had little effect on BIM expression, though AKT downregulation increased, to some extent, ATO-induced nuclear fragmentation and followed cell apoptosis (Figure 4A). Similar results were also found in ATO-treated ovarian cancer cells when FOXO3A expression was downregulated using siRNAs targeting FOXO3A (Figure 4B). These results suggest that the AKT pathway may not be involved in regulating BIM expression during ATO-induced apoptosis.


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)

BIM mediated-AKT dephosphorylation regulates apoptosis induced by ATO.A. Effect of AKT siRNA on the BIM expression. Cells were transfected with either control siRNA or AKT1 siRNA for 48 h and then were exposed to ATO for 72 h. Cells were lysed and assayed for individual protein levels by Western blot. CF is referred to cleaved PARP. β-Actin was used as a protein loading control. Up, protein levels of AKT, BIM, cleaved caspase-3, and PARP were detected by Western blot. Relative amount of individual protein level was set as described in Figure 3B. p-AKT and AKT levels of Ctrl siRNA transfected and treated A2780 cells were considered as 1. Down, analysis of apoptotic cells. as described in Material and Methods. All data are depicted graphically as the means ± standard errors of the means for at least three independent experiments. P>0.05 (not significant). B. Effect of FOXO3A siRNA on the BIM expression and cell apoptosis. Cells were transfected with either control siRNA or FOXO3A siRNA for 48 h and then were exposed to ATO for 72 h. Up, Following transfection for 48 h, Immunoblot for BIM and FOXO3A protein expression was performed on whole cell lysates. Relative amount of individual protein level was set as described in Figure 3B. FOXO3A and BIM levels of Ctrl siRNA transfected COC1 cells were considered as 1. Down, protein levels of BIM, cytosol cyt c and PARP were assayed by Western blot in FOXO3A siRNA OVCAR-3 cells. For cyt c detection, cells were treated following Figure 1E. As described in Figure 3B, relative amount of BIM and cyt c in ATO treated cells (72 h) were regarded as 1. C. Effect of AKT on BIM phosphorylation. Cells were metabolically labeled with [32P] orthophosphoric acid and treated with ATO (2 µM) for 48 h, and BIM was immunoprecipitated by using an agarose-conjugated BIM antibody, then detection of phosphorylation of BIM. Phosphorylation of BIM was determined by autoradiography (upper panel). Western blot analysis was performed to confirm and quantify BIM protein (lower panel). Left, the effect of LY294002 on BIM phosphorylation. Cells were treated with ATO and/or LY294002 (25 µM), then detection of BIM expression and phosphorylation. As described in Figure 3B, relative amount of 32p-BIM and BIM in ATO treated cells were regarded as 1. Right, the effect of AKT siRNA on BIM phosphorylation. Cells were transfected with either control siRNA or AKT1 siRNA for 48 h and then were exposed to ATO for 48 h. Detection of BIM expression and phosphorylation as described. Relative amount of 32p-BIM and BIM in AKT transfected and ATO treated cells were set as 1. D. Effect of BIM shRNA on AKT phosphorylation. Cells were transfected with either control shRNA or BIM shRNA for 48 h and then were exposed to ATO for 48 h. Western blot examined BIM expression, caspase-3 cleavage, protein levels of p-AKT (Ser473) and AKT in cells. Relative amount of p-AKT, AKT and BIM in Ctrl shRNA transfected and ATO treated cells were set as 1. All data are representative of three independent experiments.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3105099&req=5

pone-0020586-g004: BIM mediated-AKT dephosphorylation regulates apoptosis induced by ATO.A. Effect of AKT siRNA on the BIM expression. Cells were transfected with either control siRNA or AKT1 siRNA for 48 h and then were exposed to ATO for 72 h. Cells were lysed and assayed for individual protein levels by Western blot. CF is referred to cleaved PARP. β-Actin was used as a protein loading control. Up, protein levels of AKT, BIM, cleaved caspase-3, and PARP were detected by Western blot. Relative amount of individual protein level was set as described in Figure 3B. p-AKT and AKT levels of Ctrl siRNA transfected and treated A2780 cells were considered as 1. Down, analysis of apoptotic cells. as described in Material and Methods. All data are depicted graphically as the means ± standard errors of the means for at least three independent experiments. P>0.05 (not significant). B. Effect of FOXO3A siRNA on the BIM expression and cell apoptosis. Cells were transfected with either control siRNA or FOXO3A siRNA for 48 h and then were exposed to ATO for 72 h. Up, Following transfection for 48 h, Immunoblot for BIM and FOXO3A protein expression was performed on whole cell lysates. Relative amount of individual protein level was set as described in Figure 3B. FOXO3A and BIM levels of Ctrl siRNA transfected COC1 cells were considered as 1. Down, protein levels of BIM, cytosol cyt c and PARP were assayed by Western blot in FOXO3A siRNA OVCAR-3 cells. For cyt c detection, cells were treated following Figure 1E. As described in Figure 3B, relative amount of BIM and cyt c in ATO treated cells (72 h) were regarded as 1. C. Effect of AKT on BIM phosphorylation. Cells were metabolically labeled with [32P] orthophosphoric acid and treated with ATO (2 µM) for 48 h, and BIM was immunoprecipitated by using an agarose-conjugated BIM antibody, then detection of phosphorylation of BIM. Phosphorylation of BIM was determined by autoradiography (upper panel). Western blot analysis was performed to confirm and quantify BIM protein (lower panel). Left, the effect of LY294002 on BIM phosphorylation. Cells were treated with ATO and/or LY294002 (25 µM), then detection of BIM expression and phosphorylation. As described in Figure 3B, relative amount of 32p-BIM and BIM in ATO treated cells were regarded as 1. Right, the effect of AKT siRNA on BIM phosphorylation. Cells were transfected with either control siRNA or AKT1 siRNA for 48 h and then were exposed to ATO for 48 h. Detection of BIM expression and phosphorylation as described. Relative amount of 32p-BIM and BIM in AKT transfected and ATO treated cells were set as 1. D. Effect of BIM shRNA on AKT phosphorylation. Cells were transfected with either control shRNA or BIM shRNA for 48 h and then were exposed to ATO for 48 h. Western blot examined BIM expression, caspase-3 cleavage, protein levels of p-AKT (Ser473) and AKT in cells. Relative amount of p-AKT, AKT and BIM in Ctrl shRNA transfected and ATO treated cells were set as 1. All data are representative of three independent experiments.
Mentions: AKT mediates BIM activation through two primary pathways: (1) AKT phosphorylates BIM directly and inhibits BIM activation [19], [33], or (2) AKT phosphorylates FOXO3A, leading to its cytoplasmic retention by 14-3-3 proteins. Thereby, AKT could not translocate into the nucleus to induce BIM transcription, meaning that AKT regulates BIM activation indirectly. On the basis of these observations, we decided to examine whether AKT affects BIM activation in ovarian cancer cells. We first downregulated the expression of AKT1 by siRNA in ATO-treated A2780 and OVCAR-3 cells and found that the downregulation of AKT increased caspase-3 and PARP cleavage during ATO treatment. However, AKT downregulation had little effect on BIM expression, though AKT downregulation increased, to some extent, ATO-induced nuclear fragmentation and followed cell apoptosis (Figure 4A). Similar results were also found in ATO-treated ovarian cancer cells when FOXO3A expression was downregulated using siRNAs targeting FOXO3A (Figure 4B). These results suggest that the AKT pathway may not be involved in regulating BIM expression during ATO-induced apoptosis.

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