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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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Caspase-3 mediates PP2A-related AKT dephosphorylation.A. Different cells were pre-incubated with indicated concentrations of OA for 1 h and exposed to ATO for 48 h, then lysed in NP40 buffer for detection. Western blot analysis of total AKT and p-AKT (Ser473) levels was shown. β-Actin was used as a protein loading control. Relative amount of p-AKT and AKT in untreated cells were set as 1. B. Analysis of the effect of caspase-3 on PP2A activation. Up, Cells were pre-incubated with indicated concentrations of DEVD-CHO for 1 h and exposed to ATO for 48 h, then lysed in NP40 buffer for detection. Western blot analysis of total AKT and p-AKT (Ser473) levels was shown. Relative amount of p-AKT and AKT in untreated cells were set as 1. Middle, the treated cells were lysed with sample buffer and subjected to immunoblot assay with an A subunit-specific anti-PP2A/A antibody. CF is referred to cleaved PP2A/A. The membrane was then stripped and reprobed with a C subunit-specific anti-PP2A/C antibody. Relative amount of PP2A/C in untreated cells (0 h) were set as 1. Down, Western blot analysis of the effect of DEVD-CHO (100 µM) in PP2A cleavage. C. Detection of PP2A activity. Cells were pre-incubated with or without 0.2 µM of OA or 100 µM of DEVD-CHO for 1 h and exposed to drug for 48 h. PP2A activity with the cellular proteins extracted from RIPA buffer-lysed cells was measured using phosphopeptide KIpTIRR as a substrate as described in Materials and Methods. Each column represents the mean ± S.D. of triplicate assays. *, P<0.05. D. Detection of the binding of AKT with PP2A. Cells were incubated with or without ATO for 48 h. In some groups of cells, 0.2 µM of OA or 100 µM of DEVD-CHO was added 1 h prior to the addition of drug. The cells were then lysed with RIPA buffer for immunoprecipitation with anti-AKT antibody followed by immunoblot assay with anti-PP2A/C and anti-AKT antibodies. Relative amount of individual protein in untreated cells were set as 1. Data are representative of at least three independent experiments.
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pone-0020586-g005: Caspase-3 mediates PP2A-related AKT dephosphorylation.A. Different cells were pre-incubated with indicated concentrations of OA for 1 h and exposed to ATO for 48 h, then lysed in NP40 buffer for detection. Western blot analysis of total AKT and p-AKT (Ser473) levels was shown. β-Actin was used as a protein loading control. Relative amount of p-AKT and AKT in untreated cells were set as 1. B. Analysis of the effect of caspase-3 on PP2A activation. Up, Cells were pre-incubated with indicated concentrations of DEVD-CHO for 1 h and exposed to ATO for 48 h, then lysed in NP40 buffer for detection. Western blot analysis of total AKT and p-AKT (Ser473) levels was shown. Relative amount of p-AKT and AKT in untreated cells were set as 1. Middle, the treated cells were lysed with sample buffer and subjected to immunoblot assay with an A subunit-specific anti-PP2A/A antibody. CF is referred to cleaved PP2A/A. The membrane was then stripped and reprobed with a C subunit-specific anti-PP2A/C antibody. Relative amount of PP2A/C in untreated cells (0 h) were set as 1. Down, Western blot analysis of the effect of DEVD-CHO (100 µM) in PP2A cleavage. C. Detection of PP2A activity. Cells were pre-incubated with or without 0.2 µM of OA or 100 µM of DEVD-CHO for 1 h and exposed to drug for 48 h. PP2A activity with the cellular proteins extracted from RIPA buffer-lysed cells was measured using phosphopeptide KIpTIRR as a substrate as described in Materials and Methods. Each column represents the mean ± S.D. of triplicate assays. *, P<0.05. D. Detection of the binding of AKT with PP2A. Cells were incubated with or without ATO for 48 h. In some groups of cells, 0.2 µM of OA or 100 µM of DEVD-CHO was added 1 h prior to the addition of drug. The cells were then lysed with RIPA buffer for immunoprecipitation with anti-AKT antibody followed by immunoblot assay with anti-PP2A/C and anti-AKT antibodies. Relative amount of individual protein in untreated cells were set as 1. Data are representative of at least three independent experiments.

Mentions: To validate our hypothesis, we first determined whether PP2A mediated AKT phosphorylation in our study. Pretreatment of the cells with OA, an inhibitor of PP2A, resulted in a gradual reversal of ATO-induced AKT dephosphorylation in a dose-dependent manner in COC1/CP cells (Figure 5A). At the same time, OA also rescued AKT phosphorylation in A2780 and OVCAR-3 cells during ATO treatment.


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)

Caspase-3 mediates PP2A-related AKT dephosphorylation.A. Different cells were pre-incubated with indicated concentrations of OA for 1 h and exposed to ATO for 48 h, then lysed in NP40 buffer for detection. Western blot analysis of total AKT and p-AKT (Ser473) levels was shown. β-Actin was used as a protein loading control. Relative amount of p-AKT and AKT in untreated cells were set as 1. B. Analysis of the effect of caspase-3 on PP2A activation. Up, Cells were pre-incubated with indicated concentrations of DEVD-CHO for 1 h and exposed to ATO for 48 h, then lysed in NP40 buffer for detection. Western blot analysis of total AKT and p-AKT (Ser473) levels was shown. Relative amount of p-AKT and AKT in untreated cells were set as 1. Middle, the treated cells were lysed with sample buffer and subjected to immunoblot assay with an A subunit-specific anti-PP2A/A antibody. CF is referred to cleaved PP2A/A. The membrane was then stripped and reprobed with a C subunit-specific anti-PP2A/C antibody. Relative amount of PP2A/C in untreated cells (0 h) were set as 1. Down, Western blot analysis of the effect of DEVD-CHO (100 µM) in PP2A cleavage. C. Detection of PP2A activity. Cells were pre-incubated with or without 0.2 µM of OA or 100 µM of DEVD-CHO for 1 h and exposed to drug for 48 h. PP2A activity with the cellular proteins extracted from RIPA buffer-lysed cells was measured using phosphopeptide KIpTIRR as a substrate as described in Materials and Methods. Each column represents the mean ± S.D. of triplicate assays. *, P<0.05. D. Detection of the binding of AKT with PP2A. Cells were incubated with or without ATO for 48 h. In some groups of cells, 0.2 µM of OA or 100 µM of DEVD-CHO was added 1 h prior to the addition of drug. The cells were then lysed with RIPA buffer for immunoprecipitation with anti-AKT antibody followed by immunoblot assay with anti-PP2A/C and anti-AKT antibodies. Relative amount of individual protein in untreated cells were set as 1. Data are representative of at least three independent experiments.
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Related In: Results  -  Collection

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pone-0020586-g005: Caspase-3 mediates PP2A-related AKT dephosphorylation.A. Different cells were pre-incubated with indicated concentrations of OA for 1 h and exposed to ATO for 48 h, then lysed in NP40 buffer for detection. Western blot analysis of total AKT and p-AKT (Ser473) levels was shown. β-Actin was used as a protein loading control. Relative amount of p-AKT and AKT in untreated cells were set as 1. B. Analysis of the effect of caspase-3 on PP2A activation. Up, Cells were pre-incubated with indicated concentrations of DEVD-CHO for 1 h and exposed to ATO for 48 h, then lysed in NP40 buffer for detection. Western blot analysis of total AKT and p-AKT (Ser473) levels was shown. Relative amount of p-AKT and AKT in untreated cells were set as 1. Middle, the treated cells were lysed with sample buffer and subjected to immunoblot assay with an A subunit-specific anti-PP2A/A antibody. CF is referred to cleaved PP2A/A. The membrane was then stripped and reprobed with a C subunit-specific anti-PP2A/C antibody. Relative amount of PP2A/C in untreated cells (0 h) were set as 1. Down, Western blot analysis of the effect of DEVD-CHO (100 µM) in PP2A cleavage. C. Detection of PP2A activity. Cells were pre-incubated with or without 0.2 µM of OA or 100 µM of DEVD-CHO for 1 h and exposed to drug for 48 h. PP2A activity with the cellular proteins extracted from RIPA buffer-lysed cells was measured using phosphopeptide KIpTIRR as a substrate as described in Materials and Methods. Each column represents the mean ± S.D. of triplicate assays. *, P<0.05. D. Detection of the binding of AKT with PP2A. Cells were incubated with or without ATO for 48 h. In some groups of cells, 0.2 µM of OA or 100 µM of DEVD-CHO was added 1 h prior to the addition of drug. The cells were then lysed with RIPA buffer for immunoprecipitation with anti-AKT antibody followed by immunoblot assay with anti-PP2A/C and anti-AKT antibodies. Relative amount of individual protein in untreated cells were set as 1. Data are representative of at least three independent experiments.
Mentions: To validate our hypothesis, we first determined whether PP2A mediated AKT phosphorylation in our study. Pretreatment of the cells with OA, an inhibitor of PP2A, resulted in a gradual reversal of ATO-induced AKT dephosphorylation in a dose-dependent manner in COC1/CP cells (Figure 5A). At the same time, OA also rescued AKT phosphorylation in A2780 and OVCAR-3 cells during ATO treatment.

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