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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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Related in: MedlinePlus

AKT is necessary for ATO-treated cell apoptosis.A. Detection of AKT phosphorylation, expression and caspase-9 activation. Western blot analysis of total AKT, p-AKT (Ser473) levels and caspase-9 cleavage in COC1 cells. Cells were treated with ATO (2 µM) at different time and lysed in NP40 buffer for detection. CF is referred to cleaved caspase-9. The same detection was employed in COC1/CP, A2780 and OVCAR-3. These cells were treated for 72 h, then lysed and assayed for individual protein levels by Western blot. β-Actin was used as a protein loading control. p-AKT and AKT levels were measured by densitometric analysis of the Western blots and compared to actin levels. Relative amount of p-AKT or AKT from untreated cells was considered as 1. B. Protein levels of the PI-3K signaling pathway in ATO-treated cells. Cells were exposed to drug for different periods of time. Cell lysates were prepared and assayed for individual protein levels by Western blot. Individual protein levels were measured by densitometric analysis of the Western blots and compared to actin levels. Relative amount of individual protein from untreated cells (0 h) was set as 1. C. Effects of ATO on protein levels of mTOR2, including p-mTOR (Ser 2448and 2481), mTOR, RICTOR, SIN1, in A2780 cells. Cells were exposed to drug as described in B, and then cell lysates were assayed by Western blot. Relative amount of protein levels were set as described in B. Cell lysates were assayed by western blot. Relative amount of protein levels were set as described in B. D. Protective effect of constitutive AKT on ATO-induced apoptosis in ovarian cells. OVCAR-3 and A2780 cells were transfected with constitutive AKT1 and were selected for 8 weeks by G-418 and treated with ATO for 72 h. Cells were lysed and assayed for individual protein levels by Western blot. Left, protein levels of p-AKT (Ser473) and AKT (AKT1) in both Ctrl or AKT vector transfected OVCAR-3 and A2780 cells were detected. Relative amount of protein levels were set as described in B. p-AKT and AKT levels of AKT1 transfected A2780 cells were considered as 1. Right, analysis of apoptosis in Ctrl or AKT1 vector transfected OVCAR-3 and A2780 cells. Cell apoptosis was quantitatively detected by a cell death ELISA kit 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<0.01. E. Detection of the role of LY294002 on apoptosis of cells. Cells were treated with ATO and/or 25 µM LY294002 for 72 h, and then cells were lysed and assayed for individual protein levels by Western blot. Left, protein levels of p-AKT (Ser473) and total AKT in COC1 cells were detected. Cell apoptosis was quantitatively detected by a cell death ELISA kit. Relative amount of protein levels were set as described in B. p-AKT and AKT levels from untreated cells were set as 1. Right, analysis of apoptotic cells as describe in Materials and Methods. All data are depicted graphically as the means ± standard errors of the means for at least three independent experiments. **, P<0.01.
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pone-0020586-g003: AKT is necessary for ATO-treated cell apoptosis.A. Detection of AKT phosphorylation, expression and caspase-9 activation. Western blot analysis of total AKT, p-AKT (Ser473) levels and caspase-9 cleavage in COC1 cells. Cells were treated with ATO (2 µM) at different time and lysed in NP40 buffer for detection. CF is referred to cleaved caspase-9. The same detection was employed in COC1/CP, A2780 and OVCAR-3. These cells were treated for 72 h, then lysed and assayed for individual protein levels by Western blot. β-Actin was used as a protein loading control. p-AKT and AKT levels were measured by densitometric analysis of the Western blots and compared to actin levels. Relative amount of p-AKT or AKT from untreated cells was considered as 1. B. Protein levels of the PI-3K signaling pathway in ATO-treated cells. Cells were exposed to drug for different periods of time. Cell lysates were prepared and assayed for individual protein levels by Western blot. Individual protein levels were measured by densitometric analysis of the Western blots and compared to actin levels. Relative amount of individual protein from untreated cells (0 h) was set as 1. C. Effects of ATO on protein levels of mTOR2, including p-mTOR (Ser 2448and 2481), mTOR, RICTOR, SIN1, in A2780 cells. Cells were exposed to drug as described in B, and then cell lysates were assayed by Western blot. Relative amount of protein levels were set as described in B. Cell lysates were assayed by western blot. Relative amount of protein levels were set as described in B. D. Protective effect of constitutive AKT on ATO-induced apoptosis in ovarian cells. OVCAR-3 and A2780 cells were transfected with constitutive AKT1 and were selected for 8 weeks by G-418 and treated with ATO for 72 h. Cells were lysed and assayed for individual protein levels by Western blot. Left, protein levels of p-AKT (Ser473) and AKT (AKT1) in both Ctrl or AKT vector transfected OVCAR-3 and A2780 cells were detected. Relative amount of protein levels were set as described in B. p-AKT and AKT levels of AKT1 transfected A2780 cells were considered as 1. Right, analysis of apoptosis in Ctrl or AKT1 vector transfected OVCAR-3 and A2780 cells. Cell apoptosis was quantitatively detected by a cell death ELISA kit 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<0.01. E. Detection of the role of LY294002 on apoptosis of cells. Cells were treated with ATO and/or 25 µM LY294002 for 72 h, and then cells were lysed and assayed for individual protein levels by Western blot. Left, protein levels of p-AKT (Ser473) and total AKT in COC1 cells were detected. Cell apoptosis was quantitatively detected by a cell death ELISA kit. Relative amount of protein levels were set as described in B. p-AKT and AKT levels from untreated cells were set as 1. Right, analysis of apoptotic cells as describe in Materials and Methods. All data are depicted graphically as the means ± standard errors of the means for at least three independent experiments. **, P<0.01.

Mentions: Recent studies have indicated that AKT modulates BIM activation either directly or indirectly [19], [33]. Moreover, ATO induces apoptosis in myeloma cells by decreasing AKT activity and expression in cells [34]. Based on the above observations, we speculate that AKT is probably involved in ATO-induced apoptosis by regulating BIM expression. Accordingly, we first measured whether AKT is involved in ATO-induced apoptosis in ovarian cancer cells. As shown in Figure 3A, ATO induced the downregulation of p-AKT at Ser473 in cisplatin-sensitive COC1 cells in a time-dependent manner, although the expression level of total AKT protein exhibited no change. Downregulation of p-AKT induced activation of caspase-9 in sensitive COC1 cells. Similarly, ATO induced the reduction of p-AKT and the cleavage of caspase-9 in other ovarian cancer cells, in spite of their differences in chemo-sensitivity.


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)

AKT is necessary for ATO-treated cell apoptosis.A. Detection of AKT phosphorylation, expression and caspase-9 activation. Western blot analysis of total AKT, p-AKT (Ser473) levels and caspase-9 cleavage in COC1 cells. Cells were treated with ATO (2 µM) at different time and lysed in NP40 buffer for detection. CF is referred to cleaved caspase-9. The same detection was employed in COC1/CP, A2780 and OVCAR-3. These cells were treated for 72 h, then lysed and assayed for individual protein levels by Western blot. β-Actin was used as a protein loading control. p-AKT and AKT levels were measured by densitometric analysis of the Western blots and compared to actin levels. Relative amount of p-AKT or AKT from untreated cells was considered as 1. B. Protein levels of the PI-3K signaling pathway in ATO-treated cells. Cells were exposed to drug for different periods of time. Cell lysates were prepared and assayed for individual protein levels by Western blot. Individual protein levels were measured by densitometric analysis of the Western blots and compared to actin levels. Relative amount of individual protein from untreated cells (0 h) was set as 1. C. Effects of ATO on protein levels of mTOR2, including p-mTOR (Ser 2448and 2481), mTOR, RICTOR, SIN1, in A2780 cells. Cells were exposed to drug as described in B, and then cell lysates were assayed by Western blot. Relative amount of protein levels were set as described in B. Cell lysates were assayed by western blot. Relative amount of protein levels were set as described in B. D. Protective effect of constitutive AKT on ATO-induced apoptosis in ovarian cells. OVCAR-3 and A2780 cells were transfected with constitutive AKT1 and were selected for 8 weeks by G-418 and treated with ATO for 72 h. Cells were lysed and assayed for individual protein levels by Western blot. Left, protein levels of p-AKT (Ser473) and AKT (AKT1) in both Ctrl or AKT vector transfected OVCAR-3 and A2780 cells were detected. Relative amount of protein levels were set as described in B. p-AKT and AKT levels of AKT1 transfected A2780 cells were considered as 1. Right, analysis of apoptosis in Ctrl or AKT1 vector transfected OVCAR-3 and A2780 cells. Cell apoptosis was quantitatively detected by a cell death ELISA kit 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<0.01. E. Detection of the role of LY294002 on apoptosis of cells. Cells were treated with ATO and/or 25 µM LY294002 for 72 h, and then cells were lysed and assayed for individual protein levels by Western blot. Left, protein levels of p-AKT (Ser473) and total AKT in COC1 cells were detected. Cell apoptosis was quantitatively detected by a cell death ELISA kit. Relative amount of protein levels were set as described in B. p-AKT and AKT levels from untreated cells were set as 1. Right, analysis of apoptotic cells as describe in Materials and Methods. All data are depicted graphically as the means ± standard errors of the means for at least three independent experiments. **, P<0.01.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3105099&req=5

pone-0020586-g003: AKT is necessary for ATO-treated cell apoptosis.A. Detection of AKT phosphorylation, expression and caspase-9 activation. Western blot analysis of total AKT, p-AKT (Ser473) levels and caspase-9 cleavage in COC1 cells. Cells were treated with ATO (2 µM) at different time and lysed in NP40 buffer for detection. CF is referred to cleaved caspase-9. The same detection was employed in COC1/CP, A2780 and OVCAR-3. These cells were treated for 72 h, then lysed and assayed for individual protein levels by Western blot. β-Actin was used as a protein loading control. p-AKT and AKT levels were measured by densitometric analysis of the Western blots and compared to actin levels. Relative amount of p-AKT or AKT from untreated cells was considered as 1. B. Protein levels of the PI-3K signaling pathway in ATO-treated cells. Cells were exposed to drug for different periods of time. Cell lysates were prepared and assayed for individual protein levels by Western blot. Individual protein levels were measured by densitometric analysis of the Western blots and compared to actin levels. Relative amount of individual protein from untreated cells (0 h) was set as 1. C. Effects of ATO on protein levels of mTOR2, including p-mTOR (Ser 2448and 2481), mTOR, RICTOR, SIN1, in A2780 cells. Cells were exposed to drug as described in B, and then cell lysates were assayed by Western blot. Relative amount of protein levels were set as described in B. Cell lysates were assayed by western blot. Relative amount of protein levels were set as described in B. D. Protective effect of constitutive AKT on ATO-induced apoptosis in ovarian cells. OVCAR-3 and A2780 cells were transfected with constitutive AKT1 and were selected for 8 weeks by G-418 and treated with ATO for 72 h. Cells were lysed and assayed for individual protein levels by Western blot. Left, protein levels of p-AKT (Ser473) and AKT (AKT1) in both Ctrl or AKT vector transfected OVCAR-3 and A2780 cells were detected. Relative amount of protein levels were set as described in B. p-AKT and AKT levels of AKT1 transfected A2780 cells were considered as 1. Right, analysis of apoptosis in Ctrl or AKT1 vector transfected OVCAR-3 and A2780 cells. Cell apoptosis was quantitatively detected by a cell death ELISA kit 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<0.01. E. Detection of the role of LY294002 on apoptosis of cells. Cells were treated with ATO and/or 25 µM LY294002 for 72 h, and then cells were lysed and assayed for individual protein levels by Western blot. Left, protein levels of p-AKT (Ser473) and total AKT in COC1 cells were detected. Cell apoptosis was quantitatively detected by a cell death ELISA kit. Relative amount of protein levels were set as described in B. p-AKT and AKT levels from untreated cells were set as 1. Right, analysis of apoptotic cells as describe in Materials and Methods. All data are depicted graphically as the means ± standard errors of the means for at least three independent experiments. **, P<0.01.
Mentions: Recent studies have indicated that AKT modulates BIM activation either directly or indirectly [19], [33]. Moreover, ATO induces apoptosis in myeloma cells by decreasing AKT activity and expression in cells [34]. Based on the above observations, we speculate that AKT is probably involved in ATO-induced apoptosis by regulating BIM expression. Accordingly, we first measured whether AKT is involved in ATO-induced apoptosis in ovarian cancer cells. As shown in Figure 3A, ATO induced the downregulation of p-AKT at Ser473 in cisplatin-sensitive COC1 cells in a time-dependent manner, although the expression level of total AKT protein exhibited no change. Downregulation of p-AKT induced activation of caspase-9 in sensitive COC1 cells. Similarly, ATO induced the reduction of p-AKT and the cleavage of caspase-9 in other ovarian cancer cells, in spite of their differences in chemo-sensitivity.

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