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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 regulated BIM expression is important for cell apoptosis in ovarian cancer cells.A and B. Western blots analysis of the expression level of BCL-2 family protein. Time-dependent analysis of BCL-2 member's expression levels in COC1/CP and COC1 cells. Cells were treated with ATO (2 µM) at indicated time, then lysed in NP40 buffer for detection. β-Actin was used as a protein loading control. Individual protein levels were measured by densitometric analysis of the Western blots and compared to actin levels. Relative amount of individual protein from treated cells (72 h) was set as 1. C. Effect of BIM shRNA on cell apoptosis. In the same conditions with A, cells were transfected with BIM or Ctrl shRNA for 48 h, and then treated with or without ATO for 48 h. Cell lysates were prepared and assayed for BIM, caspase-9 and cleaved caspase-3 by western blot. CF is referred to cleaved caspase-9. Relative fold means amount of BIM compared to actin and Ctrl condition was regarded as 1. D. Detection the effect of BIM shRNA on cell death with nuclear staining. Cells were transfected with BIM or Ctrl shRNA for 48 h, and then transfected cells were treated with ATO for 48 h. Cell death was assessed by measuring cells with condensed and fragmented nuclear by microscopy. Arrows indicate the condensed, fragmented, brightly stained nuclei, which are the hallmark of apoptosis. All data are representative of three independent experiments.
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pone-0020586-g002: ATO regulated BIM expression is important for cell apoptosis in ovarian cancer cells.A and B. Western blots analysis of the expression level of BCL-2 family protein. Time-dependent analysis of BCL-2 member's expression levels in COC1/CP and COC1 cells. Cells were treated with ATO (2 µM) at indicated time, then lysed in NP40 buffer for detection. β-Actin was used as a protein loading control. Individual protein levels were measured by densitometric analysis of the Western blots and compared to actin levels. Relative amount of individual protein from treated cells (72 h) was set as 1. C. Effect of BIM shRNA on cell apoptosis. In the same conditions with A, cells were transfected with BIM or Ctrl shRNA for 48 h, and then treated with or without ATO for 48 h. Cell lysates were prepared and assayed for BIM, caspase-9 and cleaved caspase-3 by western blot. CF is referred to cleaved caspase-9. Relative fold means amount of BIM compared to actin and Ctrl condition was regarded as 1. D. Detection the effect of BIM shRNA on cell death with nuclear staining. Cells were transfected with BIM or Ctrl shRNA for 48 h, and then transfected cells were treated with ATO for 48 h. Cell death was assessed by measuring cells with condensed and fragmented nuclear by microscopy. Arrows indicate the condensed, fragmented, brightly stained nuclei, which are the hallmark of apoptosis. All data are representative of three independent experiments.

Mentions: Mitochondrial dysfunction plays an important role in apoptosis in ovarian cells. Previous studies reported that changes in the gene expression of BCL-2-family proteins was involved in ATO-induced apoptosis [32] and that the BH3-only proteins were necessary for ATO-induced apoptosis in myeloma cells. However, it is unclear whether BH3 proteins might function in ovarian cancer cells following ATO treatment. Therefore, we investigated the expression of BCL-2-family proteins in cisplatin-sensitive and -resistant cells after ATO treatment. As depicted in Figure 2A, pro-apoptotic proteins, such as BAX, PUMA and NOXA, were not significantly changed in COC1 cells at various time points after ATO treatment. Anti-apoptotic BCL-2 and BCL-XL proteins also exhibited no major alterations. However, in contrast to other proteins, the expression level of BIM was markedly increased after ATO treatment, providing evidence that BIM was involved in apoptotic cell death in ovarian cancer cells. Meanwhile, ATO induced BIM expression equally in COC1/CP cells (Figure 2B). Similar results were also observed in other cell lines (data not shown). These results suggest that BIM plays an important role in ATO-induced apoptosis in ovarian cancer cells.


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 regulated BIM expression is important for cell apoptosis in ovarian cancer cells.A and B. Western blots analysis of the expression level of BCL-2 family protein. Time-dependent analysis of BCL-2 member's expression levels in COC1/CP and COC1 cells. Cells were treated with ATO (2 µM) at indicated time, then lysed in NP40 buffer for detection. β-Actin was used as a protein loading control. Individual protein levels were measured by densitometric analysis of the Western blots and compared to actin levels. Relative amount of individual protein from treated cells (72 h) was set as 1. C. Effect of BIM shRNA on cell apoptosis. In the same conditions with A, cells were transfected with BIM or Ctrl shRNA for 48 h, and then treated with or without ATO for 48 h. Cell lysates were prepared and assayed for BIM, caspase-9 and cleaved caspase-3 by western blot. CF is referred to cleaved caspase-9. Relative fold means amount of BIM compared to actin and Ctrl condition was regarded as 1. D. Detection the effect of BIM shRNA on cell death with nuclear staining. Cells were transfected with BIM or Ctrl shRNA for 48 h, and then transfected cells were treated with ATO for 48 h. Cell death was assessed by measuring cells with condensed and fragmented nuclear by microscopy. Arrows indicate the condensed, fragmented, brightly stained nuclei, which are the hallmark of apoptosis. All data are representative of three independent experiments.
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Related In: Results  -  Collection

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pone-0020586-g002: ATO regulated BIM expression is important for cell apoptosis in ovarian cancer cells.A and B. Western blots analysis of the expression level of BCL-2 family protein. Time-dependent analysis of BCL-2 member's expression levels in COC1/CP and COC1 cells. Cells were treated with ATO (2 µM) at indicated time, then lysed in NP40 buffer for detection. β-Actin was used as a protein loading control. Individual protein levels were measured by densitometric analysis of the Western blots and compared to actin levels. Relative amount of individual protein from treated cells (72 h) was set as 1. C. Effect of BIM shRNA on cell apoptosis. In the same conditions with A, cells were transfected with BIM or Ctrl shRNA for 48 h, and then treated with or without ATO for 48 h. Cell lysates were prepared and assayed for BIM, caspase-9 and cleaved caspase-3 by western blot. CF is referred to cleaved caspase-9. Relative fold means amount of BIM compared to actin and Ctrl condition was regarded as 1. D. Detection the effect of BIM shRNA on cell death with nuclear staining. Cells were transfected with BIM or Ctrl shRNA for 48 h, and then transfected cells were treated with ATO for 48 h. Cell death was assessed by measuring cells with condensed and fragmented nuclear by microscopy. Arrows indicate the condensed, fragmented, brightly stained nuclei, which are the hallmark of apoptosis. All data are representative of three independent experiments.
Mentions: Mitochondrial dysfunction plays an important role in apoptosis in ovarian cells. Previous studies reported that changes in the gene expression of BCL-2-family proteins was involved in ATO-induced apoptosis [32] and that the BH3-only proteins were necessary for ATO-induced apoptosis in myeloma cells. However, it is unclear whether BH3 proteins might function in ovarian cancer cells following ATO treatment. Therefore, we investigated the expression of BCL-2-family proteins in cisplatin-sensitive and -resistant cells after ATO treatment. As depicted in Figure 2A, pro-apoptotic proteins, such as BAX, PUMA and NOXA, were not significantly changed in COC1 cells at various time points after ATO treatment. Anti-apoptotic BCL-2 and BCL-XL proteins also exhibited no major alterations. However, in contrast to other proteins, the expression level of BIM was markedly increased after ATO treatment, providing evidence that BIM was involved in apoptotic cell death in ovarian cancer cells. Meanwhile, ATO induced BIM expression equally in COC1/CP cells (Figure 2B). Similar results were also observed in other cell lines (data not shown). These results suggest that BIM plays an important role in ATO-induced apoptosis in ovarian cancer cells.

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