Limits...
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.

Show MeSH

Related in: MedlinePlus

JNK pathway upregulates BIM expression and phosphorylation.A. Detection of p-JNK, and p-c-Jun in cells. COC1 cells were treated with ATO (2 µM) for different periods of time, then lysed with sample buffer and subjected to immunoblot assay with antibodies for phosphor-c-Jun (Ser 63) or polyclonal-phospho-JNK. COC1/CP cells were treatd with ATO for 72 h, then lysed for detection. Relative amount of p-JNK and p-c-Jun in untreated cells (72 h) were set as 1. B. JNK inhibitor prevents BIM expression and phosphorylation. Cells were treated with ATO (2 µM) for 72 h in the presence of JNK inhibitors SP600125 (10 µM). Lysated cells were immunoblotted with antibodies for phospho-c-Jun (Ser 63), polyclonal-phospho-JNK, phospho-AKT (Ser 473) or polyclonal-BIM. BIM phosphorylation was detected as described in Figure 4. Relative amount of p-JNK, p-c-Jun, 32p-BIM and BIM in ATO treated cells were set as 1. Relative fold means amount of p-AKT from untreated cells were regarded as 1. C. Dominant negative c-Jun inhibits BIM expression and phosphorylation. Left, OVCAR-3 cells were transiently transfected with pEGFP-TAM67 mutant or control vector (Ctrl vector). The mutant location was detected by microscopy. Middle, COC1/CP cells were transfected with pEGFP-C2 or pEGFP-TAM67, then lysed with antibodies for GFP or c-Jun. lane 1: cells with pEGFP-C2 vector. 2: untransfected cells. 3: cells with pEGFP-TAM67. Relative fold means amount of GFP in pEGFP-C2 vector transfected cells was regarded as 1. Right, A2780 cells were transfected with pEGFP-TAM67 or Ctrl vector. Stable positive clones were selected with 1 mg/ml G418 for several weeks. Ctrl vector transfected cells were treated with ATO (2 µM) and/or SP600125 for 72 h. pEGFP-TAM67 transfected cells were also incubated with ATO for 72 h. Lysated cells were immunoblotted with antibodies. BIM phosphorylation was detected as described in Figure 4. In all immunoblot analysis, β-Actin was used as a protein loading control. Relative amount of 32p-BIM and BIM in ATO treated cells were set as 1. All data are representative of three independent experiments.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3105099&req=5

pone-0020586-g006: JNK pathway upregulates BIM expression and phosphorylation.A. Detection of p-JNK, and p-c-Jun in cells. COC1 cells were treated with ATO (2 µM) for different periods of time, then lysed with sample buffer and subjected to immunoblot assay with antibodies for phosphor-c-Jun (Ser 63) or polyclonal-phospho-JNK. COC1/CP cells were treatd with ATO for 72 h, then lysed for detection. Relative amount of p-JNK and p-c-Jun in untreated cells (72 h) were set as 1. B. JNK inhibitor prevents BIM expression and phosphorylation. Cells were treated with ATO (2 µM) for 72 h in the presence of JNK inhibitors SP600125 (10 µM). Lysated cells were immunoblotted with antibodies for phospho-c-Jun (Ser 63), polyclonal-phospho-JNK, phospho-AKT (Ser 473) or polyclonal-BIM. BIM phosphorylation was detected as described in Figure 4. Relative amount of p-JNK, p-c-Jun, 32p-BIM and BIM in ATO treated cells were set as 1. Relative fold means amount of p-AKT from untreated cells were regarded as 1. C. Dominant negative c-Jun inhibits BIM expression and phosphorylation. Left, OVCAR-3 cells were transiently transfected with pEGFP-TAM67 mutant or control vector (Ctrl vector). The mutant location was detected by microscopy. Middle, COC1/CP cells were transfected with pEGFP-C2 or pEGFP-TAM67, then lysed with antibodies for GFP or c-Jun. lane 1: cells with pEGFP-C2 vector. 2: untransfected cells. 3: cells with pEGFP-TAM67. Relative fold means amount of GFP in pEGFP-C2 vector transfected cells was regarded as 1. Right, A2780 cells were transfected with pEGFP-TAM67 or Ctrl vector. Stable positive clones were selected with 1 mg/ml G418 for several weeks. Ctrl vector transfected cells were treated with ATO (2 µM) and/or SP600125 for 72 h. pEGFP-TAM67 transfected cells were also incubated with ATO for 72 h. Lysated cells were immunoblotted with antibodies. BIM phosphorylation was detected as described in Figure 4. In all immunoblot analysis, β-Actin was used as a protein loading control. Relative amount of 32p-BIM and BIM in ATO treated cells were set as 1. All data are representative of three independent experiments.

Mentions: Although we have confirmed that BIM mediates AKT activation in ATO-induced apoptosis, the mechanism of how ATO induces BIM is obscure. Recent studies demonstrated that ATO triggered the JNK pathway in cell apoptosis [41] and that JNK activation modulated BIM phosphorylation and expression [42], [43]. Accordingly, we wondered if JNK is activated and mediates BIM activation during ATO-induced apoptosis in ovarian cancer cells. To determine the function of the JNK pathway in BIM activation during ATO-induced apoptosis, we analyzed the phosphorylation of JNK (p-JNK) and c-Jun (p-c-Jun). The results demonstrated that ATO triggered the phosphorylation of JNK and c-Jun in a time-dependent manner, suggesting that the JNK pathway is activated during ATO-induced apoptosis (Figure 6A). In addition, our data also revealed that JNK activation increased BIM expression and phosphorylation, while the JNK inhibitor SP600125 prevented BIM activation (Figure 6B). These results suggest that the JNK pathway contributes not only to BIM expression, but also to the phosphorylation of BIM.


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)

JNK pathway upregulates BIM expression and phosphorylation.A. Detection of p-JNK, and p-c-Jun in cells. COC1 cells were treated with ATO (2 µM) for different periods of time, then lysed with sample buffer and subjected to immunoblot assay with antibodies for phosphor-c-Jun (Ser 63) or polyclonal-phospho-JNK. COC1/CP cells were treatd with ATO for 72 h, then lysed for detection. Relative amount of p-JNK and p-c-Jun in untreated cells (72 h) were set as 1. B. JNK inhibitor prevents BIM expression and phosphorylation. Cells were treated with ATO (2 µM) for 72 h in the presence of JNK inhibitors SP600125 (10 µM). Lysated cells were immunoblotted with antibodies for phospho-c-Jun (Ser 63), polyclonal-phospho-JNK, phospho-AKT (Ser 473) or polyclonal-BIM. BIM phosphorylation was detected as described in Figure 4. Relative amount of p-JNK, p-c-Jun, 32p-BIM and BIM in ATO treated cells were set as 1. Relative fold means amount of p-AKT from untreated cells were regarded as 1. C. Dominant negative c-Jun inhibits BIM expression and phosphorylation. Left, OVCAR-3 cells were transiently transfected with pEGFP-TAM67 mutant or control vector (Ctrl vector). The mutant location was detected by microscopy. Middle, COC1/CP cells were transfected with pEGFP-C2 or pEGFP-TAM67, then lysed with antibodies for GFP or c-Jun. lane 1: cells with pEGFP-C2 vector. 2: untransfected cells. 3: cells with pEGFP-TAM67. Relative fold means amount of GFP in pEGFP-C2 vector transfected cells was regarded as 1. Right, A2780 cells were transfected with pEGFP-TAM67 or Ctrl vector. Stable positive clones were selected with 1 mg/ml G418 for several weeks. Ctrl vector transfected cells were treated with ATO (2 µM) and/or SP600125 for 72 h. pEGFP-TAM67 transfected cells were also incubated with ATO for 72 h. Lysated cells were immunoblotted with antibodies. BIM phosphorylation was detected as described in Figure 4. In all immunoblot analysis, β-Actin was used as a protein loading control. Relative amount of 32p-BIM and BIM in ATO treated cells were set as 1. All data are representative of three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0020586-g006: JNK pathway upregulates BIM expression and phosphorylation.A. Detection of p-JNK, and p-c-Jun in cells. COC1 cells were treated with ATO (2 µM) for different periods of time, then lysed with sample buffer and subjected to immunoblot assay with antibodies for phosphor-c-Jun (Ser 63) or polyclonal-phospho-JNK. COC1/CP cells were treatd with ATO for 72 h, then lysed for detection. Relative amount of p-JNK and p-c-Jun in untreated cells (72 h) were set as 1. B. JNK inhibitor prevents BIM expression and phosphorylation. Cells were treated with ATO (2 µM) for 72 h in the presence of JNK inhibitors SP600125 (10 µM). Lysated cells were immunoblotted with antibodies for phospho-c-Jun (Ser 63), polyclonal-phospho-JNK, phospho-AKT (Ser 473) or polyclonal-BIM. BIM phosphorylation was detected as described in Figure 4. Relative amount of p-JNK, p-c-Jun, 32p-BIM and BIM in ATO treated cells were set as 1. Relative fold means amount of p-AKT from untreated cells were regarded as 1. C. Dominant negative c-Jun inhibits BIM expression and phosphorylation. Left, OVCAR-3 cells were transiently transfected with pEGFP-TAM67 mutant or control vector (Ctrl vector). The mutant location was detected by microscopy. Middle, COC1/CP cells were transfected with pEGFP-C2 or pEGFP-TAM67, then lysed with antibodies for GFP or c-Jun. lane 1: cells with pEGFP-C2 vector. 2: untransfected cells. 3: cells with pEGFP-TAM67. Relative fold means amount of GFP in pEGFP-C2 vector transfected cells was regarded as 1. Right, A2780 cells were transfected with pEGFP-TAM67 or Ctrl vector. Stable positive clones were selected with 1 mg/ml G418 for several weeks. Ctrl vector transfected cells were treated with ATO (2 µM) and/or SP600125 for 72 h. pEGFP-TAM67 transfected cells were also incubated with ATO for 72 h. Lysated cells were immunoblotted with antibodies. BIM phosphorylation was detected as described in Figure 4. In all immunoblot analysis, β-Actin was used as a protein loading control. Relative amount of 32p-BIM and BIM in ATO treated cells were set as 1. All data are representative of three independent experiments.
Mentions: Although we have confirmed that BIM mediates AKT activation in ATO-induced apoptosis, the mechanism of how ATO induces BIM is obscure. Recent studies demonstrated that ATO triggered the JNK pathway in cell apoptosis [41] and that JNK activation modulated BIM phosphorylation and expression [42], [43]. Accordingly, we wondered if JNK is activated and mediates BIM activation during ATO-induced apoptosis in ovarian cancer cells. To determine the function of the JNK pathway in BIM activation during ATO-induced apoptosis, we analyzed the phosphorylation of JNK (p-JNK) and c-Jun (p-c-Jun). The results demonstrated that ATO triggered the phosphorylation of JNK and c-Jun in a time-dependent manner, suggesting that the JNK pathway is activated during ATO-induced apoptosis (Figure 6A). In addition, our data also revealed that JNK activation increased BIM expression and phosphorylation, while the JNK inhibitor SP600125 prevented BIM activation (Figure 6B). These results suggest that the JNK pathway contributes not only to BIM expression, but also to the phosphorylation of BIM.

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