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Cytoplasmic p21 induced by p65 prevents doxorubicin-induced cell death in pancreatic carcinoma cell line.

Zhou Y, Li G, Ji Y, Liu C, Zhu J, Lu Y - J. Biomed. Sci. (2012)

Bottom Line: Here we showed that over-expression of p65 decreased the cytotoxic effect of DOX on PANC1 cells, correlating with increased induction of cytoplasmic p21.We observed that pro-caspase-3 physically associated with cytoplasmic p21, which may be contribution to prevent p21 translocation into the nucleus.Likewise, down-regulation of p65 expression enhanced the cytotoxic effect of DOX, due to a significant decrease of mRNA levels of anti-apoptotic genes, such as the cellular inhibitor of apoptosis-1 (c-IAP1), and the long isoform of B cell leukemia/lymphoma-2 (Bcl-2), leading to efficient induction of caspase-3 cleavage in the cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Tongji University School of Medicine, Shanghai, China.

ABSTRACT

Background: Studies have shown the existence of p21 induction in a p53-dependent and -independent pathway. Our previous study indicates that DOX-induced p65 is able to bind the p21 promoter to activate its transactivation in the cells.

Methods: Over-expression and knock-down experiments were performed in Human Pancreatic Carcinoma (PANC1) cells. Cell cycle and cell death related proteins were assessed by Western Blotting. Cytotoxicity assay was checked by CCK-8 kit. Cell growth was analyzed by flow cytometers.

Results: Here we showed that over-expression of p65 decreased the cytotoxic effect of DOX on PANC1 cells, correlating with increased induction of cytoplasmic p21. We observed that pro-caspase-3 physically associated with cytoplasmic p21, which may be contribution to prevent p21 translocation into the nucleus. Our data also suggested that no clear elevation of nuclear p21 by p65 provides a survival advantage by progression cell cycle after treatment of DOX. Likewise, down-regulation of p65 expression enhanced the cytotoxic effect of DOX, due to a significant decrease of mRNA levels of anti-apoptotic genes, such as the cellular inhibitor of apoptosis-1 (c-IAP1), and the long isoform of B cell leukemia/lymphoma-2 (Bcl-2), leading to efficient induction of caspase-3 cleavage in the cells. More, we present evidence that over-expression of p53 or p53/p65 in the PANC1 cells were more sensitive to DOX treatment, correlated with activation of caspase-3 and clear elevation of nuclear p21 level. Our previous data suggested that expression of p21 increases Gefitinib-induced cell death by blocking the cell cycle at the G1 and G2 phases. The present findings here reinforced this idea by showing p21's ability of potentiality of DOX-induced cell death correlated with its inhibition of cell cycle progression after over-expression of p53 or p53/p65.

Conclusion: Our data suggested p65 could increase p53-mediated cell death in response to DOX in PANC1 cells. Thus, it is worth noting that in p53 or defective tumors, targeting in down-regulation of p65 may well be useful, leading to the potentiality of chemotherapeutic drugs.

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Pro-caspase-3 physically associated with cytoplasmic p21 induction by p65 in PANC1 cells. (A) Cells were transiently transfected with or without over-expression of p65 and p65-targeted shRNA followed by DOX treatment (2 μg/ml) for 24 h. Whole cell extract was subjected to immunoprecipitation with anti-p21 antibody and then immunoblotted with anti-pro-caspase-3 or -active-caspase-3 antibody. (B) Cells were transfected with vector expressing wild-type (1-585), N-terminal (1-372) and C-terminal (373-585) region of p21 followed by DOX treatment (2 μg/ml) for 24 h. Immunoprecipitation with an antibody against myc peptide. The pro-caspase-3 and p21 amounts were assessed by Western blotting.
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Figure 2: Pro-caspase-3 physically associated with cytoplasmic p21 induction by p65 in PANC1 cells. (A) Cells were transiently transfected with or without over-expression of p65 and p65-targeted shRNA followed by DOX treatment (2 μg/ml) for 24 h. Whole cell extract was subjected to immunoprecipitation with anti-p21 antibody and then immunoblotted with anti-pro-caspase-3 or -active-caspase-3 antibody. (B) Cells were transfected with vector expressing wild-type (1-585), N-terminal (1-372) and C-terminal (373-585) region of p21 followed by DOX treatment (2 μg/ml) for 24 h. Immunoprecipitation with an antibody against myc peptide. The pro-caspase-3 and p21 amounts were assessed by Western blotting.

Mentions: A family of aspartate-specific cysteine proteases (caspases) plays a pivotal role in the execution of programmed cell death. To gain insight into the involvement of caspases in DOX induced cell death in this study, we investigated the effects of p65 expression status on the activation of caspase-3. The protein expression of pro-caspase-3 and active caspase-3 was assessed by Western blotting in PANC1 cells that were treated with DOX at 24 h following p65 over-expression or down-regulation transfection. As shown in Figure 1C again, pro-caspase-3 was significantly elevated in PANC1 cells after transfection with p65 expression vector followed by DOX treatment, as compared to control vector. More, activation of caspase-3 protease cleavage after DOX treatment was suppressed in this condition. In contrast, when the cells were transfected with p65-targeted shRNA, the activation of caspase-3 was increased (Figure 2A). These results demonstrated that activation of caspase-3 was associated with DOX-induced cell death in PANC1 cells (Figure 1B and 1C).


Cytoplasmic p21 induced by p65 prevents doxorubicin-induced cell death in pancreatic carcinoma cell line.

Zhou Y, Li G, Ji Y, Liu C, Zhu J, Lu Y - J. Biomed. Sci. (2012)

Pro-caspase-3 physically associated with cytoplasmic p21 induction by p65 in PANC1 cells. (A) Cells were transiently transfected with or without over-expression of p65 and p65-targeted shRNA followed by DOX treatment (2 μg/ml) for 24 h. Whole cell extract was subjected to immunoprecipitation with anti-p21 antibody and then immunoblotted with anti-pro-caspase-3 or -active-caspase-3 antibody. (B) Cells were transfected with vector expressing wild-type (1-585), N-terminal (1-372) and C-terminal (373-585) region of p21 followed by DOX treatment (2 μg/ml) for 24 h. Immunoprecipitation with an antibody against myc peptide. The pro-caspase-3 and p21 amounts were assessed by Western blotting.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3298465&req=5

Figure 2: Pro-caspase-3 physically associated with cytoplasmic p21 induction by p65 in PANC1 cells. (A) Cells were transiently transfected with or without over-expression of p65 and p65-targeted shRNA followed by DOX treatment (2 μg/ml) for 24 h. Whole cell extract was subjected to immunoprecipitation with anti-p21 antibody and then immunoblotted with anti-pro-caspase-3 or -active-caspase-3 antibody. (B) Cells were transfected with vector expressing wild-type (1-585), N-terminal (1-372) and C-terminal (373-585) region of p21 followed by DOX treatment (2 μg/ml) for 24 h. Immunoprecipitation with an antibody against myc peptide. The pro-caspase-3 and p21 amounts were assessed by Western blotting.
Mentions: A family of aspartate-specific cysteine proteases (caspases) plays a pivotal role in the execution of programmed cell death. To gain insight into the involvement of caspases in DOX induced cell death in this study, we investigated the effects of p65 expression status on the activation of caspase-3. The protein expression of pro-caspase-3 and active caspase-3 was assessed by Western blotting in PANC1 cells that were treated with DOX at 24 h following p65 over-expression or down-regulation transfection. As shown in Figure 1C again, pro-caspase-3 was significantly elevated in PANC1 cells after transfection with p65 expression vector followed by DOX treatment, as compared to control vector. More, activation of caspase-3 protease cleavage after DOX treatment was suppressed in this condition. In contrast, when the cells were transfected with p65-targeted shRNA, the activation of caspase-3 was increased (Figure 2A). These results demonstrated that activation of caspase-3 was associated with DOX-induced cell death in PANC1 cells (Figure 1B and 1C).

Bottom Line: Here we showed that over-expression of p65 decreased the cytotoxic effect of DOX on PANC1 cells, correlating with increased induction of cytoplasmic p21.We observed that pro-caspase-3 physically associated with cytoplasmic p21, which may be contribution to prevent p21 translocation into the nucleus.Likewise, down-regulation of p65 expression enhanced the cytotoxic effect of DOX, due to a significant decrease of mRNA levels of anti-apoptotic genes, such as the cellular inhibitor of apoptosis-1 (c-IAP1), and the long isoform of B cell leukemia/lymphoma-2 (Bcl-2), leading to efficient induction of caspase-3 cleavage in the cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Tongji University School of Medicine, Shanghai, China.

ABSTRACT

Background: Studies have shown the existence of p21 induction in a p53-dependent and -independent pathway. Our previous study indicates that DOX-induced p65 is able to bind the p21 promoter to activate its transactivation in the cells.

Methods: Over-expression and knock-down experiments were performed in Human Pancreatic Carcinoma (PANC1) cells. Cell cycle and cell death related proteins were assessed by Western Blotting. Cytotoxicity assay was checked by CCK-8 kit. Cell growth was analyzed by flow cytometers.

Results: Here we showed that over-expression of p65 decreased the cytotoxic effect of DOX on PANC1 cells, correlating with increased induction of cytoplasmic p21. We observed that pro-caspase-3 physically associated with cytoplasmic p21, which may be contribution to prevent p21 translocation into the nucleus. Our data also suggested that no clear elevation of nuclear p21 by p65 provides a survival advantage by progression cell cycle after treatment of DOX. Likewise, down-regulation of p65 expression enhanced the cytotoxic effect of DOX, due to a significant decrease of mRNA levels of anti-apoptotic genes, such as the cellular inhibitor of apoptosis-1 (c-IAP1), and the long isoform of B cell leukemia/lymphoma-2 (Bcl-2), leading to efficient induction of caspase-3 cleavage in the cells. More, we present evidence that over-expression of p53 or p53/p65 in the PANC1 cells were more sensitive to DOX treatment, correlated with activation of caspase-3 and clear elevation of nuclear p21 level. Our previous data suggested that expression of p21 increases Gefitinib-induced cell death by blocking the cell cycle at the G1 and G2 phases. The present findings here reinforced this idea by showing p21's ability of potentiality of DOX-induced cell death correlated with its inhibition of cell cycle progression after over-expression of p53 or p53/p65.

Conclusion: Our data suggested p65 could increase p53-mediated cell death in response to DOX in PANC1 cells. Thus, it is worth noting that in p53 or defective tumors, targeting in down-regulation of p65 may well be useful, leading to the potentiality of chemotherapeutic drugs.

Show MeSH
Related in: MedlinePlus