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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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P53 promoted DOX-induced cell death was partly due to its ability to stop cell cycle progression. Cells were transiently transfected with or without over-expression of p53 alone or p53 and p65 together followed by DOX treatment (2 μg/ml) for 24 h. (A) The level of CDK2, CDK4, cyclinD1 or cyclinE protein were detected by Western blotting. GAPDH was used as controls. (B) Cell cycle pattern. Error bar indicates the standard error of the mean of three independent experiments.
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Figure 6: P53 promoted DOX-induced cell death was partly due to its ability to stop cell cycle progression. Cells were transiently transfected with or without over-expression of p53 alone or p53 and p65 together followed by DOX treatment (2 μg/ml) for 24 h. (A) The level of CDK2, CDK4, cyclinD1 or cyclinE protein were detected by Western blotting. GAPDH was used as controls. (B) Cell cycle pattern. Error bar indicates the standard error of the mean of three independent experiments.

Mentions: Next, we sought to test some other key cell cycle regulators which also showed in Figure 6A. Thus, we observed remarkably reduction in the levels of cdk2, cdk4, cyclinE and cyclinD1 by DOX after transfection with p53 or p53/p65 over-expression vector. To examine the change in the cell cycle pattern of these cells, the flow cytometry was carried out (Figure 6B). As expected, in PANC1 (p53++) and (p53++/p65++) cells fractions of G1-phase cells were increased from 54 and 49 to 79% and 76%, indicating the cell cycle is arrested at G1-phase due to increasing of nuclear p21 expression. These data suggest data that nuclear p21 induction by p53 promotes cell death may also due to its ability to stop cell cycle progression, which at least partly contributes to DOX-induced cell death in p53-dependent way.


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)

P53 promoted DOX-induced cell death was partly due to its ability to stop cell cycle progression. Cells were transiently transfected with or without over-expression of p53 alone or p53 and p65 together followed by DOX treatment (2 μg/ml) for 24 h. (A) The level of CDK2, CDK4, cyclinD1 or cyclinE protein were detected by Western blotting. GAPDH was used as controls. (B) Cell cycle pattern. Error bar indicates the standard error of the mean of three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 6: P53 promoted DOX-induced cell death was partly due to its ability to stop cell cycle progression. Cells were transiently transfected with or without over-expression of p53 alone or p53 and p65 together followed by DOX treatment (2 μg/ml) for 24 h. (A) The level of CDK2, CDK4, cyclinD1 or cyclinE protein were detected by Western blotting. GAPDH was used as controls. (B) Cell cycle pattern. Error bar indicates the standard error of the mean of three independent experiments.
Mentions: Next, we sought to test some other key cell cycle regulators which also showed in Figure 6A. Thus, we observed remarkably reduction in the levels of cdk2, cdk4, cyclinE and cyclinD1 by DOX after transfection with p53 or p53/p65 over-expression vector. To examine the change in the cell cycle pattern of these cells, the flow cytometry was carried out (Figure 6B). As expected, in PANC1 (p53++) and (p53++/p65++) cells fractions of G1-phase cells were increased from 54 and 49 to 79% and 76%, indicating the cell cycle is arrested at G1-phase due to increasing of nuclear p21 expression. These data suggest data that nuclear p21 induction by p53 promotes cell death may also due to its ability to stop cell cycle progression, which at least partly contributes to DOX-induced cell death in p53-dependent way.

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