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Differential effects of hypoxia on etoposide-induced apoptosis according to the cancer cell lines.

Cosse JP, Sermeus A, Vannuvel K, Ninane N, Raes M, Michiels C - Mol. Cancer (2007)

Bottom Line: Etoposide increased p53 activity in all cell lines while hypoxia alone decreased it only in HepG2 cells.Hypoxia had no influence on the etoposide-induced p53 activity in A549, increased p53 abundance in MCF-7 cells but markedly decreased p53 activity in HepG2 cells.These results evidenced that there was a striking parallelism between the effect of hypoxia on the etoposide-induced p53 stabilization as well as p53 target gene expression and its effect on the etoposide-induced apoptosis according to the cell type.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory of Biochemistry and Cellular Biology (URBC), FUNDP-University of Namur, 61 rue de Bruxelles, 5000 Namur, Belgium. jean-philippe.cosse@fundp.ac.be

ABSTRACT

Background: It is more and more recognized that hypoxia plays a role in the resistance of cancer cells to chemotherapy. However, the mechanisms underlying this resistance still need deeper understanding. The aim of this study was to investigate the effect of hypoxia on this process since hypoxia is one of the hallmarks of tumor environment.

Results: The effect of hypoxia on the apoptosis induced by etoposide, one drug commonly used in chemotherapy, was investigated using three different cancer cell lines. Gene expression changes were also studied in order to delineate the mechanisms responsible for the hypoxia-induced chemoresistance. We observed that hypoxia differentially influenced etoposide-induced cell death according to the cancer cell type. While hypoxia inhibited apoptosis in hepatoma HepG2 cells, it had no influence in lung carcinoma A549 cells and further enhanced it in breast cancer MCF-7 cells. Etoposide increased p53 activity in all cell lines while hypoxia alone decreased it only in HepG2 cells. Hypoxia had no influence on the etoposide-induced p53 activity in A549, increased p53 abundance in MCF-7 cells but markedly decreased p53 activity in HepG2 cells. Using low density DNA arrays to detect the expression of genes involved in the regulation of apoptosis, etoposide and hypoxia were shown to each influence the expression of numerous genes, many of the ones influenced by etoposide being p53 target genes. Again, the influence of hypoxia on the etoposide-induced changes was different according to the cell type.

Conclusion: These results evidenced that there was a striking parallelism between the effect of hypoxia on the etoposide-induced p53 stabilization as well as p53 target gene expression and its effect on the etoposide-induced apoptosis according to the cell type. They are very interesting not only because they provide one possible mechanism for the induction of chemoresistance under hypoxic conditions in cells like HepG2 but also because they indicate that not all cell types respond the same way. This knowledge is of importance in designing adequate treatment according to the type of tumors.

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Effect of hypoxia on the etoposide-induced apoptosis. A549, MCF-7 or HepG2 cells were incubated under normoxic (N) or hypoxic (H) conditions with or without etoposide (e, 50 μM) for 40 hours. A, PARP-1 and cleaved 85 kDa fragment were detected in total cell extracts by western blotting, using a specific anti-PARP-1 antibody. a-tubulin was used to assess the total amount of proteins loaded on the gel. B, LDH release was assessed. Results are presented in percentages, as means ± 1 SD (n = 3).
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Figure 2: Effect of hypoxia on the etoposide-induced apoptosis. A549, MCF-7 or HepG2 cells were incubated under normoxic (N) or hypoxic (H) conditions with or without etoposide (e, 50 μM) for 40 hours. A, PARP-1 and cleaved 85 kDa fragment were detected in total cell extracts by western blotting, using a specific anti-PARP-1 antibody. a-tubulin was used to assess the total amount of proteins loaded on the gel. B, LDH release was assessed. Results are presented in percentages, as means ± 1 SD (n = 3).

Mentions: Cell apoptosis and cell death were followed for a longer period of incubation in order to investigate whether the effect of hypoxia is sustained. Cells were incubated in the presence of etoposide under normoxic or hypoxic conditions for 40 h (Fig. 2). As an indication for apoptosis, PARP cleavage was assessed by western blot. Results (Fig. 2A) show that PARP cleavage induced by etoposide was more pronounced after 40 h incubation than after 16 h, in all three cell lines. Hypoxia prevented etoposide-induced PARP cleavage in HepG2 cells while it enhanced it in MCF-7 cells and had no effect in A549 cells. Cell death was also detected, as evidenced by an increase in LDH release, but again hypoxia differently influenced etoposide-induced cell death according to the cell type, with similar effects as observed for PARP cleavage. It has to be noted that hypoxia also induced a slight increase in cell death after 40 h incubation in A549 and MCF-7 cells. All together, these results indicate that the effect of hypoxia was sustained and translated in effect on cell viability.


Differential effects of hypoxia on etoposide-induced apoptosis according to the cancer cell lines.

Cosse JP, Sermeus A, Vannuvel K, Ninane N, Raes M, Michiels C - Mol. Cancer (2007)

Effect of hypoxia on the etoposide-induced apoptosis. A549, MCF-7 or HepG2 cells were incubated under normoxic (N) or hypoxic (H) conditions with or without etoposide (e, 50 μM) for 40 hours. A, PARP-1 and cleaved 85 kDa fragment were detected in total cell extracts by western blotting, using a specific anti-PARP-1 antibody. a-tubulin was used to assess the total amount of proteins loaded on the gel. B, LDH release was assessed. Results are presented in percentages, as means ± 1 SD (n = 3).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2099441&req=5

Figure 2: Effect of hypoxia on the etoposide-induced apoptosis. A549, MCF-7 or HepG2 cells were incubated under normoxic (N) or hypoxic (H) conditions with or without etoposide (e, 50 μM) for 40 hours. A, PARP-1 and cleaved 85 kDa fragment were detected in total cell extracts by western blotting, using a specific anti-PARP-1 antibody. a-tubulin was used to assess the total amount of proteins loaded on the gel. B, LDH release was assessed. Results are presented in percentages, as means ± 1 SD (n = 3).
Mentions: Cell apoptosis and cell death were followed for a longer period of incubation in order to investigate whether the effect of hypoxia is sustained. Cells were incubated in the presence of etoposide under normoxic or hypoxic conditions for 40 h (Fig. 2). As an indication for apoptosis, PARP cleavage was assessed by western blot. Results (Fig. 2A) show that PARP cleavage induced by etoposide was more pronounced after 40 h incubation than after 16 h, in all three cell lines. Hypoxia prevented etoposide-induced PARP cleavage in HepG2 cells while it enhanced it in MCF-7 cells and had no effect in A549 cells. Cell death was also detected, as evidenced by an increase in LDH release, but again hypoxia differently influenced etoposide-induced cell death according to the cell type, with similar effects as observed for PARP cleavage. It has to be noted that hypoxia also induced a slight increase in cell death after 40 h incubation in A549 and MCF-7 cells. All together, these results indicate that the effect of hypoxia was sustained and translated in effect on cell viability.

Bottom Line: Etoposide increased p53 activity in all cell lines while hypoxia alone decreased it only in HepG2 cells.Hypoxia had no influence on the etoposide-induced p53 activity in A549, increased p53 abundance in MCF-7 cells but markedly decreased p53 activity in HepG2 cells.These results evidenced that there was a striking parallelism between the effect of hypoxia on the etoposide-induced p53 stabilization as well as p53 target gene expression and its effect on the etoposide-induced apoptosis according to the cell type.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory of Biochemistry and Cellular Biology (URBC), FUNDP-University of Namur, 61 rue de Bruxelles, 5000 Namur, Belgium. jean-philippe.cosse@fundp.ac.be

ABSTRACT

Background: It is more and more recognized that hypoxia plays a role in the resistance of cancer cells to chemotherapy. However, the mechanisms underlying this resistance still need deeper understanding. The aim of this study was to investigate the effect of hypoxia on this process since hypoxia is one of the hallmarks of tumor environment.

Results: The effect of hypoxia on the apoptosis induced by etoposide, one drug commonly used in chemotherapy, was investigated using three different cancer cell lines. Gene expression changes were also studied in order to delineate the mechanisms responsible for the hypoxia-induced chemoresistance. We observed that hypoxia differentially influenced etoposide-induced cell death according to the cancer cell type. While hypoxia inhibited apoptosis in hepatoma HepG2 cells, it had no influence in lung carcinoma A549 cells and further enhanced it in breast cancer MCF-7 cells. Etoposide increased p53 activity in all cell lines while hypoxia alone decreased it only in HepG2 cells. Hypoxia had no influence on the etoposide-induced p53 activity in A549, increased p53 abundance in MCF-7 cells but markedly decreased p53 activity in HepG2 cells. Using low density DNA arrays to detect the expression of genes involved in the regulation of apoptosis, etoposide and hypoxia were shown to each influence the expression of numerous genes, many of the ones influenced by etoposide being p53 target genes. Again, the influence of hypoxia on the etoposide-induced changes was different according to the cell type.

Conclusion: These results evidenced that there was a striking parallelism between the effect of hypoxia on the etoposide-induced p53 stabilization as well as p53 target gene expression and its effect on the etoposide-induced apoptosis according to the cell type. They are very interesting not only because they provide one possible mechanism for the induction of chemoresistance under hypoxic conditions in cells like HepG2 but also because they indicate that not all cell types respond the same way. This knowledge is of importance in designing adequate treatment according to the type of tumors.

Show MeSH
Related in: MedlinePlus