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Hypoxia counteracts taxol-induced apoptosis in MDA-MB-231 breast cancer cells: role of autophagy and JNK activation.

Notte A, Ninane N, Arnould T, Michiels C - Cell Death Dis (2013)

Bottom Line: Autophagy activation and the development of hypoxic regions within the tumors are known to promote cancer cell resistance.The role of JNK in autophagy and apoptosis induction was studied using siRNAs.In conclusion, the resistance against taxol-induced cell death observed under hypoxia can be explained by a more effective autophagic flow activated via the classical mTOR pathway and by a mechanism involving JNK, which could be dependent on Bcl2 and BclXL phosphorylation but independent of JNK-induced autophagy activation.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Biochemistry and Cellular Biology, NARILIS, University of Namur-FUNDP, Namur, Belgium.

ABSTRACT
Cancer cell resistance against chemotherapy is still a heavy burden to improve anticancer treatments. Autophagy activation and the development of hypoxic regions within the tumors are known to promote cancer cell resistance. Therefore, we sought to evaluate the role of autophagy and hypoxia on the taxol-induced apoptosis in MDA-MB-231 breast cancer cells. The results showed that taxol induced apoptosis after 16 h of incubation, and that hypoxia protected MDA-MB-231 cells from taxol-induced apoptosis. In parallel, taxol induced autophagy activation already after 2 h of incubation both under normoxia and hypoxia. Autophagy activation after taxol exposure was shown to be a protective mechanism against taxol-induced cell death both under normoxia and hypoxia. However, at longer incubation time, the autophagic process reached a saturation point under normoxia leading to cell death, whereas under hypoxia, autophagy flow still correctly took place allowing the cells to survive. Autophagy induction is induced after taxol exposure via mechanistic target of rapamycin (mTOR) inhibition, which is more important in cells exposed to hypoxia. Taxol also induced c-Jun N-terminal kinase (JNK) activation and phosphorylation of its substrates B-cell CLL/lymphoma 2 (Bcl2) and BCL2-like 1 (BclXL) under normoxia and hypoxia very early after taxol exposure. Bcl2 and BclXL phosphorylation was decreased more importantly under hypoxia after long incubation time. The role of JNK in autophagy and apoptosis induction was studied using siRNAs. The results showed that JNK activation promotes resistance against taxol-induced apoptosis under normoxia and hypoxia without being involved in induction of autophagy. In conclusion, the resistance against taxol-induced cell death observed under hypoxia can be explained by a more effective autophagic flow activated via the classical mTOR pathway and by a mechanism involving JNK, which could be dependent on Bcl2 and BclXL phosphorylation but independent of JNK-induced autophagy activation.

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JNK-dependent phosphorylation of c-jun, Bcl2 and BclXL is induced by taxol but decreased after long incubation time under hypoxia. MDA-MB-231 cells were incubated under normoxia (N) or hypoxia (H) without (C) or with taxol (T) at 50 μM. (a) After 2, 4, 8, 16 and 24 h of incubation, c-jun, Bcl2, BclXL, phospho-c-jun, phospho-Bcl2 and phospho-BclXL were detected in total cell extracts, obtained with a phospho-protein-specific lysis buffer, by western blotting analysis, using specific antibodies. β-actin was used to assess the total amount of proteins loaded on the gel. The graphs below represent the quantification of phospho-c-jun, phospho-Bcl2 and phospho-BclXL abundance normalized to β-actin relative to c-jun, Bcl2 and BclXL total abundance normalized to β-actin. (b) MDA-MB-231 cells were untransfected (X) or transfected with 25 nM of JNK1 and 25 nM of JNK2 siRNA (SI) or negative control RF siRNA at 50 nM (RF) for 24 h. The transfection media were removed and replaced by culture media for 24 h. Cells were then incubated under normoxia (N) or hypoxia (H) for 16 h, without (C) or with taxol (T) at 50 μM. Bcl2, BclXL, phospho-Bcl2, phospho-BclXL were detected in total cell extracts, obtained with a phospho-protein-specific lysis buffer, by western blotting analysis, using specific antibodies. β-actin was used to assess the total amount of proteins loaded on the gel
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fig5: JNK-dependent phosphorylation of c-jun, Bcl2 and BclXL is induced by taxol but decreased after long incubation time under hypoxia. MDA-MB-231 cells were incubated under normoxia (N) or hypoxia (H) without (C) or with taxol (T) at 50 μM. (a) After 2, 4, 8, 16 and 24 h of incubation, c-jun, Bcl2, BclXL, phospho-c-jun, phospho-Bcl2 and phospho-BclXL were detected in total cell extracts, obtained with a phospho-protein-specific lysis buffer, by western blotting analysis, using specific antibodies. β-actin was used to assess the total amount of proteins loaded on the gel. The graphs below represent the quantification of phospho-c-jun, phospho-Bcl2 and phospho-BclXL abundance normalized to β-actin relative to c-jun, Bcl2 and BclXL total abundance normalized to β-actin. (b) MDA-MB-231 cells were untransfected (X) or transfected with 25 nM of JNK1 and 25 nM of JNK2 siRNA (SI) or negative control RF siRNA at 50 nM (RF) for 24 h. The transfection media were removed and replaced by culture media for 24 h. Cells were then incubated under normoxia (N) or hypoxia (H) for 16 h, without (C) or with taxol (T) at 50 μM. Bcl2, BclXL, phospho-Bcl2, phospho-BclXL were detected in total cell extracts, obtained with a phospho-protein-specific lysis buffer, by western blotting analysis, using specific antibodies. β-actin was used to assess the total amount of proteins loaded on the gel

Mentions: Various reports showed that taxol induces JNK activation.39, 40, 41 In order to investigate whether taxol induced JNK activation and Bcl2/BclXL phosphorylation, the abundance of c-jun, Bcl2, BclXL and the phosphorylated forms of these proteins was assessed by western blotting using specific antibodies raised against JNK phosphorylation sites (Figure 5a). Taxol induced c-jun, Bcl2 and BclXL phosphorylation at early time point under normoxia and hypoxia, whereas a decrease in the abundance of these phosphorylation forms was observed after 16 and 24 h under hypoxia. JNK invalidation with siRNAs showed that Bcl2 and BclXL phosphorylation was JNK-dependent, as JNK invalidation (Supplementary data 9) resulted in a decrease in phospho-Bcl2 and phospho-BclXL abundance (Figure 5b).


Hypoxia counteracts taxol-induced apoptosis in MDA-MB-231 breast cancer cells: role of autophagy and JNK activation.

Notte A, Ninane N, Arnould T, Michiels C - Cell Death Dis (2013)

JNK-dependent phosphorylation of c-jun, Bcl2 and BclXL is induced by taxol but decreased after long incubation time under hypoxia. MDA-MB-231 cells were incubated under normoxia (N) or hypoxia (H) without (C) or with taxol (T) at 50 μM. (a) After 2, 4, 8, 16 and 24 h of incubation, c-jun, Bcl2, BclXL, phospho-c-jun, phospho-Bcl2 and phospho-BclXL were detected in total cell extracts, obtained with a phospho-protein-specific lysis buffer, by western blotting analysis, using specific antibodies. β-actin was used to assess the total amount of proteins loaded on the gel. The graphs below represent the quantification of phospho-c-jun, phospho-Bcl2 and phospho-BclXL abundance normalized to β-actin relative to c-jun, Bcl2 and BclXL total abundance normalized to β-actin. (b) MDA-MB-231 cells were untransfected (X) or transfected with 25 nM of JNK1 and 25 nM of JNK2 siRNA (SI) or negative control RF siRNA at 50 nM (RF) for 24 h. The transfection media were removed and replaced by culture media for 24 h. Cells were then incubated under normoxia (N) or hypoxia (H) for 16 h, without (C) or with taxol (T) at 50 μM. Bcl2, BclXL, phospho-Bcl2, phospho-BclXL were detected in total cell extracts, obtained with a phospho-protein-specific lysis buffer, by western blotting analysis, using specific antibodies. β-actin was used to assess the total amount of proteins loaded on the gel
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fig5: JNK-dependent phosphorylation of c-jun, Bcl2 and BclXL is induced by taxol but decreased after long incubation time under hypoxia. MDA-MB-231 cells were incubated under normoxia (N) or hypoxia (H) without (C) or with taxol (T) at 50 μM. (a) After 2, 4, 8, 16 and 24 h of incubation, c-jun, Bcl2, BclXL, phospho-c-jun, phospho-Bcl2 and phospho-BclXL were detected in total cell extracts, obtained with a phospho-protein-specific lysis buffer, by western blotting analysis, using specific antibodies. β-actin was used to assess the total amount of proteins loaded on the gel. The graphs below represent the quantification of phospho-c-jun, phospho-Bcl2 and phospho-BclXL abundance normalized to β-actin relative to c-jun, Bcl2 and BclXL total abundance normalized to β-actin. (b) MDA-MB-231 cells were untransfected (X) or transfected with 25 nM of JNK1 and 25 nM of JNK2 siRNA (SI) or negative control RF siRNA at 50 nM (RF) for 24 h. The transfection media were removed and replaced by culture media for 24 h. Cells were then incubated under normoxia (N) or hypoxia (H) for 16 h, without (C) or with taxol (T) at 50 μM. Bcl2, BclXL, phospho-Bcl2, phospho-BclXL were detected in total cell extracts, obtained with a phospho-protein-specific lysis buffer, by western blotting analysis, using specific antibodies. β-actin was used to assess the total amount of proteins loaded on the gel
Mentions: Various reports showed that taxol induces JNK activation.39, 40, 41 In order to investigate whether taxol induced JNK activation and Bcl2/BclXL phosphorylation, the abundance of c-jun, Bcl2, BclXL and the phosphorylated forms of these proteins was assessed by western blotting using specific antibodies raised against JNK phosphorylation sites (Figure 5a). Taxol induced c-jun, Bcl2 and BclXL phosphorylation at early time point under normoxia and hypoxia, whereas a decrease in the abundance of these phosphorylation forms was observed after 16 and 24 h under hypoxia. JNK invalidation with siRNAs showed that Bcl2 and BclXL phosphorylation was JNK-dependent, as JNK invalidation (Supplementary data 9) resulted in a decrease in phospho-Bcl2 and phospho-BclXL abundance (Figure 5b).

Bottom Line: Autophagy activation and the development of hypoxic regions within the tumors are known to promote cancer cell resistance.The role of JNK in autophagy and apoptosis induction was studied using siRNAs.In conclusion, the resistance against taxol-induced cell death observed under hypoxia can be explained by a more effective autophagic flow activated via the classical mTOR pathway and by a mechanism involving JNK, which could be dependent on Bcl2 and BclXL phosphorylation but independent of JNK-induced autophagy activation.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Biochemistry and Cellular Biology, NARILIS, University of Namur-FUNDP, Namur, Belgium.

ABSTRACT
Cancer cell resistance against chemotherapy is still a heavy burden to improve anticancer treatments. Autophagy activation and the development of hypoxic regions within the tumors are known to promote cancer cell resistance. Therefore, we sought to evaluate the role of autophagy and hypoxia on the taxol-induced apoptosis in MDA-MB-231 breast cancer cells. The results showed that taxol induced apoptosis after 16 h of incubation, and that hypoxia protected MDA-MB-231 cells from taxol-induced apoptosis. In parallel, taxol induced autophagy activation already after 2 h of incubation both under normoxia and hypoxia. Autophagy activation after taxol exposure was shown to be a protective mechanism against taxol-induced cell death both under normoxia and hypoxia. However, at longer incubation time, the autophagic process reached a saturation point under normoxia leading to cell death, whereas under hypoxia, autophagy flow still correctly took place allowing the cells to survive. Autophagy induction is induced after taxol exposure via mechanistic target of rapamycin (mTOR) inhibition, which is more important in cells exposed to hypoxia. Taxol also induced c-Jun N-terminal kinase (JNK) activation and phosphorylation of its substrates B-cell CLL/lymphoma 2 (Bcl2) and BCL2-like 1 (BclXL) under normoxia and hypoxia very early after taxol exposure. Bcl2 and BclXL phosphorylation was decreased more importantly under hypoxia after long incubation time. The role of JNK in autophagy and apoptosis induction was studied using siRNAs. The results showed that JNK activation promotes resistance against taxol-induced apoptosis under normoxia and hypoxia without being involved in induction of autophagy. In conclusion, the resistance against taxol-induced cell death observed under hypoxia can be explained by a more effective autophagic flow activated via the classical mTOR pathway and by a mechanism involving JNK, which could be dependent on Bcl2 and BclXL phosphorylation but independent of JNK-induced autophagy activation.

Show MeSH
Related in: MedlinePlus