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. 2008 Aug;12(4):1265-71.
doi: 10.1111/j.1582-4934.2008.00282.x. Epub 2008 Feb 8.

Inhibition of ERK attenuates autophagy and potentiates tumour necrosis factor-alpha-induced cell death in MCF-7 cells

U Sivaprasad  1 , A Basu
Affiliations

Inhibition of ERK attenuates autophagy and potentiates tumour necrosis factor-alpha-induced cell death in MCF-7 cells

U Sivaprasad et al. J Cell Mol Med. 2008 Aug.

Abstract

The role of autophagy in cell death is under considerable debate. The process of autophagy has been shown to lead to either cell survival or cell death depending on cell type and stimulus. In the present study, we determined the contribution of ERK1/2 signalling to autophagy and cell death induced by tumour necrosis factor-alpha (TNF) in MCF-7 breast cancer cells. Treatment of MCF-7 cells with TNF caused a time-dependent increase in ERK1/2 activity. There was an induction of autophagy and cleavage of caspase-7, -8, -9 and PARP. Pharmacological inhibition of ERK1/2 phosphorylation with U0126 or PD98059 resulted in a decrease in TNF-induced autophagy that was accompanied by an increase in cleavage of caspase-7, -8, -9 and PARP Furthermore, inhibition of ERK1/2 signalling resulted in decreased clonogenic capacity of MCF-7 cells. These data suggest that TNF-induces autophagy through ERK1/2 and that inhibition of autophagy increases cellular sensitivity to TNF.

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Figures

Fig. 1
Fig. 1
TNF induces apoptosis and autophagy in MCF-7 cells. Cells were treated with or without 0.1 nM TNF for the indicated time periods. Cell lysates were analysed by Western blotting performed with the indicated antibodies. The arrows indicate LC3-II form and processed forms of caspases. GAPDH was used to control for loading differences.
Fig. 2
Fig. 2
TNF induces formation of EGFP-LC3 puncta. (A) MCF-7 cells were stably transfected with EGFP vector alone or EGFP-LC3. Cell lysates were analysed by Western blotting performed with the indicated antibodies. GAPDH was used to control for loading differences. (B) MCF-7/EGFP and MCF-7/LC3 cells were treated with 0.1 nM TNF for 16 hrs. Formation of LC3 puncta was observed using a fluorescence microscope.
Fig. 3
Fig. 3
TNF activates ERK1/2. MCF-7 cells were treated with or without 0.1 nM TNF for the indicated time periods. Cell lysates were analysed by Western blotting performed with the indicated antibodies. GAPDH was used to control for loading differences.
Fig. 4
Fig. 4
MAPK inhibitors decrease LC3-II levels and increase PARP cleavage induced by TNF. MCF-7 cells were pre-treated with indicated concentrations of U0126 (A) or PD98059 (B) for 1 h prior to treatment with or without TNF (0.1 nM) for 16 hrs. Cell lysates were analysed by Western blotting performed with the indicated antibodies. GAPDH was used to control for loading differences.
Fig. 4
Fig. 4
MAPK inhibitors decrease LC3-II levels and increase PARP cleavage induced by TNF. MCF-7 cells were pre-treated with indicated concentrations of U0126 (A) or PD98059 (B) for 1 h prior to treatment with or without TNF (0.1 nM) for 16 hrs. Cell lysates were analysed by Western blotting performed with the indicated antibodies. GAPDH was used to control for loading differences.
Fig. 5
Fig. 5
ERK inhibition attenuates TNF-induced autophagy. MCF-7 cells were pre-treated with 10 μM U0126 for 1 h prior to treatment with or without TNF (0.1 nM) for 16 hrs. (A) Cell lysates were analysed by Western blotting performed with the indicated antibodies. The arrow indicates the LC3-II form. (B) Arrows indicate processed forms of caspases. GAPDH was used to control for loading differences. (C) LC3-II levels and percentage PARP cleavage were determined by densitometric quantification of Western blots. Data are mean ± S.E.M. of three separate experiments. * denotes significant difference from untreated controls (P < 0.05); ** denotes significant difference from TNF-treated samples (P <0.05) using Student's paired t-test. (D) MCF-7/EGFP and MCF-7/LC3 cells were treated with 0.1 nM TNF for 16 hrs. Formation of LC3 puncta was observed using a fluorescence microscope.
Fig. 5
Fig. 5
ERK inhibition attenuates TNF-induced autophagy. MCF-7 cells were pre-treated with 10 μM U0126 for 1 h prior to treatment with or without TNF (0.1 nM) for 16 hrs. (A) Cell lysates were analysed by Western blotting performed with the indicated antibodies. The arrow indicates the LC3-II form. (B) Arrows indicate processed forms of caspases. GAPDH was used to control for loading differences. (C) LC3-II levels and percentage PARP cleavage were determined by densitometric quantification of Western blots. Data are mean ± S.E.M. of three separate experiments. * denotes significant difference from untreated controls (P < 0.05); ** denotes significant difference from TNF-treated samples (P <0.05) using Student's paired t-test. (D) MCF-7/EGFP and MCF-7/LC3 cells were treated with 0.1 nM TNF for 16 hrs. Formation of LC3 puncta was observed using a fluorescence microscope.
Fig. 5
Fig. 5
ERK inhibition attenuates TNF-induced autophagy. MCF-7 cells were pre-treated with 10 μM U0126 for 1 h prior to treatment with or without TNF (0.1 nM) for 16 hrs. (A) Cell lysates were analysed by Western blotting performed with the indicated antibodies. The arrow indicates the LC3-II form. (B) Arrows indicate processed forms of caspases. GAPDH was used to control for loading differences. (C) LC3-II levels and percentage PARP cleavage were determined by densitometric quantification of Western blots. Data are mean ± S.E.M. of three separate experiments. * denotes significant difference from untreated controls (P < 0.05); ** denotes significant difference from TNF-treated samples (P <0.05) using Student's paired t-test. (D) MCF-7/EGFP and MCF-7/LC3 cells were treated with 0.1 nM TNF for 16 hrs. Formation of LC3 puncta was observed using a fluorescence microscope.
Fig. 6
Fig. 6
MAPK inhibition sensitizes cells to TNF-induced cell death. MCF-7 cells were pre-treated with indicated concentrations of U0126 for 1 h and then treated with or without TNF (0.001 or 0.003 nM). Clonogenic assay was performed as described in Materials and Methods. The bar graph represents average colonies + S.E.M. of three separate experiments. a, denotes significant difference from untreated control cells (P < 0.05); b, denotes significant difference from TNF-treated cells (P <0.05); c, denotes significant difference from U0126-treated cells (P < 0.05).
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