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Down-regulation of estrogen receptor-alpha and rearranged during transfection tyrosine kinase is associated with withaferin a-induced apoptosis in MCF-7 breast cancer cells.

Zhang X, Mukerji R, Samadi AK, Cohen MS - BMC Complement Altern Med (2011)

Bottom Line: Cell cycle effects were analyzed by PI flow cytometry.WA resulted in growth inhibition and decreased viability in MCF-7 cells with an IC50 of 576 nM for 72 h.WA-induced apoptosis was associated with down-regulation of ERα, REarranged during Transfection (RET) tyrosine kinase, and heat shock factor-1 (HSF1), as well as up-regulation of phosphorylated p38 mitogen-activated protein kinase (phospho-p38 MAPK), p53 and p21 protein expression.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Surgery, University of Kansas School of Medicine, Kansas City, Kansas, USA. xzhang2@kumc.edu

ABSTRACT

Background: Withaferin A (WA), a naturally occurring withanolide, induces apoptosis in both estrogen-responsive MCF-7 and estrogen-independent MDA-MB-231 breast cancer cell lines with higher sensitivity in MCF-7 cells, but the underlying mechanisms are not well defined. The purpose of this study was to determine the anti-cancer effects of WA in MCF-7 breast cancer cells and explore alterations in estrogen receptor alpha (ERα) and its associated molecules in vitro as novel mechanisms of WA action.

Methods: The effects of WA on MCF-7 viability and proliferation were evaluated by 3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay and trypan blue exclusion assays. Apoptosis was evaluated by Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) flow cytometry and Western blot analysis of poly (ADP-ribose) polymerase (PARP) cleavage. Cell cycle effects were analyzed by PI flow cytometry. Western blotting was also conducted to examine alterations in the expression of ERα and pathways that are associated with ERα function.

Results: WA resulted in growth inhibition and decreased viability in MCF-7 cells with an IC50 of 576 nM for 72 h. It also caused a dose- and time-dependent apoptosis and G2/M cell cycle arrest. WA-induced apoptosis was associated with down-regulation of ERα, REarranged during Transfection (RET) tyrosine kinase, and heat shock factor-1 (HSF1), as well as up-regulation of phosphorylated p38 mitogen-activated protein kinase (phospho-p38 MAPK), p53 and p21 protein expression. Co-treatment with protein synthesis inhibitor cycloheximide or proteasome inhibitor MG132 revealed that depletion of ERα by WA is post-translational, due to proteasome-dependent ERα degradation.

Conclusions: Taken together, down-regulation of ERα, RET, HSF1 and up-regulation of phospho-p38 MAPK, p53, p21 are involved in the pro-apoptotic and growth-inhibitory effects of WA in MCF-7 breast cancer cells in vitro. Down-regulation of ERα protein levels by WA is caused by proteasome-dependent ERα degradation.

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Effect of WA on MCF-7 cell protein expression as measured by Western analysis. A, Western analysis of the expression of ERα, RET, p53, p21, HSF-1, survivin, PARP, pro-caspase3, pro-caspase7 at 24 h after treatments with increasing concentrations of WA in MCF-7 cells. B, Western analysis of the expression of p38, phospho-p38, ERα, RET, p53, p21, HSF-1, survivin, PARP, pro-caspase3, pro-caspase7 at 0, 1, 3, 6, 12, 24, and 48 h after treatment with 2.5 μM of WA in MCF-7 cells.
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Figure 5: Effect of WA on MCF-7 cell protein expression as measured by Western analysis. A, Western analysis of the expression of ERα, RET, p53, p21, HSF-1, survivin, PARP, pro-caspase3, pro-caspase7 at 24 h after treatments with increasing concentrations of WA in MCF-7 cells. B, Western analysis of the expression of p38, phospho-p38, ERα, RET, p53, p21, HSF-1, survivin, PARP, pro-caspase3, pro-caspase7 at 0, 1, 3, 6, 12, 24, and 48 h after treatment with 2.5 μM of WA in MCF-7 cells.

Mentions: To address whether WA causes apoptosis, we first did Annexin V-FITC/PI flow cytometry. As shown in Figure 4, WA significantly induces apoptosis in a dose- and time-dependent manner. In the dose response study, MCF-7 cells were treated with 0, 1, 2.5 and 5 μM of WA for 24 h. Early apoptosis (defined as Annexin V-positive/PI-negative) was induced by 1 μM WA at 24 h (12.7% increase compared to controls). 2.5 μM of WA induced more late apoptosis (23.5%) than early apoptosis (4.6%), while 5 μM of WA mainly induced late apoptosis (67.4%). In the time course study, cells were treated with 2.5 μM of WA for 0, 6, 12 and 24 h. No significant increase in apoptosis could be seen at 6 h. At 12 and 24 h, increase in both early apoptosis (3.3% and 3.4%, respectively,) and late apoptosis (13.9% and 29.5%, respectively) was induced. The WA-induced apoptosis in MCF-7 cells was also confirmed by Western analysis of PARP cleavage as well as caspase-3 and caspase-7 activation (Figure 5A and 5B). In the dose response study, 2.5 or 5 μM of WA significantly increased PARP cleavage at 24 h, which is associated with a decrease in pro-caspase3 and pro-caspase7 levels. In the time course study, slight but significant increase in PARP cleavage could be seen at 12 h after treatment with 2.5 μM of WA. By 24 h, PARP cleavage became more apparent.


Down-regulation of estrogen receptor-alpha and rearranged during transfection tyrosine kinase is associated with withaferin a-induced apoptosis in MCF-7 breast cancer cells.

Zhang X, Mukerji R, Samadi AK, Cohen MS - BMC Complement Altern Med (2011)

Effect of WA on MCF-7 cell protein expression as measured by Western analysis. A, Western analysis of the expression of ERα, RET, p53, p21, HSF-1, survivin, PARP, pro-caspase3, pro-caspase7 at 24 h after treatments with increasing concentrations of WA in MCF-7 cells. B, Western analysis of the expression of p38, phospho-p38, ERα, RET, p53, p21, HSF-1, survivin, PARP, pro-caspase3, pro-caspase7 at 0, 1, 3, 6, 12, 24, and 48 h after treatment with 2.5 μM of WA in MCF-7 cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3198756&req=5

Figure 5: Effect of WA on MCF-7 cell protein expression as measured by Western analysis. A, Western analysis of the expression of ERα, RET, p53, p21, HSF-1, survivin, PARP, pro-caspase3, pro-caspase7 at 24 h after treatments with increasing concentrations of WA in MCF-7 cells. B, Western analysis of the expression of p38, phospho-p38, ERα, RET, p53, p21, HSF-1, survivin, PARP, pro-caspase3, pro-caspase7 at 0, 1, 3, 6, 12, 24, and 48 h after treatment with 2.5 μM of WA in MCF-7 cells.
Mentions: To address whether WA causes apoptosis, we first did Annexin V-FITC/PI flow cytometry. As shown in Figure 4, WA significantly induces apoptosis in a dose- and time-dependent manner. In the dose response study, MCF-7 cells were treated with 0, 1, 2.5 and 5 μM of WA for 24 h. Early apoptosis (defined as Annexin V-positive/PI-negative) was induced by 1 μM WA at 24 h (12.7% increase compared to controls). 2.5 μM of WA induced more late apoptosis (23.5%) than early apoptosis (4.6%), while 5 μM of WA mainly induced late apoptosis (67.4%). In the time course study, cells were treated with 2.5 μM of WA for 0, 6, 12 and 24 h. No significant increase in apoptosis could be seen at 6 h. At 12 and 24 h, increase in both early apoptosis (3.3% and 3.4%, respectively,) and late apoptosis (13.9% and 29.5%, respectively) was induced. The WA-induced apoptosis in MCF-7 cells was also confirmed by Western analysis of PARP cleavage as well as caspase-3 and caspase-7 activation (Figure 5A and 5B). In the dose response study, 2.5 or 5 μM of WA significantly increased PARP cleavage at 24 h, which is associated with a decrease in pro-caspase3 and pro-caspase7 levels. In the time course study, slight but significant increase in PARP cleavage could be seen at 12 h after treatment with 2.5 μM of WA. By 24 h, PARP cleavage became more apparent.

Bottom Line: Cell cycle effects were analyzed by PI flow cytometry.WA resulted in growth inhibition and decreased viability in MCF-7 cells with an IC50 of 576 nM for 72 h.WA-induced apoptosis was associated with down-regulation of ERα, REarranged during Transfection (RET) tyrosine kinase, and heat shock factor-1 (HSF1), as well as up-regulation of phosphorylated p38 mitogen-activated protein kinase (phospho-p38 MAPK), p53 and p21 protein expression.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Surgery, University of Kansas School of Medicine, Kansas City, Kansas, USA. xzhang2@kumc.edu

ABSTRACT

Background: Withaferin A (WA), a naturally occurring withanolide, induces apoptosis in both estrogen-responsive MCF-7 and estrogen-independent MDA-MB-231 breast cancer cell lines with higher sensitivity in MCF-7 cells, but the underlying mechanisms are not well defined. The purpose of this study was to determine the anti-cancer effects of WA in MCF-7 breast cancer cells and explore alterations in estrogen receptor alpha (ERα) and its associated molecules in vitro as novel mechanisms of WA action.

Methods: The effects of WA on MCF-7 viability and proliferation were evaluated by 3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay and trypan blue exclusion assays. Apoptosis was evaluated by Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) flow cytometry and Western blot analysis of poly (ADP-ribose) polymerase (PARP) cleavage. Cell cycle effects were analyzed by PI flow cytometry. Western blotting was also conducted to examine alterations in the expression of ERα and pathways that are associated with ERα function.

Results: WA resulted in growth inhibition and decreased viability in MCF-7 cells with an IC50 of 576 nM for 72 h. It also caused a dose- and time-dependent apoptosis and G2/M cell cycle arrest. WA-induced apoptosis was associated with down-regulation of ERα, REarranged during Transfection (RET) tyrosine kinase, and heat shock factor-1 (HSF1), as well as up-regulation of phosphorylated p38 mitogen-activated protein kinase (phospho-p38 MAPK), p53 and p21 protein expression. Co-treatment with protein synthesis inhibitor cycloheximide or proteasome inhibitor MG132 revealed that depletion of ERα by WA is post-translational, due to proteasome-dependent ERα degradation.

Conclusions: Taken together, down-regulation of ERα, RET, HSF1 and up-regulation of phospho-p38 MAPK, p53, p21 are involved in the pro-apoptotic and growth-inhibitory effects of WA in MCF-7 breast cancer cells in vitro. Down-regulation of ERα protein levels by WA is caused by proteasome-dependent ERα degradation.

Show MeSH
Related in: MedlinePlus