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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 viability as measured by MTS assay. Cells (1 × 103 per well) were cultured in 96-well plates for 24 h, and treated with indicated concentrations of WA for 72 h. The viable cells were quantified by MTS assay in triplicates.
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Figure 1: Effect of WA on MCF-7 cell viability as measured by MTS assay. Cells (1 × 103 per well) were cultured in 96-well plates for 24 h, and treated with indicated concentrations of WA for 72 h. The viable cells were quantified by MTS assay in triplicates.

Mentions: The growth-inhibitory effect of WA on MCF-7 cells was demonstrated by MTS assay (Figure 1) and trypan blue exclusion assay (Figure 2). As shown in Figure 1, treatments with WA for 72 h at concentrations between 313 nM and 5 μM reduced the number of viable cells. 50% inhibition of cell viability was induced with 576 nM of WA at 72 h. Similarly, Trypan blue exclusion assay showed that WA significantly decreased the number of viable MCF-7 cells at 24 h after treatment in a dose-dependent manner. WA decreased the percentage of viable cells in total cell population to 50% at between 1 to 2.5 μM, and decreased the number of viable cells to 50% compared to controls at between 0.5 to 1 μM.


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 viability as measured by MTS assay. Cells (1 × 103 per well) were cultured in 96-well plates for 24 h, and treated with indicated concentrations of WA for 72 h. The viable cells were quantified by MTS assay in triplicates.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Effect of WA on MCF-7 cell viability as measured by MTS assay. Cells (1 × 103 per well) were cultured in 96-well plates for 24 h, and treated with indicated concentrations of WA for 72 h. The viable cells were quantified by MTS assay in triplicates.
Mentions: The growth-inhibitory effect of WA on MCF-7 cells was demonstrated by MTS assay (Figure 1) and trypan blue exclusion assay (Figure 2). As shown in Figure 1, treatments with WA for 72 h at concentrations between 313 nM and 5 μM reduced the number of viable cells. 50% inhibition of cell viability was induced with 576 nM of WA at 72 h. Similarly, Trypan blue exclusion assay showed that WA significantly decreased the number of viable MCF-7 cells at 24 h after treatment in a dose-dependent manner. WA decreased the percentage of viable cells in total cell population to 50% at between 1 to 2.5 μM, and decreased the number of viable cells to 50% compared to controls at between 0.5 to 1 μM.

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