Diallyl trisulfide inhibits estrogen receptor-α activity in human breast cancer cells

Abstract

Organosulfur compounds from garlic effectively inhibit growth of transplanted as well as spontaneous cancers in preclinical animal models without any adverse side effects. However, the mechanisms underlying anticancer effect of this class of compounds are not fully understood. This study reports, for the first time, that garlic organosulfide diallyl trisulfide (DATS) inhibits estrogen receptor-α (ER-α) activity in human breast cancer cells. Exposure of MCF-7 and T47D cells to DATS resulted in downregulation of ER-α protein, which peaked between 12- and 24-h post-treatment. DATS was relatively more effective in suppressing ER-α protein expression compared with its mono and disulfide analogs. The 17β-estradiol (E2)-induced expression of pS2 and cyclin D1, ER-α target gene products, was also decreased in the presence of DATS. Downregulation of ER-α protein expression resulting from DATS treatment was accompanied by a decrease in nuclear levels of ER-α protein, ER-α mRNA suppression, and inhibition of ERE2e1b-luciferase reporter activity. DATS-mediated inhibition of cell viability and apoptosis induction were not affected in the presence of E2. In agreement with these results, ectopic expression of ER-α in MDA-MB-231 cell line failed to confer any protection against cell proliferation inhibition or apoptosis induction resulting from DATS exposure. DATS treatment caused a decrease in protein levels of peptidyl-prolyl cis-trans isomerase (Pin1), and overexpression of Pin1 partially attenuated ER-α downregulation by DATS. DATS-induced apoptosis was modestly but significantly augmented by overexpression of Pin1. In conclusion, this study identifies ER-α as a novel target of DATS in mammary cancer cells.

Fig. 1

DATS treatment suppresses ER-α protein expression in MCF-7 and T47D cells. a Chemical structures of the organosulfur compounds used in this study. b Western blotting for ER-α protein using lysates from MCF-7 cells treated for 24 h with DMSO or the indicated doses of DAS, DADS, and DATS. c Western blotting for estrogen-inducible proteins in MCF-7 and T47D cells after 24 h treatment with DATS (20 μM) and/or E2 (10 nM). d Western blotting for time-course kinetic effect of DATS treatment on ER-α protein level in MCF-7 and T47D cells. Densitometric quantitation of band intensity relative to corresponding DMSO-treated control after normalization for loading control is shown on top of the band. e Effect of DATS treatment (6 and 12 h) on viability of MCF-7 and T47D cells. Combined results from two independent experiments are shown as mean ± SD (n = 6). *Significantly different between the indicated groups by one-way ANOVA followed by Dunnett’s test. Each experiment was performed at least twice and the results were consistent

Fig. 2

DATS treatment decreases nuclear levels of ER-α protein in MCF-7 and T47D cells. Immunocytochemical analysis of ER-α protein level in a MCF-7 cells and b T47D cells after 6 or 12 h treatment with DMSO or 20 μM DATS (×100 objective magnification). Similar results were observed in replicate experiments

Fig. 3

DATS treatment decreases ER-α mRNA expression in MCF-7 and T47D cells. a ERE2e1b-associated luciferase reporter activity in MCF-7 and T47D cells after 12 h treatment with 10 nM E2 alone or E2 plus DATS (10 or 20 μM). Results shown are mean ± SD (n = 3). *Significantly different (P < 0.05) between the indicated groups by one-way ANOVA followed by Bonferroni’s test. b RT-PCR for ER-α mRNA expression in MCF-7 and T47D cells after 6, 12, or 24 h treatment with DMSO or the indicated concentrations of DATS. Number above band indicates change in mRNA level relative to corresponding DMSO-treated control after normalization to GAPDH mRNA level. Each experiment was repeated at least twice

Fig. 4

Effect of E2 on DATS-induced apoptosis. a Western blotting for estrogen-inducible pS2 protein in MCF-7 cells with or without treatment with 10 nM E2. b Cell viability was determined by trypan blue dye exclusion assay using MCF-7 and T47D cells treated for 24 h with DMSO or indicated doses of DATS in the absence or presence of E2. Results shown are mean ± SD (n = 3–6). *Significantly different (P < 0.05) compared with corresponding DMSO-treated control by one-way ANOVA followed by Bonferroni’s test. c Representative flow histograms showing early (Annexin V positive) and late apoptotic fractions (Annexin V + propidium iodide positive) in MCF-7 cells treated for 24 h with DMSO or 20 μM of DATS in the absence or presence of 10 nM E2. d Quantitation of early + late apoptosis from data shown in c. Results shown are mean ± SD (n = 3). *Significantly different (P < 0.05) between the indicated groups by one-way ANOVA followed by Bonferroni’s test. Similar results were observed in replicate experiments

Fig. 5

Overexpression of ER-α fails to confer protection against DATS-induced apoptosis in MDA-MB-231 cells. a Western blotting for ER-α protein using lysates from empty vector transfected cells (lane 1) and ER-α overexpressing MDA-MB-231 cells (lane 2). b Cell proliferation assay was done using empty vector-transfected control cells and ER-α overexpressing MDA-MB-231 cells after 72 h treatment with DMSO or 20 μM of DATS. Results shown are mean ± SD (n = 3). *Significantly different (P < 0.05) between the indicated groups by one-way ANOVA followed by Bonferroni’s test. c Representative flow histograms depicting sub-G₀–G₁ phase populations in empty vector transfected and ER-α overexpressing MDA-MB-231 cells treated for 24 h with DMSO (control) or 20 μM of DATS. d Quantitation of sub-G₀–G₁ phase population from data shown in c. *Significantly different (P < 0.05) between the indicated groups by one-way ANOVA followed by Bonferroni’s test. e Representative flow histograms showing early and late apoptosis in empty vector transfected and ER-α-overexpressing MDA-MB-231 cells treated for 24 h with DMSO or 20 μM of DATS. Each experiment was performed at least twice and the results were consistent

Fig. 6

DATS-induced apoptosis is modestly augmented by Pin1. a Western blotting for Pin1 and ER-α proteins using lysates from empty vector transfected and Pin1-overexpressing MCF-7 cells treated with the indicated doses of DATS or DMSO for 6 and 12 h. Densitometric quantitation of band intensity relative to corresponding DMSO-treated control after normalization to loading control is shown on top of the band. b Representative flow histograms showing early and late apoptosis in empty vector transfected and Pin1-overexpressing MCF-7 cells treated for 24 h with DMSO or 20 μM of DATS. c Quantitation of apoptosis from results shown in panel b. Combined results from two independent experiments are shown as mean ± SD (n = 6). *Significantly different (P < 0.05) between the indicated groups by one-way ANOVA followed by Bonferroni’s test