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. 2013 Feb;138(1):69-79.
doi: 10.1007/s10549-013-2440-2. Epub 2013 Feb 15.

Critical Role for Reactive Oxygen Species in Apoptosis Induction and Cell Migration Inhibition by Diallyl Trisulfide, a Cancer Chemopreventive Component of Garlic

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Free PMC article

Critical Role for Reactive Oxygen Species in Apoptosis Induction and Cell Migration Inhibition by Diallyl Trisulfide, a Cancer Chemopreventive Component of Garlic

Kumar Chandra-Kuntal et al. Breast Cancer Res Treat. .
Free PMC article

Abstract

Diallyl trisulfide (DATS) is a structurally simple but biologically active constituent of processed garlic with in vivo activity against chemically induced as well as oncogene-driven cancer in experimental rodents. This study offers novel insights into the mechanisms underlying anticancer effects of DATS using human breast cancer cells as a model. Exposure of human breast cancer cells (MCF-7 and MDA-MB-231) and a cell line derived from spontaneously developing mammary tumor of a transgenic mouse (BRI-JM04) to DATS resulted in a dose-dependent inhibition of cell viability that was accompanied by apoptosis induction. A non-tumorigenic normal human mammary cell line (MCF-10A) was resistant to growth inhibition and apoptosis induction by DATS. The DATS-induced apoptosis in MDA-MB-231, MCF-7, and BRI-JM04 cells was associated with reactive oxygen species (ROS) production as evidenced by fluorescence microscopy and flow cytometry using a chemical probe (MitoSOX Red). Overexpression of Cu,Zn-superoxide dismutase (Cu,Zn-SOD) as well as Mn-SOD conferred significant protection against DATS-induced ROS production and apoptotic cell death in MDA-MB-231 and MCF-7 cells. Activation of Bak, but not Bax, resulting from DATS treatment was markedly suppressed by overexpression of Mn-SOD. The DATS treatment caused ROS generation, but not activation of Bax or Bak, in MCF-10A cells. Furthermore, the DATS-mediated inhibition of cell migration was partially but significantly attenuated by Cu,Zn-SOD and Mn-SOD overexpression in association with changes in levels of proteins involved in epithelial-mesenchymal transition. The DATS-mediated induction of heme oxygenase-1 was partially attenuated by overexpression of Mn-SOD. These results provide novel mechanistic insights indicating a critical role for ROS in anticancer effects of DATS.

Conflict of interest statement

Conflict of interests

KC-K, JL, and SVS declare no conflict of interest.

Figures

Figure 1
Figure 1
DATS inhibits viability of breast cancer cells in association with apoptosis induction. a Effect of DATS treatment (24 h exposure) on viability of breast cancer cells (MDA-MB-231, MCF-7, and BRI-JM04) and MCF-10A cells as determined by trypan blue dye exclusion assay. b Effect of DATS treatment (24 h exposure) on histone-associated DNA fragment release into the cytosol, a measure of apoptotic cell death, in breast cancer cells (MDA-MB-231, MCF-7, and BRI-JM04) and MCF-10A cells. Results shown are mean ± SD (n=3). *Significantly different (P < 0.05) compared with DMSO-treated control by one-way ANOVA followed by Dunnett’s test. Each experiment was performed at least twice.
Figure 2
Figure 2
DATS treatment caused ROS production in cancerous and normal breast cells. a Immunofluorescence microscopy for MitoSOX Red and MitoTracker Green fluorescence in MDA-MB-231, MCF-7, and MCF-10A cells following 3 h treatment with DMSO (control) or DATS (20 or 40 μM). b Flow cytometric measurement of MitoSOX Red fluorescence in MDA-MB-231, MCF-7, BRI-JM04, and MCF-10A cells following 3 or 6 h treatment with DMSO (control) or DATS (20 or 40 μM). Results shown are mean ± SD (n=3). *Significantly different (P < 0.05) compared with corresponding DMSO-treated control by one-way ANOVA followed by Dunnett’s test. Each experiment was performed at least twice and the results were consistent.
Figure 3
Figure 3
Overexpression of SOD confers protection against DATS-induced apoptosis. a Western blotting for Cu,Zn-SOD or Mn-SOD in MDA-MB-231 or MCF-7 cells stably transfected with empty pcDNA3.1 vector (lane 1) or the same vector encoding for Cu,Zn-SOD or Mn-SOD (lane 2). The blots were stripped and reprobed with anti-actin antibody to normalize for differences in protein level. Number on top of the immunoreactive band represents change in protein level relative to empty vector transfected cells. b ROS generation after 3 h treatment with DMSO (control) or the indicated concentration of DATS in MDA-MB-231 or MCF-7 cells stably transfected with empty vector or corresponding SOD plasmid. c Cell viability after 24 h treatment with DMSO (control) or the indicated concentration of DATS in MDA-MB-231 or MCF-7 cells stably transfected with empty vector or SOD (Cu,Zn-SOD or Mn-SOD) plasmid. d Histone-associated DNA fragment release into the cytosol after 24 h treatment with DMSO (control) or the indicated concentration of DATS in MDA-MB-231 or MCF-7 cells stably transfected with empty vector or SOD (Cu,Zn-SOD or Mn-SOD) plasmid. Results shown are mean ± SD (n=3). Significantly different (P < 0.05) compared with * DMSO-treated control, and # between empty vector transfected cells and SOD overexpressing cells by one-way ANOVA followed by Bonferroni’s multiple comparison test.
Figure 4
Figure 4
Overexpression of Mn-SOD confers protection against DATS-mediated Bak activation. a Immunocytochemical analysis for activated Bax in MCF-7 cells transfected with empty pcDNA3.1 vector or the same vector encoding for Mn-SOD and treated for 8 h with DMSO (control) or the indicated concentrations of DATS. b Immunocytochemical analysis for activated Bak in MCF-7 cells transfected with empty pcDNA3.1 vector or the same vector encoding for Mn-SOD and treated for 8 h with DMSO (control) or the indicated concentrations of DATS. c Immunocytochemical analysis for activated Bax and activated Bak in MCF-10A cells after 8 h treatment with DMSO or the indicated concentrations of DATS. Images were acquired at 100 objective magnification. Each experiment was repeated twice, and representative data from one such experiment are shown.
Figure 5
Figure 5
Overexpression of Mn-SOD confers protection against DATS-mediated inhibition of cell migration. a Representative images depicting in vitro migration by MDA-MB-231 cells transfected with the empty pcDNA3.1 vector or the same vector encoding for Cu,Zn-SOD or Mn-SOD and treated for 24 h with DMSO (control) or the indicated concentrations of DATS (×10 objective magnification). b Quantitation of the results shown in panel a. Results shown are mean ± SD (n=3). Significantly different (P < 0.05) compared with * DMSO-treated control, and # between empty vector transfected cells and SOD overexpressing cells by one-way ANOVA followed by Bonferroni’s multiple comparison test. Each experiment was performed at least twice and the results were consistent.
Figure 6
Figure 6
Overexpression of Mn-SOD confers protection against DATS-mediated suppression of vimentin. a Immunoblotting for E-cadherin and vimentin using lysates from MDA-MB-231 cells treated for 8 h with DMSO or the indicated concentrations of DATS. The blots were stripped and reprobed with anti-actin antibody to normalize for differences in protein level. Numbers on top of the immunoreactive bands represent change in protein level relative to DMSO-treated control. Immunoblotting for each protein was performed at least twice using independently prepared lysates, and representative data from one such experiment are shown. b Immunocytochemical analysis for E-cadherin in MDA-MB-231 cells transfected with empty pcDNA3.1 vector or the same vector encoding for Mn-SOD and treated for 8 h with DMSO (control) or the indicated concentrations of DATS. c Immunocytochemical analysis for vimentin in MDA-MB-231 cells transfected with empty vector or the same vector encoding for Mn-SOD and treated for 24 h with DMSO (control) or the indicated concentrations of DATS. Images were acquired at 100 objective magnification. d Western blotting for heme oxygenase-1 using lysates from MCF-7 cells transfected with empty pcDNA3.1 vector or the same vector encoding for Mn-SOD and treated for 24 h with DMSO (control) or the indicated concentrations of DATS. Quantitation relative to respective DMSO-treated control is shown.

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