The standardized Korean Red Ginseng extract and its ingredient ginsenoside Rg3 inhibit manifestation of breast cancer stem cell-like properties through modulation of self-renewal signaling

Abstract

Background: The ginsenoside Rg3, one of active components of red ginseng, has chemopreventive and anticancer potential. Cancer stem cells retain self-renewal properties which account for cancer recurrence and resistance to anticancer therapy. In our present study, we investigated whether the standardized Korean Red Ginseng extract (RGE) and Rg3 could modulate the manifestation of breast cancer stem cell-like features through regulation of self-renewal activity.

Methods: The effects of RGE and Rg3 on the proportion of CD44^high/CD24^low cells, as representative characteristics of stem-like breast cancer cells, were determined by flow cytometry. The mammosphere formation assay was performed to assess self-renewal capacities of breast cancer cells. Aldehyde dehydrogenase activity of MCF-7 mammospheres was measured by the ALDEFLUOR assay. The expression levels of Sox-2, Bmi-1, and P-Akt and the nuclear localization of hypoxia inducible factor-1α in MCF-7 mammospheres were verified by immunoblot analysis.

Results: Both RGE and Rg3 decreased the viability of breast cancer cells and significantly reduced the populations of CD44^high/CD24^low in MDA-MB-231 cells. RGE and Rg3 treatment attenuated the expression of Sox-2 and Bmi-1 by inhibiting the nuclear localization of hypoxia inducible factor-1α in MCF-7 mammospheres. Suppression of the manifestation of breast cancer stem cell-like properties by Rg3 was mediated through the blockade of Akt-mediated self-renewal signaling.

Conclusion: This study suggests that Rg3 has a therapeutic potential targeting breast cancer stem cells.

Keywords: Breast cancer stem cells; Ginseng; Ginsenoside Rg3; Red ginseng extract; Self-renewal.

Fig. 1

Effects of RGE on viability and stemness properties of breast cancer cells. (A) MCF-7, MDA-MB-231, and MCF-10A cells were treated with RGE (0.5, 1 or 5 mg/mL) or vehicle for 48 h. The data are presented as means ± SD. (B) At the tertiary mammosphere state, cells cultured in a 96-well ultralow attachment surface plate were treated with RGE (1 or 2.5 mg/mL) for 5 days. The number, the size, and the shape of mammospheres were examined by phase-contrast microscopy. Illustrations are the representative phase-contrast photomicrographs of mammospheres, and the graph bars are presented based on the number of mammospheres bigger than 100 μm. The values are presented as means ± SD (n = 3). (C) MDA-MB-231 cells treated with 1 or 2.5 mg/mL of RGE for 48 h were stained with anti-CD44-APC and anti-CD24-PE. Flow cytometric dot plots represent changes in the proportion of CD44^high/CD24^low cells. Quadrant analysis of fluorescence intensity of gated cells in FL2 and FL4 channels was from 10,000 events. Numerical values in the cytogram indicate the percentage of gated cells in each quadrant. The values are expressed as means ± SD (n = 3). APC, allophycocyanin; RGE, red ginseng extract; SD, standard deviation.

Fig. 2

Effects of Rg3 on viability and stemness properties of breast cancer cells. (A) MCF-7, MDA-MB-231, and MCF-10A cells were treated with Rg3 (25, 50 or 100 μM) for 72 h. The data are presented as means ± SD. (B) On tertiary mammosphere formation, Rg3 was treated twice at an interval of 2 days during the 5 days of maintenance. The number, the size, and the shape of mammospheres were examined by phase-contrast microscopy. Pictures are the representative phase-contrast photomicrographs of mammospheres, and the graph bars are presented based on the number of mammospheres bigger than 100 μm. The values are presented as means ± SD (n = 3). (C) MCF-7 mammospheres were subjected to the ALDFLUOR assay, followed by flow cytometry to detect cells with the ALDH activity. Tertiary MCF-7 mammospheres were treated with Rg3 (25 μM) twice at an interval of 2 days during the 5 days of maintenance. An ALDH inhibitor, DEAB was used to assess the background fluorescence. Quadrant analysis of fluorescence intensity of gated cells in FL1 channels was from 10,000 events. Numerical values in the plots indicate the percentage of gated cells in R1 quadrant. (D) MDA-MB-231 cells treated with Rg3 (10, 25 or 50 μM) for 48 h were stained with anti-CD44-APC and anti-CD24-PE. Flow cytometric dot plots represent changes in the proportion of CD44^high/CD24^low cells. Quadrant analysis of fluorescence intensity of gated cells in FL2 and FL4 channels was from 10,000 events. Numerical values in the cytogram indicate the percentage of gated cells in each quadrant. The values are expressed as means ± SD (n = 3).ALDH, aldehyde dehydrogenase; APC, allophycocyanin; DEAB, diethylaminobenzaldehyde; SD, standard deviation.

Fig. 3

Effects of RGE and Rg3 on self-renewal signaling of stem-like breast cancer cells. (A) On tertiary mammosphere formation, MCF-7 mammospheres were treated with RGE (1 mg/mL) twice at an interval of 2 days during the 5 days of maintenance. (B, C) MCF-7 and MDA-MB-231 mammospheres were treated with Rg3 (25 μM) in the same manner. The protein levels of Sox-2 and Bmi-1 were measured by Western blot analysis. The values are presented means ± SD (n = 3). RGE, red ginseng extract; SD, standard deviation.

Fig. 4

Effects of RGE and Rg3 on phosphorylation of Akt in MCF-7 mammospheres. Tertiary MCF-7 mammospheres were generated. The expression and phosphorylation of Akt were determined after treatment with RGE or Rg3. (A) With RGE (1 mg/mL) for 5 days. (B) With Rg3 (25 μM) for 5 days. The whole lysates were subjected to Western blot analysis. The values are presented means ± SD (n = 3). n.s., nonsignificant; RGE, red ginseng extract; SD, standard deviation.

Fig. 5

Involvement of the PI3K-Akt axis and a possible role of HIF-1α in self-renewal signaling of MCF-7 mammospheres. (A) LY294002, an Akt inhibitor was treated for 3 days to tertiary MCF-7 mammospheres. The expression of Sox-2 and Bmi-1 was measured by Western blot analysis. (B) The effect of LY294002 on mammosphere formation was confirmed by the mammosphere assay. The number, the size, and the shape of mammospheres were examined by phase-contrast microscopy. The graph represents the number of spheres that were bigger than 100 μm. The values are indicated as means ± SD (n = 3). (C) Tertiary MCF-7 mammospheres were treated with Rg3 (25 μM) or LY294002 (20 μM) for 5 and 3 days, respectively. Nuclear and cytosolic fractions were isolated from the cells and the localization of HIF-1α in MCF-7 mammospheres was measured by Western blot analysis. (D) Tertiary MCF-7 mammospheres were transfected with HIF-1α siRNA on the tertiary mammosphere formation for 72 h before the sample collection. The expression of Sox-2 and Bmi-1 was measured by Western blot analysis. The effect of HIF-1α knockdown on the formation of mammospheres was examined by phase-contrast microscopy. Pictures are the representative phase-contrast photomicrographs of mammospheres. HIF-1α, hypoxia inducible factor-1α; PI3K, phosphoinositide 3-kinase; SD, standard deviation.

Fig. 6

Schematic representation of a proposed mechanism underlying the inhibitory effects of Rg3 on self-renewal signaling. Rg3, a major anticancer component of RGE, inhibits the Akt-mediated self-renewal signaling which, in turn, modulates stem-like properties. HIF-1α, hypoxia inducible factor-1α; RGE, red ginseng extract.