Stereoselective Anti-Cancer Activities of Ginsenoside Rg3 on Triple Negative Breast Cancer Cell Models

Abstract

Ginsenoside Rg3 (Rg3) has two epimers, 20(S)-ginsenoside Rg3 (SRg3) and 20(R)-ginsenoside Rg3 (RRg3), and while Rg3 itself has been reported to have anti-cancer properties, few studies have been reported on the anti-cancer effects of the different epimers. The aim was to investigate the stereoselective effects of the Rg3 epimers on triple negative breast cancer (TNBC) cell lines, tested using cell-based assays for proliferation, apoptosis, cell cycle arrest, migration and invasion. Molecular docking showed that Rg3 interacted with the aquaporin 1 (AQP1) water channel (binding score -9.4 kJ mol^-1). The Xenopus laevis oocyte expression system was used to study the effect of Rg3 epimers on the AQP1 water permeability. The AQP1 expression in TNBC cell lines was compared with quantitative-polymerase chain reaction (PCR). The results showed that only SRg3 inhibited the AQP1 water flux and inhibited the proliferation of MDA-MB-231 (100 μM), due to cell cycle arrest at G0/G1. SRg3 inhibited the chemoattractant-induced migration of MDA-MB-231. The AQP1 expression in MDA-MB-231 was higher than in HCC1143 or DU4475 cell lines. These results suggest a role for AQP1 in the proliferation and chemoattractant-induced migration of this cell line. Compared to SRg3, RRg3 had more potency and efficacy, inhibiting the migration and invasion of MDA-MB-231. Rg3 has stereoselective anti-cancer effects in the AQP1 high-expressing cell line MDA-MB-231.

Keywords: Ginsenoside Rg3; breast cancer; epimer; stereoselective; triple negative breast cancer.

Figure 1

(A) The role of aquaporin 1 (AQP1) in cell migration and invasion (as reviewed in [39]). AQPs are redistributed to the leading edge of the migrating cell, leading water, ions and gases inside the cell; hence, along with changes in actin polymerisation, they play a role in the forward movement of the cell. AQP1 is a tetramer. Water passes through the pore of each monomer, and ions and gases pass through the central pore of the tetramer. (B) Top view of an AQP1 monomer, being blocked with Rg3, the black structure, (C) Side view of an AQP1 monomer, blocked with Rg3, and (D) H-bonding between Rg3 and Gly 65.

Figure 2

(A) Double swelling assay showing the swelling rates for the first and the second swelling on a single oocyte. Eight oocytes per treatment were measured for swelling in a hypotonic medium, before and 2 h after exposure to a vehicle or epimers of Rg3. The results were analysed and presented with a linear regression. (B) The AQP1 transcript expression in MDA-MB-231, HCC1143 and DU4475 cell lines. Each data point represents a mean ± SD value of 3 replicates, and comparisons were made with the vehicle control group (**** p < 0.0001).

Figure 3

Effect of SRg3 and RRg3 on the proliferation of MDA-MB-231, HCC1143 and DU4475 triple negative breast cancer cell lines, after 3 days, with (A) a crystal violet assay on the adherent cell lines and (B) an MTS assay. Only 100 µM of SRg3 showed an inhibition of proliferation of MDA-MB-231 in both assays, indicating the cell line selectivity and stereoselective effect of Rg3 for the inhibition of proliferation. Each data point represents a mean ± SD value of 6 replicates, and comparisons are made with the vehicle control group (**** p < 0.0001).

Figure 4

The effect of SRg3 on apoptosis and cell cycle arrest in the MDA-MB-231 cell line. (A) Total apoptotic cells (%) induced by vehicle or 100 µM SRg3 after 3 days of exposure; (B) Scatter plots of untreated cells or the cells treated with vehicle or SRg3. The left lower quadrant, right lower quadrant, right upper quadrant and left upper quadrant indicate viable cells, early apoptotic cells, late apoptotic cells and necrotic cells, respectively. (C) Cell population (%) in each of the G0/G1, S and G2/M phases of the cell cycle. (D) Histograms of the untreated, vehicle and SRG3-treated cells, following staining with PI. The violet, yellow and green curves represent events in the G0/G1, S and G2/M phases, respectively. The data presented is representative of 3 repeats.

Figure 5

Migration assays on the MDA-MB-231 cell line following exposure to RRg3 and SRg3. (A) The percentage of wound closure following exposure of the MDA-MB-231 cell line to RRg3 and SRg3. Data is presented as mean ± SD of 6 repeats, and comparisons are made with the vehicle control group (** p = 0.001). (B) The transwell migration assay on the MDA-MB-231 cell line following exposure to RRg3 and SRg3. Data are presented as mean ± SD of 3 replicates, and comparisons are made with the vehicle control group (**** p < 0.0001).

Figure 6

Spheroid invasion assay on the MDA-MB-231 cell line following exposure to RRg3 and SRg3. (A) The percentage of increase in the area, as an indicator of invasion to the extracellular matrix, following exposure of the MDA-MB-231 spheroids to RRg3 and SRg3. Data are presented as mean ± SD of 3 replicates, and comparisons are made with the vehicle control group (***) p = 0.0001. (B) Representative images of the vehicle control and RRg3-treated spheroids after 7 days, indicating the inhibition of spheroid invasion in the RRg3-treated spheroids.