The apoptotic effect of D Rhamnose β-hederin, a novel oleanane-type triterpenoid saponin on breast cancer cells

Abstract

There is growing interest in development of natural products as anti-cancer and chemopreventive agents. Many triterpenoids have been proved as potential agents for chemoprevention and therapy of breast cancer. Ginsenosides from ginseng, which mostly belong to dammarane-type triterpenoids, have gained great attention for their anti-breast cancer activity with diverse mechanisms. However, studies of other kinds of triterpenoid saponins on breast cancer are limited. Previously, we purified and identified a novel oleanane-type triterpene saponin named D Rhamnose β-hederin (DRβ-H) from Clematis ganpiniana, a Chinese traditional anti-tumor herb. In the present study, DRβ-H showed strong inhibitory activity on the growth of various breast cancer cells and induced apoptosis in these cells. DRβ-H inhibited PI3K/AKT and activated ERK signaling pathway. PI3K inhibitor LY294002 synergistically enhanced DRβ-H-induced apoptosis whereas MEK inhibitor U0126 reduced the apoptosis rate. Moreover, DRβ-H regulated the ratio of pro-apoptotic and anti-apoptotic Bcl-2 family proteins. Furthermore, DRβ-H induced depolarization of mitochondrial membrane potential which released Apaf-1 and Cytochrome C from the inter membrane space into the cytosol, where they promoted caspase-9 and caspase-3 activation. This is the first report on the pro-apoptotic effects of DRβ-H, a novel oleanane-type triterpenoid saponin, on breast cancer cells and its comprehensive apoptosis pathways. It implied that oleanane-type triterpenoid saponin DRβ-H could be a promising candidate for chemotherapy of breast cancer.

Figure 1. DRβ-H inhibited growth of various breast cancer cells.

(A) MTT assay of MCF-7, MDA-MB-231, BT474 and SUM1315 cells treated by DRβ-H. (B, C) Colony formation assay of MCF-7 and MDA-MB-231 cells following treatment with DRβ-H for two weeks. (D, E) Colony numbers of three independent experiments were shown in column statistics. Data are mean±standard error of mean (SEM) of three independent experiments. *p<0.05, **p<0.01, ***p<0.001 vs. DRβ-H -untreated group. The # shows that the two groups had statistically significant difference.

Figure 2. DRβ-H induced apoptosis in breast cancer cells.

(A, B) The apoptosis rate of MCF-7 and MDA-MB-231 cells measured by flow cytometry. DRβ-H induced early apoptosis in MCF-7 and MDA-MB-231 cells in a dose-dependent manner. (C, D) Early apoptosis cells of three independent experiments were shown in column statistics. Data are mean±SEM of three independent experiments. *p<0.05, **p<0.01, ***p<0.001 vs. DRβ-H-untreated group. The # shows that the two groups had statistically significant difference.

Figure 3. DRβ-H induced mitochondria-mediated apoptosis of MCF-7 and MDA-MB-231 cells.

(A) Effects of DRβ-H on the mitochondrial depolarization in MCF-7 and MDA-MB-231 cells. After the application of DRβ-H, JC-1 fluorescence shifted from red-orange to greenish yellow, which indicated the depolarization of mitochondrial membrane potential. (B) Effect of DRβ-H on Apaf-1 and Cytochrome C release. Cyto C: Cytochorme C. (C) Effect of DRβ-H on caspase-3 and caspase-9 activation. DRβ-H increased the activity of caspase-3 and caspase-9 in both MCF-7 and MDA-MB-231 cells. This activation could be reversed by the caspase inhibitors respectively. (D) Effect of DRβ-H on the expression of Bax, Bak, Bad, Bcl-xL, Mcl-1 and Bcl-2. Data are mean±SEM of three independent experiments. *p<0.05, **p<0.01, ***p<0.001 vs. DRβ-H-untreated group.

Figure 4. DRβ-H induced apoptosis of MCF-7 and MDA-MB-231 cells by inhibiting PI3K/AKT signaling pathway.

(A) Effect of DRβ-H on the phosphorylation of PI3K, PDK1, and AKT in MCF-7 and MDA-MB-231 cells. (B) LY294002 enhanced the DRβ-H-induced inhibition of p-AKT. (C, D) LY294002 markedly increased the DRβ-H-induced apoptosis rate in MCF-7 and MDA-MB-231 cells. (E, F) Early apoptosis rate of cells treated with DRβ-H and/or LY294002 of three independent experiments were shown in column statistics. LY: LY294002. Data are mean±SEM of three independent experiments. *p<0.05, **p<0.01, ***p<0.001 vs. DRβ-H-treated alone group.

Figure 5. DRβ-H induced apoptosis of MCF-7 and MDA-MB-231 cells by activating ERK.

(A) Effect of DRβ-H on the phosphorylation of ERK, P38 and JNK in MCF-7 and MDA-MB-231 cells. (B) U0126 reduced the DRβ-H-induced activation of p-ERK. (C, D) U0126 significantly reduced the DRβ-H–induced apoptosis rate in MCF-7 and MDA-MB-231 cells. (E, F) Early apoptosis rate of cells treated with DRβ-H and/or U0126 of three independent experiments were shown in column statistics. Data are mean±SEM of three independent experiments. *p<0.05, **p<0.01, ***p<0.001 vs. DRβ-H-treated alone group.