D Rhamnose β-Hederin against human breast cancer by reducing tumor-derived exosomes

Abstract

D Rhamnose β-hederin (DRβ-H), a novel oleanane-type triterpenoid saponin isolated from the traditional Chinese medicinal plant Clematis ganpiniana, has been demonstrated to be effective against various types of tumor. However, the exact role of DRβ-H on breast cancer remains largely unresolved. In the present study, it was observed that DRβ-H exhibited anti-proliferative and pro-apoptotic activity in human breast cancer cells (MCF-7/S). DRβ-H was able to inhibit exosome secretion, and the level of exosomes was positively associated with cell growth after absorption and internalization by target breast cancer cells. By analyzing the miRNA profiles of exosomes and MCF-7/S, it was identified that several miRNAs were detected exclusively in exosomes. Knockdown of the top five exosomal miRNAs and an MCF-7/S proliferation assay indicated that exosomal miR-130a and miR-425 may enhance MCF-7/S cell viability. Target gene prediction and pathway analysis revealed the involvement of miR-130a and miR-425 in pathways associated with malignant cell proliferation. These results demonstrated that DRβ-H inhibited MCF-7/S cell growth through reducing exosome release.

Keywords: D Rhamnose β-Hederin; breast cancer; exosomes; microRNAs.

Figure 1.

Effects of DRβ-H on the proliferation and apoptosis of MCF-7/S cells. (A) The chemical structure of DRβ-H. (B) MTT assay of MCF-7/S treated by different concentrations of DRβ-H (0, 5, 10, 20, 40 and 80 µg/ml) for 24 h. DRβ-H inhibited cell proliferation in a dose-dependent manner. (C) MTT assay of MCF-7/S treated by 20 µg/ml DRβ-H for different times (0, 24, 48 and 72 h). DRβ-H inhibited cell proliferation in a time-dependent manner. (D) Flow cytometry analysis of MCF-7/S treated by different concentrations of DRβ-H (0, 10, 20 and 40 µg/ml) for 24 h. DRβ-H induced cell apoptosis in a dose-dependent manner. Data are presented as the mean ± the standard deviation of three independent experiments. *P<0.05 vs. DRβ-H-untreated group. The ** and ^# shows that the two groups had statistically significant difference (P<0.05). MCF-7/s, wild-type drug-sensitive MCF-7 breast cancer cells; DRβ-H, DRhamnose β-hederin.

Figure 2.

DRβ-H reducesexosomerelease in MCF-7/S cells. (A) Transmission electron microscopic image of exosomes isolated from MCF-7/S cells. Bar indicates 200 nm. (B) Exosome quantity of MCF-7/S treated by different concentrations of DRβ-H (0, 5, 10, 20, 40 and 80 µg/ml). DRβ-H decreased exosome secretion in a dose-dependent manner. (C) Western blotting for CD63 expression in exosomes after MCF-7/S were treated by different concentrations of DRβ-H (0, 20 and 80 µg/ml). Data are presented as the mean ± the standard deviation of three independent experiments. MCF-7/s, wild-type drug-sensitive MCF-7 breast cancer cells; DRβ-H, DRhamnose β-hederin.

Figure 3.

DRβ-H suppresses MCF-7/S growth by inhibiting exosome release. (A) Confocal laser microscopic images of GFP-S exposed to PKH26-stainedexosomes for 24 h; (a) green signal from established GFP-S cells; (b) red fluorescent signal from PKH26-labeled exosomes; (c) merged image of (a) and (b) presents the internalization of exosomes (white arrows) on the cell membranes and inside the cytoplasm of GFP-S. (B) Different concentrations of exosomes from 0–200 µg/ml were added into DRβ-H-treated MCF-7/S cells. Exosomes promoted cell proliferation in a dose-dependent manner. (C) Different concentrations of exosomesfrom 0–200 µg/ml were added into DRβ-H-treated MCF-7/S cells. Exosomes decreased cell apoptosis in a dose-dependent manner. Data are presented as the mean ± the standard deviation of three independent experiments. *P<0.05 vs. exosome-untreated group. MCF-7/s, wild-type drug-sensitive MCF-7 breast cancer cells; DRβ-H, DRhamnose β-hederin.

Figure 4.

DRβ-H decreases exosomal miRNAs to regulate MCF-7/S proliferation. (A) A scatter plot of miRNA signal intensity revealed that miRNA repertoires of exosomes were different from those of their parental cells. (B) Relative expressions of the top five exosomal miRNAs (miR-130a, miR-183, miR-20b, miR-25, and miR-425) following transfecting with the corresponding miRNA inhibitors. Negative control groups were transfected with the specific inhibitors, which have the same amount as the basic group but a different sequence from those miRNAs. (C) Effects of miRNA knockdown exosomes on MCF-7/S proliferation. Data are presented as the mean ± the standard deviation of three independent experiments. *P<0.05 vs. negative control group. MCF-7/s, wild-type drug-sensitive MCF-7 breast cancer cells; DRβ-H, DRhamnose β-hederin; miR, micro RNA.