Elsevier

Biomedicine & Pharmacotherapy

Volume 95, November 2017, Pages 18-24
Biomedicine & Pharmacotherapy

Original article
Berberine activates caspase-9/cytochrome c-mediated apoptosis to suppress triple-negative breast cancer cells in vitro and in vivo

https://doi.org/10.1016/j.biopha.2017.08.045Get rights and content

Abstract

Berberine (BBR) is an isoquinoline alkaloid isolated from Cotridis rhizoma and exhibits multiple biological roles including anti-microbe, anti-inflammation and anti-tumor activities. In this study, two triple-negative breast cancer cell (TNBC) lines, MDA-MB-231 and BT549, were used to investigate the effect of BBR on growth of TNBC in vitro and in vivo. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to evaluate the viability of cells treated with BBR. After 48 h treatments, a 50% inhibitory concentration (IC50) of BBR to BT549 and MDA-MB-231 cells are at 16.575 ± 1.219 μg/ml and 18.525 ± 6.139 μg/ml respectively. BBR reduced colony formation of BT549 and MDA-MB-231 cells. The wound-healing assay showed BBR decreased breast cancer cell migrations (P < 0.01). AnnexinV-PI staining assay confirmed BBR induced cellular apoptosis. The expressions of caspase-3, caspase-9, Bcl-2 and Bax were detected by western blot, which showed BBR activated caspase-3, 9 and Bax, but down-regulated Bcl-2 expression. BBR promoted the release of cytochrome c through the immunofluorescent analysis (P < 0.01). We also found BBR increased the level of cellular γH2AX and increased the expression of Ligase4, which suggests BBR induces the double-strand breaks (DSB). These results thus demonstrated that BBR induced DSB, subsequently increased the release of cytochrome c and eventually triggered the caspase9-dependent apoptosis. In addition, we used a MDA-MB-231 mouse-xenograftmodel to evaluate the effect of BBR on tumor growth. BBR suppressed tumor growth and increased caspase-9 levels in xenograft tumors through immunohistochemistry analysis (P < 0.01). Taken together, these results demonstrate that BBR activates caspase-9/cytochrome c-mediated apoptosis to inhibit the growth of TNBC breast cancer cells in vitro and in vivo.

Introduction

Breast cancer is one of the most malignant tumors in women, approximately 1,380,000 new cases were diagnosed across the globe, with approximately 458,000 deaths each year [1], [2]. Triple negative breast cancer (TNBC) is defined by the absence of estrogen receptor, progesterone receptor and epidermal growth receptor 2 (HER2) expression at immunohistochemical examination, which accounts for 15% of all breast cancer cases [3], [4], [5]. Given lack of standard molecular targets, the current viable targeted therapies are not useful for TNBC patients [6], [7], [8]. TNBC is insensitive to the anti-hormone receptor (HR) and HER2 targeted drugs and has higher incidence than HR and HER2-positive patients [9], [10], [11]. Traditional chemotherapy with DNA damage agents are available for TNBC treatment, thus, it is needed to seek for efficient and safe chemotherapeutic drugs to treat TNBC.

Berberine (BBR) is a natural isoquinoline alkaloid extracted from plants, such as berberis aquifolium, vulgaris, aristata, and tinospora cordifolia [12]. BBR exhibits multiple biologic effects with low toxicity, and the anti-tumor activities of BBR in various human cancer cells have been reported [13], [14], [15], [16], [17], [18], [19]. More importantly, BBR has been demonstrated to directly bind with DNA and interfere with DNA replication, which suggests BBR may be involved in DNA damage repair [20]. Reports showed that BBR radiosensitized lung cancer and esophageal cancer cells by regulation of DNA repair associated protein [21], [22]. As TNBC lacks of targets for targeted therapies, in this study, we investigate the potential effect of BBR as a DNA damage agent on suppressing the growth of TNBC.

We used two TNBC cell lines, BT549 and MDA-MB-231, to investigate the apoptotic effect of BBR to TNBC. We also detected the effect of BBR to caspase9-dependent apoptosis in mice with xenografted TNBC. Our finding indicates that BBR would be a promising drug to suppress TNBC in vivo.

Section snippets

Cell culture

The human breast cancer cell lines BT549 and MDA-MB-231 were obtained from American Type Culture Collection (ATCC, Manassas, VA). Cells were cultured in RPMI 1640 (Hyclone, Massachusetts, USA) and DMEM (Gibco, Massachusetts, USA) culture media supplemented with 10% heat-inactivated fetal bovine serum (FBS, Hyclone, Massachusetts,USA), 100 μg/ml penicillin, and 100 μg/ml streptomycin (Thermo Fisher Scientific, Massachusetts,USA).

Reagents and antibodies

BBR was purchased from Sigma (St. Louis, USA). Mytomycin C was

Berberine suppressed cell proliferation

We analyzed the effect of BBR on BT549 and MDA-MB-231 cell proliferation by MTT assay. After 48 h treatment, the growth curves and IC50 values were measured. The IC50 values of BBR to BT549 and MDA-MB-23 cells are at 16.575 ± 1.219 μg/ml and 18.525 ± 6.139 μg/ml. (Fig. 1A). MTT assay showed that 5 μg/ml BBR suppressed cell proliferation in a time-dependent manner (Fig. 1B). Thus, these data demonstrated that BBR inhibited the proliferation of BT549 and MDA-MB-231 human TNBC cells.

Berberine inhibited colony formation and cell migration

Colony formation assay

Discussion

In this study, we evaluated the effect of BBR on TNBC growth and the underlying mechanism of its action. Our results showed that BBR inhibited tumor cell proliferation, migration and induced cellular apoptosis. BBR promoted the release of cyto c from mitochondria to cytosol, increased the expression of caspase-3,9 and decreased the expression of Bcl-2, which suggests BBR induced mitochondria-mediated apoptosis. As the insight mechanisms of BBR in triggering apoptosis were not well known, we

Conflict of interest

The authors do not have any conflict of interest about this paper.

Acknowledgement

This work is supported by Chinese National Scientific Foundation (grants number 81201563, 31371254) to Weifeng Mao.

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    1

    These persons contribute equally.

    2

    Current address: Department of Pediatrics, Affiliated Hospital of Guangdong Medical University, Zhanjiang, 524001, China.

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