Elsevier

Biochemical Pharmacology

Volume 161, March 2019, Pages 149-162
Biochemical Pharmacology

Betulinic acid suppresses breast cancer aerobic glycolysis via caveolin-1/NF-κB/c-Myc pathway

https://doi.org/10.1016/j.bcp.2019.01.016Get rights and content

Abstract

Emerging evidence has suggested that targeting glycolysis may be a promising strategy for cancer treatment. Betulinic acid (BA) is a natural pentacyclic terpene that has been reported to be active in inhibiting various malignancies. Here, we showed that BA could inhibit aerobic glycolysis activity in breast cancer cell lines MCF-7 and MDA-MB-231 by hampering lactate production, glucose uptake and extracellular acidification rate (ECAR), as well as suppressing aerobic glycolysis-related proteins including c-Myc, lactate dehydrogenase A (LDH-A) and p-PDK1/PDK1 (pyruvate dehydrogenase kinase 1). Mechanistic studies validated Caveolin-1 (Cav-1) as one of key targets of BA in suppressing aerobic glycolysis, as BA administration resulted in Cav-1 upregulation, whereas silencing Cav-1 abrogated the inhibitory effect of BA on aerobic glycolysis. Further investigations demonstrated that BA suppressed aerobic glycolysis in breast cancer cells by regulating the Cav-1/NF-κB/c-Myc pathway. More meaningfully, BA significantly inhibited breast cancer growth and glycolytic activity in both the transgenic MMTV-PyVT+/− breast cancer spontaneous model and the zebrafish breast cancer xenotransplantation model without any detectable side effects in vivo. Taken together, our study sheds novel insights into BA as a promising candidate drug for suppressing aerobic glycolysis, highlighting Cav-1 as a potential molecular target of BA and aerobic glycolysis regulation.

Introduction

Breast cancer is a serious threat to women’s health with high morbidity and mortality. Approximately 12.5% of women will develop breast cancer throughout their lifetime, and the age of onset is trending younger worldwide [1]. Although a number of cytotoxic drugs have been developed for breast cancer treatment such as anthraquinones, alkaloids, alkylating agents and antimetabolites, multi-drug resistance and secondary metastasis still remain major obstacles faced by oncologists [2], [3].

Aberrant metabolism is considered as one of the most important hallmarks of cancer. Cancer cells preferentially utilized glycolysis as the primary metabolic mode even in the presence of oxygen and this phenomenon was termed as Warburg effect or aerobic glycolysis [4], [5]. Currently, aerobic glycolysis has been successfully utilized for monitoring cancer recurrence or metastasis by fluorodeoxyglucose positron emission tomography (FDG-PET) [6]. Meanwhile, multiple enzymes in the glycolysis pathway have been identified as promising targets for cancer therapy and drug discovery. For example, LDH-A knockdown was demonstrated to re-sensitize taxol-resistant breast cancer cells to chemotherapy [7]. PDK1 phosphorylation was reported to promote the Warburg effect and tumor growth [8]. Hexokinase II inhibitor 2-deoxy-D-glucose (2-DG) or 3-bromopyruvate (3-BrPA) was efficient in inhibiting oncogenesis or cancer growth in various malignancies including breast cancer [9]. However, the intrinsic mechanisms of aerobic glycolysis are still far away from fully elucidation, and thus, the identification of initial oncogenic signaling is critical for the development of glycolysis inhibition strategies.

Cav-1 is the basic constituent protein of specialized plasma membrane invaginations called caveolae. Recent studies have suggested that Cav-1 acts as a molecular hub in mediating breast cancer development and metabolism reprogramming [10]. Furthermore, Cav-1 is also closely associated with cancer clinical prognosis. For example, Cav-1 expression was shown to decrease in either mammary invasive lobular carcinomas or invasive ductal carcinomas [11]. Therefore, targeting Cav-1 may be a potential strategy for breast cancer prevention and therapy. In terms of mechanisms, current evidence has indicated that Cav-1 is involved in multiple vital biological regulations, including endocytosis, transcytosis, vesicular transport, autophagy, neovascularization and angiogenesis [12]. As a membrane scaffolding protein, co-localization and co-fractionation bioassays demonstrated that Cav-1 could interacted with many signal transduction proteins and therefore inhibit their catalytic activity [13]. Importantly, Cav-1 was also reported to serve as a docking site for multiple glycolytic enzymes. For instance, the rate-limiting glycolytic enzyme phosphofructose kinase (PFK) as well as aldolase were found to co-localize with Cav-1 by binding to its scaffolding domain [14]. It is interesting to explore the regulatory role of Cav-1 in glycolytic activity and discover its small-molecule targeted drugs.

Glycolysis inhibition has been considered as a novel strategy to control cancer growth, drug resistance, and metastasis [15]. However, the application of current available glycolysis inhibitors is greatly limited due to their systemic toxicity. Therefore, discovering low-cytotoxic phytochemicals that target glycolysis is of great significance for cancer prevention and therapy. Betulinic acid (BA) is a natural pentacyclic terpene isolated from birch bark and has been reported to effectively inhibit various malignancies including breast cancer [16]. For example, BA could inhibit ER-negative breast cancer growth in vitro and in vivo by modulating transcription factor Sp1 and miRNA-27a [17]. Meanwhile, BA was capable of interacting with selective estrogen receptor modulators to target ERα in breast cancer [18]. BA was also reported to impair metastasis and reduce immunosuppressive cells in breast cancer models [19]. Notably, one of the most striking features of BA is its selective killing effect on cancer cells. BA didn’t induce cytotoxic effects on normal cells in vitro, and its systemic side effects were also not evident in vivo [20]. Therefore, BA has attracted increasing attentions worldwide with the potentiality to be developed into a promising anti-cancer drug. With regard to metabolism regulation, a recent study indicated that BA could induce metabolic reprogramming in mouse embryonic fibroblasts by activating AMPK [21]. However, until now, few studies have been conducted to validate the glycolysis regulatory effect of BA and its influence on Cav-1.

Here, we provided evidence that BA could inhibit aerobic glycolysis activity in breast cancer via modulating the Cav-1/NF-κB/c-Myc pathway. More importantly, BA significantly delayed breast cancer growth in both the transgenic MMTV-PyVT+/− breast cancer spontaneous model and the zebrafish xenotransplantation model via Cav-1-medicated glycolysis inhibition. Our study not only sheds novel insights into BA as a promising candidate drug for aerobic glycolysis inhibition but also uncovers the novel function and molecular mechanism of Cav-1 in controlling breast cancer glycolysis.

Section snippets

Cell culture

Non-malignant mammary epithelial cell line MCF-10A and breast cancer cell lines MDA-MB-231 and MCF-7 were obtained from the American Type Culture Collection (ATCC, Maryland, USA). The identities of all the above cell lines have been authenticated by short tandem repeat profiling. MCF-10A cells were cultured in DMEM/F12 medium supplemented with 5% horse serum, 1% penicillin and streptomycin (Gibco, Grand Island, NY, USA), 20 ng/ml recombinant human epidermal growth factor (EGF), 0.5 μg/ml

BA inhibits breast cancer growth and triggers apoptosis

To confirm the inhibitory effects of BA on the viability of breast cancer cell lines MCF-7 and MDA-MB-231, CCK-8 assay was performed. Cells were treated with different concentrations of BA for 12 h, 24 h, 48 h, or 72 h. Both MCF-7 and MDA-MB-231 cells exhibited a time- and dose-dependent decline in viability after BA treatment. The half maximal inhibitory concentration (IC50) of BA was 19.06 μM in MCF-7 cells and 48.55 μM in MDA-MB-231 cells when they were treated with BA for 48 h (Fig. 1A).

Discussion

The switch from oxidative phosphorylation to glycolysis provides cancer cells enough energy to meet their increased consumption and biosynthetic demands. In recent years, glycolytic inhibition has attracted increasing attentions worldwide for cancer prevention and targeted therapy [26]. Understanding the molecular mechanism of aerobic glycolysis is of great value for identifying novel therapeutic targets for cancer treatment. A number of glycolysis-related proteins, such as glucose transporter

Acknowledgements

This work was supported by the National Natural Science Foundation of China (81703749, 81703764, 81573651 and 81873306), Guangdong Science and Technology Department (2016A030306025), Guangzhou Science, Technology and Innovation Commission (805296345055), Pearl River S&T Nova Program of Guangzhou (201506010098), Combined Scientific Project Funded by Guangdong Provincial Science and Technology Agency and Guangdong Provincial Academy of Traditional Chinese Medicine (2014A020221047), the Natural

Conflicts of interest

The authors declare no conflicts of interest.

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    Lin Jiao and Shengqi Wang contributed equally to this work.

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