Cancer Letters

Cancer Letters

Volume 362, Issue 1, 28 June 2015, Pages 25-35
Cancer Letters

Original Articles
Selective targeting of FAK–Pyk2 axis by alpha-naphthoflavone abrogates doxorubicin resistance in breast cancer cells

https://doi.org/10.1016/j.canlet.2015.03.009Get rights and content

Highlights

  • Defined a new role for ANF in abrogating Dox resistance in breast cancer cells.

  • ANF decreases survival via inhibition of FAK–Pyk2 axis in Dox resistant cells.

  • ANF increases Dox retention through inhibition of P-gp pumps Dox resistant cells.

  • ANF has broad safety profile and inhibits growth of Dox-resistant cells in vivo.

  • ANF is an attractive anti-cancer drug in patients presenting with Dox resistance.

Abstract

Despite an initial positive response, breast cancer cells inevitably acquire resistance to doxorubicin (Dox). Alpha-naphthoflavone (ANF) is a well-known chemopreventive agent; however, its anti-cancer properties have not been established. We examined the therapeutic efficacy of ANF in doxorubicin-resistant MCF-7 (MCF-7/Dox) breast cancer cells and investigated its underlying molecular mechanisms of action. MCF-7/Dox cells expressed constitutively active forms of the tyrosine kinases: focal adhesion kinase (FAK-Y397) and protein tyrosine kinase 2 beta (Pyk2- Y579/580) compared with parental MCF-7 cells. ANF significantly enhanced the sensitivity of MCF-7/Dox cells to Dox cytotoxicity in vitro and when co-administered in vivo. This ANF-mediated chemosensitization has dual mechanisms of action: (a) intracellular Dox retention via suppression of P-glycoprotein pump activity, and (b) inhibition of clonogenic cell survival via de-phosphorylation of FAK, Pyk2, and EGF-induced Akt in MCF-7/Dox cells and tumor xenografts. Because of its strong chemosensitization action, broad safety profile, and health benefits, ANF is an attractive anti-cancer drug with therapeutic implications to circumvent drug resistance in breast cancer patients.

Introduction

Doxorubicin (Dox) is an FDA approved chemotherapeutic drug of the cell cycle non-specific class of drugs: the anthracycline antitumor antibiotics. Doxorubicin is commonly used to treat many types of cancer including leukemia, lymphoma, neuroblastoma, sarcoma, and Wilms' tumor as well as cancers of the lung, breast, stomach, ovary, thyroid, and bladder. In the treatment of breast cancer (BCa), Dox is highly effectively in early-stage as well as advanced-stage tumors, administered as single-drug or combination therapy [1], [2], [3], [4], [5]. However, over time, the cancer cells develop resistance to Dox which impairs treatment [6], [7]. Although the mechanisms leading to this resistance are not fully established, increased drug efflux via overexpression and increased activity of multidrug resistance pumps, such as P-glycoprotein (P-gp), are well known [8], [9], [10], [11], [12]. Unfortunately, drug efflux pump inhibitors like cyclosporin A, ketoconazole, and verapamil add to the toxic side effects associated with Dox treatment, thus decreasing the quality of life of cancer patients [13]. Therefore, Dox must be co-administered with a chemotherapeutic agent that abrogates Dox resistance and has no overlapping benefits or side effects.

Flavonoids are a group of polyphenolic phytochemical compounds that occur ubiquitously in foods of plant origin [14]. They are capable of modulating enzyme activity, inhibiting transporters and receptors, and regulating several signaling molecules in many different cell systems [15], [16]. Flavonoids have been classified as effective anti-microbial, anti-inflammatory, and anti-viral agents and in the past three decades have been effective against a wide range of diseases such as gastric ulcers, rheumatoid arthritis, diabetes mellitus, and cardiovascular diseases among others [17]. They have also been implicated in the inhibition of ABC transporters including breast cancer resistance protein (BCRP), P-gp, and multidrug resistance-associated protein 1 (MRP-1) [18], [19]. Flavonoids also exhibit protective effects against anthracycline-induced cardiotoxicity (very common in patients on a Dox regimen), have potent anxiolytic activity, and improve general health [20], [21], [22]. Alpha-naphthoflavone (ANF), a synthetic flavone, is also known as 7,8-benzoflavone. Historically, the role of ANF as a chemopreventive agent has been emphasized [23], [24]. ANF is also one of the most potent flavonoids for inhibiting BCRP mediated mitoxantrone efflux [18], [19]. Previous studies have outlined the role of ANF as a potent aromatase inhibitor and modulator of both aryl hydrocarbon receptor and cytochrome P450 (CYP450) enzymes [25], [26], [27], [28], [29], [30].

We have tested whether ANF can abrogate Dox resistance and enhance breast cancer cell response to treatment with Dox. We also investigated, for the first time, the molecular mechanisms and targets of ANF's action in breast cancer cells. As a flavone, we also expect that ANF may confer health benefits, protect against Dox-induced cardiotoxicity, and abate anxiety in patients undergoing chemotherapy; our study shows that ANF protects mice from cancer and Dox related weight loss. We have also shown that MCF-7/Dox cells overexpress P-gp and have endogenously high levels of phosphorylated FAK and Pyk-2 (tyrosine kinases implicated in inhibition of apoptosis and prevalence of metastatic tumors) and that ANF inhibits P-gp pump activity and selectively targets the FAK-Pyk-2 axis leading to abrogation of Dox resistance both in vitro and in vivo. Our results suggest that the combined use of ANF with a chemotherapy agent such as Dox may enhance the effectiveness of chemotherapy and confer health benefits in breast cancer patients.

Section snippets

Cell culture and drugs

The Doxorubicin-resistant MCF-7/Dox cells were a kind gift from Dr. Kapil Mehta (MD Anderson Cancer Center, TX). MCF-7/Dox cells and their parental MCF-7 cells (American Type Culture Collection, ATCC, Manassas, VA) were maintained in MEM (Gibco-BRL, Grand Island, NY) supplemented with 10% FBS, 0.01 mg/mL insulin, and 1% penicillin/streptomycin. Dox resistance was maintained via selective pressure by culturing MCF-7/Dox cells in a medium supplemented with 50 ng/mL of Dox (Sigma-Aldrich, St.

Determination of half maximal effective concentrations (EC50) of Dox and ANF in MCF-7/Dox cells

Time course (24, 48, or 72 h) dependent half maximal effective concentrations (EC50) of Dox and ANF in parental (MCF-7) and Dox resistant (MCF-7/Dox) cells are tabulated in Supplementary Table S1. The EC50 of Dox in MCF-7/Dox cells was 8.5 times higher than the parental MCF-7 cells at 72 h (Fig. 1A and B). In MCF-7/Dox cells, 5.884 µM Dox was required to induce 50% cell death compared with a much lower concentration of 0.6887 µM in parental MCF-7 cells at 72 h. In Fig. 1C and D, dose–response

Discussion

Resistance to chemotherapy is a major obstacle in the treatment of cancer. Investigating the underlying mechanisms of drug resistance continues to be an area of intense research targeted toward overcoming drug resistance, enhancing therapeutic effect, and minimizing single and combination therapy-associated side effects. Resistance to Dox in the clinic leads to poor prognosis and addition of chemotherapeutic drugs to the regimen leading to increased side effects and poor quality of life of the

Funding

This work was supported by the Departments of Pharmacology and Urology at Tulane University School of Medicine.

Conflict of interest

The authors declare no conflict of interest.

Acknowledgements

This manuscript is dedicated to the fond memory of Dr. Krishna C. Agrawal, our benevolent mentor. Dr. Agrawal, chairman of the Department of Pharmacology at Tulane University from 1999 to 2009, conceived the idea and initiated the project in his laboratory.

The authors thank Nancy Busija, M.A., SLP for editing the manuscript. The authors also thank the Pathology-Histology Laboratory and Department of Pathology, Tulane University Health Sciences Center for preparation of FFPE sections and target

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