Fulvestrant reverses doxorubicin resistance in multidrug-resistant breast cell lines independent of estrogen receptor expression

Abstract

Drug resistance, a major obstacle to successful cancer chemotherapy, frequently occurs in recurrent or metastatic breast cancer and results in poor clinical response. Fulvestrant is a new type of selective estrogen receptor (ER) downregulator and a promising endocrine therapy for breast cancer. In this study, we evaluated the combination treatment of fulvestrant and doxorubicin in ER-negative multidrug-resistant (MDR) breast cancer cell lines Bads‑200 and Bats‑72. Fulvestrant potentiated doxorubicin-induced cytotoxicity, apoptosis and G2/M arrest with upregulation of cyclin B1. It functioned as a substrate for P-glycoprotein (P-gp) without affecting its expression. Furthermore, fulvestrant not only restored the intracellular accumulation of doxorubicin but also relocalized it to the nuclei in Bats‑72 and Bads‑200 cells, which may be another potential mechanism of reversal of P-gp mediated doxorubicin resistance. These results indicated that the combination of fulvestrant and doxorubicin-based chemotherapy may be feasible and effective for patients with advanced breast cancer.

Figure 1.

Fulvestrant sensitizes doxorubicin-induced cytotoxicity in ER-negative MDR-positive cell lines. Cells were exposed to designated concentrations of doxorubicin with or without co-treatment of fulvestrant or P-gp modulators for 72 h, the cell viabilities were determined by MTT assays. (A) The reversal activity of 5 µM fulvestrant among BCap37, Bats-72 and Bads-200 cells compared with other P-gp modulators (5 µM verapamil and 5 µM tamoxifen). (B) The reversal effect of 5 µM fulvestrant on KB/KBv200 cells (known P-gp mediated resistance). (C) Potency of different fulvestrant concentrations (1 and 10 µM) in enhancing doxorubicin cytotoxicity in Bats-72, Bads-200 cells. *P<0.05 vs. DOX + ICI 1 µM. DOX, doxorubicin; ICI, fulvestrant; VRP, verapamil; TAM, tamoxifen; KBV, KBv200.

Figure 2.

Fulvestrant potentiates doxorubicin-induced apoptosis. BCap37, Bats-72 and Bads-200 cells treated with fulvestrant (5 µM), doxorubicin (1 or 5 µM) or their combination for 48 h. (A) Apoptotic cells by morphologic analysis. Cells were photographed at 400-fold magnification using a regular microscope. (B) Doxorubicin-induced apoptosis was quantified by Annexin V-FITC staining. BCap37, Bats-72 and Bads-200 cells were harvested and stained with Annexin V-FITC for flow cytometric analysis. The fractions of Annexin V-FITC positive cells indicate the ratios of apoptotic cells. CTL, control; ICI, fulvestrant; DOX, doxorubicin.

Figure 3.

Fulvestrant enhances doxorubicin-induced G2/M arrest and upregulation of cyclin B1. (A) Flow cytometric analysis of cell cycle distribution. BCap37, Bats-72 and Bads-200 cells treated with fulvestrant (5 µM), doxorubicin (0.1 µM) or their combination for 24 h were harvested and stained with propidium iodide DNA content analysis. The peaks corresponding to G0/G1 and G2/M phases of cell cycle are shown. (B) Western blot analyses for the cyclin B1, Cdc25c, Bcl-2 and P-gp. Whole-cell proteins were extracted from cells treated with fulvestrant (5 µM), doxorubicin (0.1 or 1 µM) or their combination for 24 h. Equal amounts (40 µg/lane) of cellular protein were analyzed. β-actin protein was blotted as a control. CTL, control; ICI, fulvestrant; DOX, doxorubicin.

Figure 4.

Function of fulvestrant as a substrate for transport by P-gp. 5 µM fulvestrant or 5 µM doxorubicin-treated P-gp reactions were performed according to the manufacturer's protocol. Luminescence was read on a luminometer. The decrease in the average relative light units of samples (∆RLU) represents fulvestrant or doxorubicin-stimulated P-gp ATPase activity. CTL, control; ICI, fulvestrant; DOX, doxorubicin.

Figure 5.

Fulvestrant alters intracellular doxorubicin distribution and accumulation. In vitro confocal images of doxorubicin fluorescence distribution in the nuclei and cytoplasm. Cells pretreated or non-pretreated with 5 µM fulvestrant were incubated with 5 µM doxorubicin for 0.5 and 2 h respectively. After cell nuclei was labeled with Hoechst-33342, fluorescent signals for doxorubicin (red) and Hoechst-33342 (blue) were observed by confocal microscopy at × 600 magnification. ICI, fulvestrant; DOX, doxorubicin.

Figure 6.

Fulvestrant alters intracellular doxorubicin uptake and efflux. Quantitation of doxorubicin uptake and efflux by flow cytometric analysis. Cells pretreated or non-pretreated with 5 µM fulvestrant were incubated with 5 µM doxorubicin for 2 h (uptake); After treated with the combination of doxorubicin and fulvestrant for 2 h, cells were washed three times with ice-cold PBS and then incubated with drug-free medium or medium with 5 µM fulvestrant for another 2 h (efflux). The intracellular fluorescence intensity of doxorubicin was measured by flow cytometry. The ratio of mean fluorescent intensity in BCap37 cells exposed to DOX for 2 h was 100%; *P<0.05 vs. DOX, **P<0.05 vs. medium. CTL, control, represents cells incubated without DOX; ICI, fulvestrant; DOX, doxorubicin.