Estradiol impairs the antiproliferative and proapoptotic effect of Zoledronic acid in hormone sensitive breast cancer cells in vitro

Abstract

Background: Zoledronic acid (ZA) has antiresorptive effects and protects from bone metastasis in women with early breast cancer. In addition, in postmenopausal women with endocrine responsive breast cancer ZA prolongs DFS. The exact mechanism is still unclear. We have therefore investigated the effect of increasing concentrations of ZA in breast cancer cell lines in the absence or presence of estradiol to mimic the hormonal environment in vitro.

Materials and methods: Using assays for cell proliferation (EZ4U, BrdU) and cell death (Annexin/PI), we have analyzed the dose-dependent antiproliferative and pro-apoptotic effects of ZA in two hormone sensitive cell lines (MCF-7 and T47D) and a hormone insensitive, triple negative cell line (MDA-MB-231) in the presence of 0, 1 and 10 nM estradiol.

Results: In the absence of estradiol, ZA exerts dose-dependent antiproliferative and pro-apoptotic antitumor effects in both, hormone sensitive (MCF-7, T47D) and -insensitive (MDA-MB-231) breast cancer cell lines (p<0.0001). In the presence of estradiol, the antitumoral effect of ZA was significantly decreased only in the hormone sensitive MCF-7 and T47D cell lines (p = 0.0008 and p = 0.0008, respectively).

Conclusion: We have demonstrated that estradiol impairs the antiproliferative and proapoptotic effect of ZA in hormone sensitive, but not in hormone insensitive breast cancer cell lines. Our findings provide a possible explanation for the differential effect of ZA on DFS in pre- and postmenopausal patients with hormone sensitive early breast cancer, which has been demonstrated clinically. We further hypothesize that endocrine insensitive tumors such as triple negative breast cancer (TNBC) should benefit from ZA irrespective of their menopausal status.

Fig 1. Proliferative effect of estradiol.

Proliferative effect of 0, 1 and 10nM estradiol on the three cell lines MCF-7 (black), T74D (grey) and MDA-MD-231 (white) in the absence of ZA (EZ4U). Abbreviations: OD = optical density.

Fig 2. Dose-dependent antiproliferative effect of ZA.

Dose-dependent antiproliferative effect of ZA on the three cell lines in the absence (A) and the presence (B) of 1nM estradiol (EZ4U). Abbreviations: OD = optical density.

Fig 3. Antiproliferative effect of ZA in the absence or presence of different concentrations of estradiol.

Antiproliferative effect of rising concentrations of ZA (0, 6.25 and 12.5μM) in the three cell lines MCF-7 (A), T47D (B) and MDA-MB-231 (C) in the absence (black bars) or the presence of 1nM (grey bars) and 10nM (white bars) estradiol (EZ4U).

Fig 4. Effect of ZA on DNA synthesis.

Effect of rising concentrations of ZA on DNA synthesis in the three cell lines MCF-7 (A), T74D (B) and MDA-MB-231 (C) in the absence (blue bars) and the presence (red bars) of 1nM estradiol (BrdU).

Fig 5. Cell cycle analysis by DNA content.

Dose-dependent pro-apoptotic effect of ZA in the three cell lines MCF-7 (A), T47D (B), MDA-MB-231 (C) in the absence or the presence of 1nM estradiol shown by the Sub-G1-Peak.

Fig 6. Pro-apoptotic effect of ZA measured by Annexin/PI staining.

Pro-apoptotic effect of rising concentrations of ZA in the three cell lines MCF-7 (A), T47D (B), MDA-MB-231 (C) in the absence or the presence of 1nM estradiol measured by AnnexinV/PI staining.