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

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.

背景
唑来膦酸（Zoledronic acid, ZA）具有抗骨吸收作用，可降低早期乳腺癌女性患者的骨转移风险。此外，对于内分泌敏感型乳腺癌的绝经后女性，ZA可延长其无病生存期（Disease-Free Survival, DFS）。其具体作用机制尚未阐明。因此本研究通过体外模拟激素环境，设置有无雌二醇的培养条件，探究不同浓度ZA对乳腺癌细胞系的作用。

材料与方法
本研究采用细胞增殖检测（EZ4U试剂盒、溴脱氧尿苷（BrdU））与细胞死亡检测（膜联蛋白V-碘化丙啶双染法（Annexin V/PI）），在0、1、10 nM雌二醇浓度条件下，分析ZA对两种激素敏感型细胞系（MCF-7、T47D）及一种激素不敏感型三阴性细胞系（MDA-MB-231）的剂量依赖性抗增殖与促凋亡作用。

结果
在未添加雌二醇的条件下，ZA对激素敏感型（MCF-7、T47D）与激素不敏感型（MDA-MB-231）乳腺癌细胞系均表现出剂量依赖性的抗增殖与促凋亡抗肿瘤活性（p<0.0001）。而在添加雌二醇的条件下，ZA的抗肿瘤活性仅在激素敏感型MCF-7与T47D细胞系中显著减弱（分别为p=0.0008、p=0.0008）。

结论
本研究证实，雌二醇可削弱ZA对激素敏感型乳腺癌细胞系的抗增殖与促凋亡作用，但对激素不敏感型细胞系无此影响。本研究结果可为临床已证实的ZA对激素敏感型早期乳腺癌绝经前、绝经后患者无病生存期产生差异化影响的现象提供潜在解释。本研究进一步推测，三阴性乳腺癌（Triple Negative Breast Cancer, TNBC）这类内分泌不敏感型肿瘤患者，无论绝经状态如何，均可从ZA治疗中获益。