Taxol Directly Induces Endoplasmic Reticulum-Associated Calcium Changes That Promote Apoptosis in Breast Cancer Cells

Abstract

Calcium, a key regulator of cell survival, is also important in regulating apoptosis. Although the chemotherapeutic agent Taxol employs apoptosis to induce cell death, the exact mechanism of how it induces apoptosis and the role of calcium in this process remains unclear. The main intracellular calcium storehouse, the endoplasmic reticulum, was identified as a new important gateway in apoptosis, possibly providing a target for Taxol. The goal of this study was to investigate whether calcium changes associated with the endoplasmic reticulum, were directly or indirectly generated by Taxol at clinically relevant doses, and related to Taxol-induced apoptosis in breast cancer cells. Time-lapsed imaging techniques followed by an endoplasmic reticulum-targeted construct, cameleon D1ER, were used to monitor cytosol--endoplasmic reticulum calcium dynamics in MDA-MB-468 (Bcl-2 negative) and MCF 7 (Bcl-2 positive) breast carcinoma cells. Apoptosis levels were measured with Annexin V and Propidium Iodide (PI) using flow cytometry. In both cell lines, Taxol at 2.5μM (∼10(-6) M) was observed to induce significant internal calcium changes, first a rapid endoplasmic reticulum calcium release and a transient cytosolic calcium increase upon Taxol addition. After several hours of Taxol treatment, the endoplasmic reticulum calcium store was gradually depleted, and a sustained cytosolic calcium elevation was observed before significant induction of apoptosis. Inhibition of these calcium changes decreased Taxol-induced apoptosis levels. In contrast, 0.2μM Taxol (∼10(-7)M) induced only a slight cellular calcium change, not enough to regulate apoptosis. Our findings demonstrate that endoplasmic reticulum calcium stores provide a direct target for Taxol action and are important for induction of apoptosis, independent of Bcl-2 status. Furthermore, our results show for the first time, that the role of calcium in Taxol-induced endoplasmic reticulum-mediated apoptosis is dependent on Taxol dosage.