Calcium regulates Taxol-induced apoptosis and resistance of the anti-apoptotic protein Bcl-2 in breast cancer cells

LB-50

Background and Objectives: Calcium (Ca²⁺), one of the key regulators of cell survival, is also important in regulating apoptosis. Although the chemotherapeutic agent Taxol employs apoptosis to induce cell death in breast cancer treatment, the exact mechanism of how it induces apoptosis and the role of Ca²⁺ in this process remain unclear. The endoplasmic reticulum (ER), as main intracellular Ca²⁺ store, is newly-recognized as an important gateway in apoptosis, and possibly provides a target for Taxol. This study investigated whether Ca²⁺ changes associated with the ER were generated and related to this apoptosis. This study further investigated the role of Ca²⁺ in the resistance of the anti-apoptotic protein Bcl-2 to Taxol-induced apoptosis.
 Study Design: A time-lapsed imaging technique was used to monitor Ca²⁺ dynamics in cultured cells. Fluorescent Ca²⁺ indicator Fluo4-AM was used to detect cytosolic Ca²⁺ levels. Cameleon D1ER, based on fluorescence resonance energy transfer (FRET), was used to specifically detect ER Ca²⁺ levels. MDA-MB-468, a Bcl-2 negative breast carcinoma cell line, and its Bcl-2 stable transfectant cell line, were used to evaluate Ca²⁺ changes upon Taxol treatment without or with the effect of Bcl-2, respectively. Taxol-induced apoptosis was measured using the Annexin V-FITC assay. The Ca²⁺ interfering agents BAPTA-AM and 2-APB were applied to evaluate whether Taxol-induced Ca²⁺ changes were related to this apoptotic event.
 Results: Our data showed the effect of Taxol on Ca²⁺ homeostasis depends upon dose and exposure time. Taxol, at a commonly-used dose (2.5 x10^-6 μM), induced a rapid ER Ca²⁺ release and resulted in a transient increase in cytosolic Ca²⁺ levels that peaked to 1.5 fold at about 4 min. A gradual ER Ca²⁺ depletion occurred within 3 h and contributed to the final sustained cytosolic Ca²⁺ increase after 12 h. Interfering with these Ca²⁺ changes caused a 40% decrease of the Taxol-induced apoptosis levels after 12 h, indicating that Ca²⁺ promotes Taxol-induced apoptosis. Taxol treatment on Bcl-2 stable transfectant cells overcame the inhibition of Bcl-2 on the ER Ca²⁺ and attenuated the resistance of Bcl-2 to apoptosis.
 Conclusion: This research characterized various Ca²⁺ changes induced by Taxol at commonly-used doses in breast cancer treatment after different exposure times. The results indicate: 1) Ca²⁺ is mobilized from the ER by Taxol, depending on dose and exposure time. 2) Taxol-induced Ca²⁺ changes promote apoptosis. 3) Anti-apoptotic protein Bcl-2 inhibits the basal ER Ca²⁺ homeostasis. 4) Taxol may attenuate Bcl-2’s resistance to apoptosis through up-regulating the ER Ca²⁺ release. These results suggest that Ca²⁺ plays an important regulatory role in Taxol-induced apoptosis and Bcl-2’s resistance to apoptosis. This information provides new insights into Taxol application and aid in the development of more effective breast cancer therapies.