Abstract 188: Role of ER calcium regulation in dosage and duration-related cytotoxic efficacy of Taxol in breast cancer cells
Authors: Zhi Pan, Lauren Gollahon
Journal: Cancer Research (2011)

Abstract The exact mechanism by which Taxol, a widely-used anticancer agent, induces apoptosis remains unclear. Our previous studies showed that high dose Taxol (10-6 M) induced apoptosis in breast cancer cells through an ER calcium-dependent mechanism whereas a lower dosage (10-7 M) used an ER calcium-independent mechanism. Both mechanisms induced significant apoptosis from 12h exposure on, peaking at 48h exposure. However, the higher dosage caused immediate calcium release from the ER store, resulting in exposure time-dependent ER calcium depletion; contributing to the initial apoptotic events induced by Taxol. This dose and duration-related calcium regulation in Taxol-induced apoptosis was further investigated. A range of Taxol dosages (10-9 M to 10-5 M) were analyzed in three breast cancer cell lines (MDA-MB-468, MDA-MB-231, MCF-7) after 1h Taxol exposure. Cells samples were collected before Taxol exposure, after 1h Taxol exposure, and from 3h to 72h following 1h Taxol exposure. Cells were pretreated with BAPTA-AM (cytosolic calcium chelator) and 2-APB (ER calcium release inhibitor) to determine the relationship between Taxol-induced calcium signals and Taxol cytotoxicity. Fluo4-AM and the D1ER cameleon were used to measure calcium. Annexin V-FITC and Propidium Iodide (PI) were used to measure apoptosis in living cells by flow cytometry. Microtubule changes were detected by immunocytochemistry. Calcium sensors and transporters were detected by western blot. Results showed that although Taxol did not induce significant apoptosis at the end of 1h exposure, it did induce significant apoptosis in cell samples collected 12-48h after exposure, depending on the dosage. Cell cycle analysis showed a similar result, e.g. 1h Taxol treatment effectively induced a significant mitotic arrest several hours later, depending on the dosage. This result suggested that some cellular changes induced by Taxol during 1h exposure could act as important cellular signals, inducing long-term effects contributing to apoptosis. Whether ER calcium changes induced by Taxol act as this signal, and possible targets activated to induce apoptosis, are currently being analyzed. In summary, our results show that Taxol, at different dosages, induces significant cytotoxicity 12- 48h after the end of short-term (1h) treatment. Additionally, there is no substantial difference between apoptosis levels after continuous 12h Taxol exposure and apoptosis levels 12h after short-term Taxol exposure. Our calcium studies suggest the immediate ER calcium release by Taxol may act as an early signal for subsequent apoptosis without the need of a constant supply of Taxol. This new finding provides important information about the role of calcium in dosage and duration-related Taxol antitumor activity, which helps to elucidate the exact mechanism of Taxol action with potential implications for revising treatment strategies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 188. doi:10.1158/1538-7445.AM2011-188