Paclitaxel in combination with AT-101 induces apoptosis via supressing Bcl-2, bcl-XL, mcl-1 proteins in human breast cancer cells.

Abstract

e13578 Background: Paclitaxel, a microtubule stabilizing agent, has been a standard of care for breast cancer. AT-101, is an -/- enantiomer of gossypol, inhibits the Bcl-2 family proteins which contain BH3 domain. We reported previously that Paclitaxel in combination with AT-101 showed strong synergistic cytotoxic and apoptotic effects in human breast cancer cells. In this study, to elucidate the molecular mechanisms involved in the apoptotic effect of AT-101/Paclitaxel combination treatment in breast cancer cells, we investigated the possible roles of anti-apoptotic Bcl-2, Bcl-XL and Mcl-1 proteins which contain BH3 domain. Involvement of caspase 3 and 7 activation was also investigated. Methods: Human breast cancer cells were treated with increasing concentrations of drugs alone or with the synergistic combination doses of AT-101 and Paclitaxel. Cell Death Detection Elisa Plus Kit (Roche) was used to detect apoptosis. Caspase 3/7 activity was evaluated by Caspase-Glo 3/7 (Promega, Madison, WI) kit. Changes in the mRNA levels of Bcl-2, Bcl-XL and Mcl-1 genes were evaluated by qRT-PCR. Expression levels of these proteins were also investigated by Western blot analysis. Results: Combined treatment was shown to have strong synergistic apoptotic effects in MCF-7 and MDA- MB-231 human breast cancer cells. mRNA levels of Bcl-2, Bcl-XL and Mcl-1 molecules were reduced by the combination treatment in both cell lines. In parallel with mRNA levels, Bcl-2, Bcl-XL and Mcl-1 protein levels were significantly reduced after this novel drug combination. Combined treatment also induced caspase 3/7 activation in breast cancer cells. Conclusions: These preliminary data suggest that anti-apoptotic proteins such as Bcl-2, Bcl-XL and Mcl-1 may play important role in the underlying mechanistic rationale of apoptotic effect of AT-101/paclitaxel combination, while pro-apoptotic Bcl-2 related genes (Caspase-3 and Caspase-7) also seem to regulate this synergistic interaction.