Taxol: Mechanisms of action and resistance

Abstract

Taxol is an antitumor agent that has been approved for the treatment of ovarian, breast and lung carcinomas. The major cellular target for Taxol is beta-tubulin. Taxol stabilizes microtubules leading to a mitotic block and apoptosis. Structure-activity studies have demonstrated that the cytotoxicity of this drug correlates with its ability to stabilize microtubules. Taxol can also induce tumor necrosis factor-alpha (TNF-alpha) gene expression in murine macrophages. Since TNF-alpha can be cytotoxic to tumor cells, the ability of Taxol to induce TNF-alpha may contribute to its overall efficacy. To address this question, a structure-activity study was performed using a series of Taxol analogs with varying degrees of cytotoxicity. RAW 264.7 murine macrophage cells were treated with Taxol and related taxanes, and then analyzed for TNF-alpha gene expression by northern blot analysis and a TNF bioassay. This study demonstrated that their was no correlation between the induction of TNF-alpha and cytotoxicity. Taxotere, a drug with a similar structure to Taxol, was unable to induce TNF-alpha gene expression even though it has demonstrated excellent activity against human tumors. These data suggest that TNF-alpha production does not contribute to the overall efficacy of Taxol. One obstacle to the successful use of Taxol is the development of drug resistance. In mammalian cells, there are 6--7 beta-tubulin isotypes. Our laboratory has demonstrated a dramatic increase in Mbeta2 (class II beta-tubulin) in a highly Taxol-resistant cell line, J7-T1, suggesting that increased Mbeta2 may contribute to Taxol resistance. To address this question, the E3-Mbeta2-42.3.5 cell line, which stably expresses Mbeta2 under the control of an ecdysone-inducible expression system, was developed. RT-PCR analysis demonstrated that E3-Mbeta2--42.3.5 had 3--5.5-fold more Mbeta2 than the control cell lines. An ∼2--3.5-fold increase in class II protein was apparent by western blot and FACS analysis. The effects of Taxol on D-Mbeta2--42.3.5, as determined by cytotoxicity assays, flow cytometry, and immunofluorescence, were similar to the control cell lines, suggesting that increased expression of Mbeta2 is not a significant mechanism of Taxol resistance. The two studies presented here contribute to a better understanding of the mechanisms of action and of resistance to Taxol.