Taxol: Mechanisms of action and resistance
Burkhart, Catherine Alyce
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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.