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Tubulin from paclitaxel-resistant cells as a probe for novel antimicrotubule agents
D L Sackett1, P Giannakakou, M Poruchynsky
1Medicine Branch, National Cancer Institute, NIH, Bethesda, MD 20892, USA. dsackett@helix.nih.gov
Purpose:
Treatment with paclitaxel (PTX) can lead to the appearance of drug resistance with accompanying changes in tubulin. The purpose of this study was to develop an assay for microtubule-active agents that are able to circumvent changes in tubulin that result in acquired resistance to paclitaxel.
Methods:
The assay measured the promotion of microtubule polymerization when target agents were added to solutions containing tubulin purified from cultured cells. Tubulin was prepared from PTX-sensitive 1A9 ovarian carcinoma cells and from a PTX-resistant clone. Polymerization was monitored spectrophotometrically and validated by electron microscopy.
Results:
Exposure of tubulin isolated from PTX-sensitive 1A9 ovarian carcinoma cells to substoichiometric PTX resulted in polymerization equivalent to that observed with brain tubulin. In contrast, tubulin from a PTX-resistant 1A9 clone failed to polymerize under identical conditions. If a C-2-modified analog of PTX (2-debenzoyl-2-(m-azidobenzoyl)paclitaxel) was substituted for PTX in the same experiment, the tubulins from both sensitive and resistant cells polymerized as well as brain tubulin. As predicted from these results, the PTX analog was nearly as cytotoxic to the PTX-resistant cells as it was to the parental cells: the relative resistance of the resistant cells compared to the parental is only 3-5-fold for the PTX analog versus 25-30-fold for PTX.
Conclusion:
Polymerization of purified tubulin from the paclitaxel-resistant cells provided an assay for agents able to circumvent the tubulin alterations that result in acquired paclitaxel resistance.
Insights
This study developed an assay using paclitaxel-resistant cells to identify new microtubule-active agents. The assay successfully identified a paclitaxel analog that circumvents drug resistance by targeting tubulin alterations.
Area of Science:
- Oncology
- Cell Biology
- Pharmacology
Background:
- Paclitaxel (PTX) treatment can induce drug resistance through alterations in cellular tubulin.
- Acquired resistance to paclitaxel poses a significant challenge in cancer therapy.
- Developing agents that overcome PTX resistance is crucial for improving patient outcomes.
Purpose of the Study:
- To develop a novel assay for identifying microtubule-active agents capable of circumventing paclitaxel resistance.
- To investigate the role of tubulin alterations in paclitaxel resistance.
- To screen for novel paclitaxel analogs that retain efficacy against resistant cancer cells.
Main Methods:
- An assay was developed to measure microtubule polymerization induced by agents acting on tubulin.
- Tubulin was purified from both paclitaxel-sensitive and paclitaxel-resistant ovarian carcinoma cells.
- Microtubule polymerization was assessed spectrophotometrically and validated using electron microscopy.
Main Results:
- Tubulin from paclitaxel-sensitive cells polymerized with paclitaxel, while tubulin from resistant cells did not.
- A C-2 modified paclitaxel analog induced polymerization in tubulin from both sensitive and resistant cells.
- The paclitaxel analog demonstrated significantly reduced resistance in vitro compared to paclitaxel.
Conclusions:
- Assessing tubulin polymerization from resistant cells provides a method to identify agents overcoming paclitaxel resistance.
- Tubulin alterations are a key mechanism in acquired paclitaxel resistance.
- Modified paclitaxel analogs show promise in overcoming drug resistance in ovarian cancer.
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