Taxol and discodermolide represent a synergistic drug combination in human carcinoma cell lines.

Recently, three natural products have been identified, the epothilones, eleutherobin, and discodermolide, whose mechanism of action is similar to that of Taxol in that they stabilize microtubules and block cells in the mitotic phase of the cell cycle. In this report, we have compared and contrasted the effects of these new agents in Taxol-sensitive and -resistant cell lines. We also have taken advantage of a human lung carcinoma cell line, A549-T12, that was isolated as a Taxol-resistant cell line and found to require low concentrations of Taxol (2-6 nM) for normal cell division. This study then examined the ability of these new compounds to substitute for Taxol in sustaining the growth of A549-T12 cells. Immunofluorescence and flow cytometry have both indicated that the epothilones and eleutherobin, but not discodermolide, can substitute for Taxol in this Taxol-dependent cell line. In A549-T12 cells, the presence of Taxol significantly amplified the cytotoxicity of discodermolide, and this phenomenon was not observed in combinations of Taxol with either the epothilones or eleutherobin. Median effect analysis using the combination index method revealed a schedule-independent synergistic interaction between Taxol and discodermolide in four human carcinoma cell lines, an effect that was not observed between Taxol and epothilone B. Flow cytometry revealed that concurrent exposure of A549 cells to Taxol and discodermolide at doses that do not induce mitotic arrest caused an increase in the hypodiploid population, thereby indicating that a possible mechanism for the observed synergy is the potentiation of apoptosis. Our results suggest that Taxol and discodermolide may constitute a promising chemotherapeutic combination.

Taxol-resistant epithelial ovarian tumors are associated with altered expression of specific beta-tubulin isotypes.

The treatment of advanced ovarian cancer with taxol is hindered by the development of drug resistance. The cellular target for taxol is the microtubule that is stabilized by the drug. Taxol preferentially binds to the beta subunit of tubulin of which there are six distinct isotypes in mammalian cells. We have used highly specific oligonucleotides and polymerase chain reaction to analyze expression of all six beta-tubulin genes. Human lung cancer cells (A549) were selected in 12 and 24 nM taxol resulting in cell lines that were 9- and 17-fold resistant, respectively. These cells displayed an altered ratio of classes I, II, III, and IVa beta-tubulin isotypes. Ovarian tumors, seven untreated primary and four taxol- resistant tumor-bearing ascites, displayed significant increases (P < 0.005) in classes I (3.6-fold), III (4.4-fold), and IVa (7.6-fold) isotypes in the taxol-resistant samples as compared with untreated primary ovarian tumors. The increased expression appears to be related to the resistance phenotype, as the basal levels of the class III and IVa isotypes in the untreated tumors were extremely low. This is the first report of altered expression of specific beta-tubulin genes in taxol-resistant ovarian tumors and we propose that the latter may play a role in clinical resistance to taxol.

Development of two radioimmunoassays to detect paclitaxel in sera and in cerebrospinal, ascitic, and pleural fluids.

BACKGROUND: Paclitaxel is an antimitotic agent isolated from the Pacific yew tree. It has demonstrated antitumor activity in several cancers and is the first of a new class of antineoplastic agents containing a taxane ring system. Its levels in serum and urine have been measured previously by high performance liquid chromatography (HPLC). In this study, the authors developed two competitive radioimmunoassay methods to determine whether they could reliably be used to measure levels of paclitaxel in sera and in cerebrospinal, ascitic, and pleural fluids. METHODS: A monoclonal antibody prepared against paclitaxel was employed in an immunoradiometric assay (IRMA), in which 125I-labeled antibody was used, and in a more conventional tritiated radioimmunoassay (RIA),in which 3H-paclitaxel was used. RESULTS: Both radioimmunoassays detected levels of paclitaxel in sera that were comparable to those observed with HPLC. However, the IRMA was the most sensitive. Only the IRMA was able to detect low levels of paclitaxel in cerebrospinal fluid after paclitaxel infusion and in sera 3 weeks after infusion. Both the IRMA and RIA methods were able to detect paclitaxel in ascitic and pleural fluids. CONCLUSIONS: Monitoring paclitaxel levels reliably in sera and other bodily fluids is possible with these radioimmunoassays and may be of value in predicting and preventing toxicity and optimizing paclitaxel treatments.

Altered expression of M beta 2, the class II beta-tubulin isotype, in a murine J774.2 cell line with a high level of taxol resistance.

A series of taxol- and taxotere-resistant J774.2 cell lines has been characterized with respect to altered expression of beta-tubulin, the cellular target for these drugs. Vertebrates have six classes of beta-tubulin isotypes, each displaying a distinct pattern of expression. Although the functional significance of multiple beta-tubulins has not been fully defined, there is evidence that the individual isotypes contribute to differences in microtubule dynamics and drug binding. To determine if alterations in the expression of beta-tubulin isotypes play a role in taxol resistance, a PCR-based methodology was developed that permits highly specific amplification of each of the six known murine beta-tubulin isotypes. Two isotypes, M beta 5 and M beta 3, were expressed abundantly in the drug-sensitive parental J774.2 cells. Although expressed at an extremely low level in the parental cells, expression of the M beta 2 isotype was increased 21-fold (< 0.005) in the cell line most resistant to taxol. These findings suggest that a cell can alter its relative tubulin isotype composition in response to an external stress and specifically imply that altered expression of M beta 2, the class II beta-tubulin isotype, may contribute to the development of high resistance to taxol.