Peripheral blood mononuclear and tumor cell pharmacodynamics of the novel epothilone B analogue, ixabepilone.

BACKGROUND: We previously demonstrated that peak microtubule bundle formation (MBF) in peripheral blood mononuclear cells (PBMCs) occurs at the end of drug infusion and correlates with drug pharmacokinetics (PK). In the current study, a new expanded evaluation of drug target effect was undertaken. PATIENTS AND METHODS: Patients with advanced solid malignancies were treated with ixabepilone 40 mg/m2 administered as a 1-h i.v. infusion every 3 weeks. Blood, plasma, and tumor tissue sampling was carried out to characterize pharmacodynamics and PK. RESULTS: Forty-seven patients were treated with 141 cycles of ixabepilone. In both PBMCs (n=27) and tumor cells (n=9), peak MBF occurred at the end of infusion; however, at 24-72 h after drug infusion, the number of cells with MBF was significantly greater in tumor cells, relative to PBMCs. A Hill model (EC50=109.65 ng/ml; r2=0.94) was fitted, which demonstrated a relationship between percentage of PBMCs with MBF and plasma ixabepilone concentration. The percentage of PBMCs with MBF at the end of infusion also correlated with severity of neutropenia (P=0.050). CONCLUSIONS: Plasma ixabepilone concentration and severity of neutropenia correlate with the level of MBF in PBMCs. Therefore, this technically straightforward assay should be considered as a complement to the clinical development of novel microtubule-binding agents.

Tumor targeting by covalent conjugation of a natural fatty acid to paclitaxel.

Certain natural fatty acids are taken up avidly by tumors for use as biochemical precursors and energy sources. We tested in mice the hypothesis that the conjugation of docosahexaenoic acid (DHA), a natural fatty acid, and an anticancer drug would create a new chemical entity that would target tumors and reduce toxicity to normal tissues. We synthesized DHA-paclitaxel, a 2'-O-acyl conjugate of the natural fatty acid DHA and paclitaxel. The data show that the conjugate possesses increased antitumor activity in mice when compared with paclitaxel. For example, paclitaxel at its optimum dose (20 mg/kg) caused neither complete nor partial regressions in any of 10 mice in a Madison 109 (M109) s.c. lung tumor model, whereas DHA-paclitaxel caused complete regressions that were sustained for 60 days in 4 of 10 mice at 60 mg/kg, 9 of 10 mice at 90 mg/kg, and 10 of 10 mice at the optimum dose of 120 mg/kg. The drug seems to be inactive as a cytotoxic agent until metabolized by cells to an active form. The conjugate is less toxic than paclitaxel, so that 4.4-fold higher molar doses can be delivered to mice. DHA-paclitaxel in rats has a 74-fold lower volume of distribution and a 94-fold lower clearance rate than paclitaxel, suggesting that the drug is primarily confined to the plasma compartment. DHA-paclitaxel is stable in plasma, and high concentrations are maintained in mouse plasma for long times. Tumor targeting of the conjugate was demonstrated by pharmacokinetic studies in M109 tumor-bearing mice, indicating an area under the drug concentration-time curve of DHA-paclitaxel in tumors that is 8-fold higher than paclitaxel at equimolar doses and 57-fold higher at equitoxic doses. At equimolar doses, the tumor area under the drug concentration-time curve of paclitaxel derived from i.v. DHA-paclitaxel is 6-fold higher than for paclitaxel derived from i.v. paclitaxel. Even at 2 weeks after treatment, 700 nM paclitaxel remains in the tumors after DHA-paclitaxel treatment. Low concentrations of DHA-paclitaxel or paclitaxel derived from DHA-paclitaxel accumulate in gastrocnemius muscle; which may be related to the finding that paclitaxel at 20 mg/kg caused hind limb paralysis in nude mice, whereas DHA-paclitaxel caused none, even at doses of 90 or 120 mg/kg. The dose-limiting toxicity in rats is myelosuppression, and, as in the mouse, little DHA-paclitaxel is converted to paclitaxel in plasma. Because DHA-paclitaxel remains in tumors for long times at high concentrations and is slowly converted to cytotoxic paclitaxel, DHA-paclitaxel may kill those slowly cycling or residual tumor cells that eventually come into cycle.

The relationship between Taxol and (+)-discodermolide: synthetic analogs and modeling studies.

BACKGROUND: During the past decade, Taxol has assumed an important role in cancer chemotherapy. The search for novel compounds with a mechanism of action similar to that of Taxol, but with greater efficacy particularly in Taxol-resistant cells, has led to the isolation of new natural products. One such compound, (+)-discodermolide, although structurally distinct from Taxol, has a similar ability to stabilize microtubules. In addition, (+)-discodermolide is active in Taxol-resistant cell lines that overexpress P-glycoprotein, the multidrug-resistant transporter. Interestingly, (+)-discodermolide demonstrates a profound enhancement of the initiation process of microtubule polymerization compared to Taxol. RESULTS: The synthesis of (+)-discodermolide analogs exploiting our highly efficient, triply convergent approach has permitted structure-activity relationship (SAR) studies. Small changes to the (+)-discodermolide structure resulted in a dramatic decrease in the ability of all four discodermolide analogs to initiate tubulin polymerization. Two of the analogs also demonstrated a decrease in total tubulin polymerization, while a change in the olefin geometry at the C8 position produced a significant decrease in cytotoxic activity. CONCLUSIONS: The availability of (+)-discodermolide and the analogs, and the resultant SAR analysis, have permitted an exploration of the similarities and differences between (+)-discodermolide and Taxol. Docking of the X-ray/solution structure of (+)-discodermolide into the Taxol binding site of beta-tubulin revealed two possible binding modes (models I and II). The preferred pharmacophore model (I), in which the C19 side chain of (+)-discodermolide matches with the C2 benzoyl group of Taxol and the delta-lactone ring of (+)-discodermolide overlays with the C13 side chain of Taxol, concurred with the results of the SAR analysis.

Multiple microtubule alterations are associated with Vinca alkaloid resistance in human leukemia cells.

Vinca alkaloids are used extensively in the treatment of childhood acute lymphoblastic leukemia (ALL) and despite their usefulness, drug resistance remains a serious clinical problem. Vinca alkaloids bind to the beta-tubulin subunit of the alpha/beta-tubulin heterodimer and inhibit polymerization of microtubules. Recent studies have implicated altered beta-tubulin isotype expression and mutations in resistance to microtubule-stabilizing agents. Microtubule-associated protein (MAP) MAP4 binds to and stabilizes microtubules, and increased expression is associated with decreased sensitivity to microtubule-depolymerizing agents. To address the significance of beta-tubulin and MAP4 alterations in childhood ALL, two CCRF-CEM-derived Vinca alkaloid resistant cell lines, VCR R (vincristine) and VLB100 (vinblastine), were examined. Decreased expression of class III beta-tubulin was detected in both VCR R and VLB100 cells. VCR R cells and to a lesser extent VLB100 cells expressed increased levels of MAP4 protein. Increased microtubule stability was observed in these VCR R cells as identified by the high levels of polymerized tubulin (45.6 +/- 2.6%; P < 0.005) compared with CEM and VLB100 cells (24.7 +/- 3.3% and 24.7 +/- 2.5%, respectively). Expression was associated with a single MAP4 isoform in the polymerized microtubule fraction in CEM and VCR cells. In contrast, VLB100 cells expressed a lower molecular weight isoform in the polymerized fraction. Two-dimensional-PAGE and immunoblotting revealed marked posttranslational changes in class I beta-tubulin in VCR R cells not evident in CEM cells. Sequencing of the beta-tubulin (HM40) gene identified a point mutation in VCR R cells in nucleotide 843 (CTC-->ATC; Leu(240)-->Ile) that was not present in CEM or VLB100 cells. This mutation resides in a region of beta-tubulin that lies in close proximity to the alpha/beta tubulin interface. Multiple alterations related to normal microtubule function were identified in ALL cells selected for resistance to Vinca alkaloids, and these alterations may provide important insight into mechanisms mediating resistance to Vinca alkaloids.

Selective potentiation of paclitaxel (taxol)-induced cell death by mitogen-activated protein kinase kinase inhibition in human cancer cell lines.

Activation of the mitogen-activated protein kinase (MAPK) pathway in HeLa and Chinese hamster ovary cells after treatment with paclitaxel (Taxol) and other microtubule interacting agents has been investigated. Using a trans-reporting system, the phosphorylation of the nuclear transcription factors Elk-1 and c-jun was measured. Concentration- and time-dependent activation of the Elk-1 pathway, mediated primarily by the extracellular signal-regulated kinase (ERK) component of the MAPK family, was observed. Inactive drug analogs and other cytotoxic compounds that do not target microtubules failed to induce similar levels of activation, thereby indicating that an interaction between these drugs and the microtubule is essential for the activation of MAPKs. Evaluation of the endogenous levels of MAPK expression revealed cell-dependent expression of the ERK, c-jun N-terminal kinase, and p38 pathways. In the case of HeLa cells, time-dependent activation of ERK coincided with increased poly(ADP-ribose) polymerase (PARP) cleavage, phosphatidylserine externalization, and increased accumulation of cells in G2/M. In both cell lines, inhibition of ERK activity potentiated paclitaxel-induced PARP cleavage and phosphatidylserine externalization, suggesting that ERK activity coincided with, but did not mediate, the cytotoxic effects of paclitaxel. We evaluated the nature of the interaction between paclitaxel and the MAPK kinase inhibitor U0126 in three cell lines, on the basis of a potential chemotherapeutic advantage of paclitaxel plus ERK inhibition. Our data confirmed additivity in those cells lines that undergo paclitaxel-induced ERK activation, and antagonism in cells with low ERK activity, suggesting that in tumors with high ERK activity, there may be an application for this strategy in therapy.

The role of ERK 1/2 and p38 MAP-kinase pathways in taxol-induced apoptosis in human ovarian carcinoma cells.

Taxol is an anticancer agent of natural origin with significant activity against a number of human cancers including ovarian and breast carcinomas. Its cytotoxic activity has been attributed to its ability to stabilize microtubules and to promote microtubule assembly. Recently it has become clearer that Taxol has additional activities including effects in cell signaling and gene expression. We have shown previously that Taxol activates ERK 1/2 MAP-kinases and results in the formation of GRB2/SHC complexes in murine macrophage-like RAW 267.4 cells. Here we demonstrate that Taxol activates ERK 1/2 and p38 MAP-kinases in human ovarian carcinoma cells with distinct kinetics. Activation of ERK1/2 has been observed at low concentrations of Taxol (1-100 nM) within 0.5-6 h, whereas longer exposure(24 h) to nanomolar concentrations of Taxol resulted in an abrogation of the ERK1/2 phosphorylation/activation. Higher concentrations (1-10 microM) resulted in a sharp inhibition of ERK1/2 activity. p38 kinase was activated by high concentrations (1-10 microM) of Taxol within 2 h and remained active for more than 24 h. The kinetic studies showed that these effects of Taxol coincided with an inhibition of proliferation, and the onset of apoptosis. The appearance of the fragmented chromatin visualized by DAPI staining, and DNA fragments seen on an agarose gel, coincided with the decrease in ERK1/2 activation and concomitant increase of the level of active p38 MAPK. The inhibitor PD98059 abrogated ERK 1/2 activation and increased the cytotoxic effect of Taxol. An inhibitor of p38 kinase, SB203580, protected the cells partially from Taxol and, unexpectedly, activated ERK 1/2 kinases. We conclude that the alternative use of ERK1/2 and p38 MAP-kinase pathways may be necessary for the transition from proliferation state to Taxol-induced apoptosisin human ovarian carcinoma cells.

Mutations in beta-tubulin map to domains involved in regulation of microtubule stability in epothilone-resistant cell lines.

The epothilones (Epos) are a group of natural products isolated from the myxobacterium, Sorangium cellulosum. They have a mechanism of action similar to that of Taxol, i.e., they stabilize microtubules and induce the formation of microtubule bundles in cells. Because they are simpler in structure than Taxol and preserve their activity in P-glycoprotein-expressing cells, they are being studied as potential antitumor drugs. In this work, a series of Epo-resistant A549 and HeLa cell lines have been selected and analyzed. Class I beta-tubulin, the major isotype of beta-tubulin in these Epo-resistant cell lines, has been sequenced in a search for mutations. In the Epo B-resistant A549 cells, there is a mutation at beta 292 from Gln to Glu, in the Epo A-resistant HeLa cell line there is a mutation at beta 173 from Pro to Ala, and in the Epo B-resistant HeLa cell line there is a heterozygous mutation at beta 422 from Tyr to a mixture of Tyr and Cys. These mutations are close to the M-loop, the nucleotide-binding site, and the microtubule-associated protein binding sites, respectively. It is likely that these mutations in beta-tubulin provide cells with a mechanism of resistance to the Epos and taxanes. Among these resistant cell lines, A549.EpoB40 is hypersensitive to microtubule-destabilizing drugs, such as vinblastine and colchicine, and HeLa.EpoB1.8 is dependent on the Epos or taxanes for growth. Our studies provide evidence that the M-loop, the GTP binding site, and the microtubule-associated protein binding sites at the COOH terminus in beta-tubulin are critical for the regulation of microtubule stability.

Taxol mediates serine phosphorylation of the 66-kDa Shc isoform.

In the human lung carcinoma cell line A549, Taxol (20 nM) causes a decreased electrophoretic mobility of the 66-kDa Shc isoform (p66shc), beginning 4 h after drug exposure, and reaching a maximum at 9-18 h. No shift was observed for the 52- and 46-kDa isoforms of Shc. The electrophoretic mobility shift of p66shc caused by Taxol is not the result of tyrosine phosphorylation, and there is no indication of a Shc/Grb2 complex in Taxol-treated A549 cells. This modification is blocked by the serine/threonine protein phosphatase 2A. In vivo 32P-labeling and subsequent phosphoamino acid analysis of p66shc indicated that both the original and the shifted p66shc were predominantly serine phosphorylated. Cyanogen bromide digestion of p66shc produced a phosphorylated fragment with an apparent molecular weight of approximately 7.9 kDa from the untreated cells and two phosphorylated fragments, of approximately 7.9 and approximately 9.6 kDa, from the Taxol-treated cells. The domain of Taxol-induced serine phosphorylation is thought to be in the cyanogen bromide fragment containing residues 2-65. The Taxol-induced electrophoretic mobility shift of p66shc was inhibited by the protein synthesis inhibitor, cycloheximide, but not by the mitogen-activated and extracellular signal-regulated protein kinase kinase (MEK) inhibitor, PD98059. This mobility shift did not occur in Taxol-resistant A549-T12 cells treated with 20 nM Taxol. In addition to Taxol, other microtubule-interacting drugs caused a decreased electrophoretic mobility of p66shc. This Taxol-mediated serine phosphorylation seen in p66shc may result from a MEK-independent signaling pathway that is activated in cells that have a prolonged or abnormal mitotic phase of the cell cycle and may play a role in signaling events that lead to cell death.

On the total synthesis and preliminary biological evaluations of 15(R) and 15(S) aza-dEpoB: a Mitsunobu inversion at C15 in pre-epothilone fragments.

[reaction-see text] The syntheses of two epothilone analogues, 15(S)-aza-12,13-desoxyepothilone B and the epimeric 15(R)-aza-12,13-desoxyepothilone B, are described. A Mitsunobu inversion was utilized for elaboration of pre-epothilone fragments to the corresponding macrolactam. Tubulin binding and cytotoxicity profiles of these analogues are presented.

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.

A common pharmacophore for Taxol and the epothilones based on the biological activity of a taxane molecule lacking a C-13 side chain.

Extensive structure-activity studies done with Taxol have identified the side chain at C-13 as one of the requirements for biological activity. Baccatin III, an analogue of Taxol lacking the C-13 side chain, has none of the biological characteristics of Taxol. Since 2-m-azido Taxol, a Taxol derivative with a m-azido substituent in the C-2 benzoyl ring, has greater activity than Taxol, we questioned whether 2-m-azido baccatin III might be active. 2-m-Azido baccatin III inhibited the proliferation of human cancer cells at nanomolar concentrations, blocked cells at mitosis, and reorganized the interphase microtubules into distinct bundles, a typical morphological change induced by Taxol. In contrast to 2-m-azido baccatin III, 2-p-azido baccatin III was similar to baccatin III, having no Taxol-like activity, further indicating the specificity and significance of the 2-meta position substituent. Molecular modeling studies done with the C-2 benzoyl ring of Taxol indicated that it fits into a pocket formed by His227 and Asp224 on beta-tubulin and that the 2-m-azido, in contrast to the 2-p-azido substituent, is capable of enhancing the interaction between the benzoyl group and the side chain of Asp224. The observation that the C-13 side chain is not an absolute requirement for biological activity in a taxane molecule has enabled the development of a new common pharmacophore model between Taxol and the epothilones.

Characterization of the Taxol binding site on the microtubule. Identification of Arg(282) in beta-tubulin as the site of photoincorporation of a 7-benzophenone analogue of Taxol.

Photoaffinity labeling methods have allowed a definition of the sites of interaction between Taxol and its cellular target, the microtubule, specifically beta-tubulin. Our previous studies have indicated that [(3)H]3'-(p-azidobenzamido)Taxol photolabels the N-terminal 31 amino acids of beta-tubulin (Rao, S., Krauss, N. E., Heerding, J. M., Swindell, C. S., Ringel, I., Orr, G. A., and Horwitz, S. B. (1994) J. Biol. Chem. 269, 3132-3134) and [(3)H]2-(m-azidobenzoyl)Taxol photolabels a peptide containing amino acid residues 217-233 of beta-tubulin (Rao, S., Orr, G. A., Chaudhary, A. G., Kingston, D. G. I., and Horwitz, S. B. (1995) J. Biol. Chem. 270, 20235-20238). The site of photoincorporation of a third photoaffinity analogue of Taxol, [(3)H]7-(benzoyldihydrocinnamoyl) Taxol, has been determined. This analogue stabilizes microtubules polymerized in the presence of GTP, but in contrast to Taxol, does not by itself enhance the polymerization of tubulin to its polymer form. CNBr digestion of [(3)H]7-(benzoyldihydrocinnamoyl)Taxol-labeled tubulin, with further arginine-specific cleavage by clostripain resulted in the isolation of a peptide containing amino acid residues 277-293. Amino acid sequence analysis indicated that the photoaffinity analogue cross-links to Arg(282) in beta-tubulin. Advances made by electron crystallography in understanding the structure of the tubulin dimer have allowed us to visualize the three sites of photoincorporation by molecular modeling. There is good agreement between the binding site of Taxol in beta-tubulin as determined by photoaffinity labeling and electron crystallography.

Structure-activity profiles of eleutherobin analogs and their cross-resistance in Taxol-resistant cell lines.

PURPOSE: Eleutherobin, a natural product, is an antimitotic agent that promotes the polymerization of stable microtubules. Although its mechanism of action is similar to that of Taxol, its structure is distinct. A structure-activity profile of synthetic eleutherobin derivatives that have modifications at C3, C8 and C15 was undertaken to define the structural requirements for microtubule stabilization and cross-resistance in Taxol-resistant cell lines. METHODS: The biological activity of five eleutherobin analogs was assessed using three techniques: (1) cytotoxicity and drug-resistance in three paired Taxol-sensitive and -resistant cell lines; (2) polymerization of microtubule protein in vitro in the absence of GTP and (3) induction of microtubule bundle formation in NIH3T3 cells. RESULTS: Eleutherobin had an IC50 value comparable to that of Taxol, whereas neoeleutherobin, which has a carbohydrate domain that is enantiomeric with that of the parent compound, was less cytotoxic and had 69% of the maximum microtubule polymerization ability of eleutherobin. Both of these compounds exhibited cross-resistance in MDRI-expressing cell lines. Removal or replacement of the C15 sugar moiety resulted in reduced microtubule polymerization and cytotoxicity compared to eleutherobin and loss of cross-resistance in the cell lines SKVLB and J7-T3-1.6, both of which express high levels of P-glycoprotein. By contrast, removal of the urocanic acid group at C8 resulted in virtually complete abrogation of biological activity. The compound lost its ability to polymerize microtubules, and its cytotoxicity was reduced by a minimum of 2000-fold in lung carcinoma A549 cells. CONCLUSIONS: Removal or modification of the sugar moiety alters the cytotoxic potency of eleutherobin and its pattern of cross-resistance in Taxol-resistant cells, although such compounds retain a small percentage of the microtubule-stabilizing activity of eleutherobin. The N(1)-methylurocanic acid moiety of eleutherobin, or perhaps some other substituent at the C8 position, is essential for Taxol-like activity. These findings will be important for the future design and the synthesis of new and more potent eleutherobin derivatives.

Antisense oligonucleotides to class III beta-tubulin sensitize drug-resistant cells to Taxol.

A major impediment to the successful use of Taxol in the treatment of cancer is the development of drug resistance. The major cellular target of Taxol is the microtubule that is comprised of alpha- and beta-tubulin heterodimers. Binding sites for Taxol have been delineated on the beta-tubulin subunit that has six isotypes. We have recently described increased expression of the brain-specific human class III beta-tubulin isotype, encoded by the Hbeta4 gene, in both Taxol-resistant ovarian tumours and non-small-cell lung cancer cell lines. To evaluate directly the role of the class III beta-tubulin isotype in mediating Taxol resistance, antisense phosphorothioate oligodeoxynucleotides (ODN) targeted against various regions of the Hbeta4 gene have been designed and examined for their efficacy in reducing Hbeta4 gene and protein expression. Taxol-resistant lung cancer cells, A549-T24, which are 17-fold resistant to Taxol and display a fourfold increase in Hbeta4 expression compared to the parental A549 cells, were treated with 1 microM antisense ODNs. Two ODNs, AS1 and AS3, were found to reduce mRNA expression by 40-50%, as determined by reverse transcription polymerase chain reaction. A concentration-dependent reduction in Hbeta4 mRNA expression was demonstrated with AS1 ODN. Immunofluorescence staining of cells treated with AS1 ODN revealed a decrease in class III protein expression which corresponded to a 39% increase in sensitivity to Taxol (P < 0.005). These findings support an important role for Hbeta4 (class III) beta-tubulin expression in Taxol resistance and have potential implications for the treatment of Taxol-resistant tumours.

A common pharmacophore for cytotoxic natural products that stabilize microtubules.

Taxol (paclitaxel), a complex diterpene obtained from the Pacific yew, Taxus brevifolia, is arguably the most important new drug in cancer chemotherapy. The mechanism of cytotoxic action for paclitaxel-i.e., the stabilization of microtubules leading to mitotic arrest-is now shared by four recently identified natural products, eleutherobin, epothilones A and B, and discodermolide. Their ability to competitively inhibit [3H]paclitaxel binding to microtubules strongly suggests the existence of a common binding site. Recently, we have developed nonaromatic analogues of paclitaxel that maintain high cytotoxicity and tubulin binding (e.g., nonataxel). We now propose a common pharmacophore that unites paclitaxel, nonataxel, the epothilones, eleutherobin, and discodermolide, and rationalizes the extensive structure-activity relationship data pertinent to these compounds. Insights from the common pharmacophore have enabled the development of a hybrid construct with demonstrated cytotoxic and tubulin-binding activity.

Upregulation of caveolin-1 and caveolae organelles in Taxol-resistant A549 cells.

Caveolin is a principal component of caveolae membranes. It has been demonstrated that the interaction of the caveolin scaffolding domain with signaling molecules can functionally inhibit the activity of these molecules. Taxol is an antitumor agent that suppresses microtubule dynamics and binds to microtubules thereby stabilizing them against depolymerization. The drug also has been implicated in the induction of apoptosis through activation of components in signal transduction cascades. Here we have investigated the role of caveolin in the development of drug resistance by examining the expression of caveolins in low- and high-level drug-resistant cell lines. Caveolin-1, but not caveolin-2, was upregulated in highly multidrug resistant SKVLBI cells that express high levels of P-glycoprotein, and in low-level Taxol-resistant A549 cell lines that express low amounts of P-glycoprotein. Two drug-resistant A549 cell lines (one 9-fold resistant to Taxol and the other 1.5-fold resistant to epothilone B), both of which express no P-glycoprotein, demonstrate a significant increase in the expression of caveolin-1. These results indicate that in low-level epothilone B- or Taxol-resistant A549 cells, increased caveolin-1 expression occurs independently of P-glycoprotein expression. Electron microscopic studies clearly demonstrate the upregulation of caveolae organelles in Taxol-resistant A549 cells. Upregulation of caveolin-1 expression in drug-sensitive A549 cells was observed acutely beginning 48 h after incubation with 10 nM Taxol. Thus, caveolin-1 may play a role in the development of Taxol resistance in A549 cells.

Identification of the domains of photoincorporation of the 3'- and 7-benzophenone analogues of taxol in the carboxyl-terminal half of murine mdr1b P-glycoprotein.

P-glycoprotein is an ATP-dependent drug-efflux pump that can transport a diverse range of structurally and functionally unrelated hydrophobic compounds across the plasma membrane. The transporter is composed of two homologous halves, each containing a nucleotide binding fold and six putative transmembrane spanning segments. The contact domains between the murine mdr1b P-glycoprotein and two photoreactive Taxol analogues have been mapped by a combination of CNBr digestion and immunoprecipitation studies. We had demonstrated previously that the 3'-p-benzoyldihydrocinnamoyl (BzDC) analogue of Taxol specifically photolabeled mdr1b P-glycoprotein and now show that the corresponding C-7 analogue likewise specifically photoincorporates into the transporter. CNBr digestion of both photolabeled P-glycoproteins gave rise to an approximate 10 kDa tritium-labeled peptide, each of which was a distinct polypeptide. The CNBr fragment generated from the 3'-BzDC-Taxol-photolabeled P-glycoprotein was immunoprecipitated by a polyclonal antibody (Ab7) raised against amino acid residues 1008-1019 of the mdr1b isoform. In contrast, the CNBr fragment generated from the 7-BzDC-Taxol-photolabeled P-glycoprotein was immunoprecipitated by a polyclonal antibody (Ab4) raised against amino acid residues 740-750. The specificity of these reactions was demonstrated by showing that the presence of the appropriate synthetic peptide blocked the immunoprecipitation. Moreover when the antibodies were reversed, no immunoprecipitation occurred. Based on the deduced amino acid sequence of mdr1b P-glycoprotein, and its hydropathy plot analysis, our data indicated that the 3'-BzDC group photoincorporates into amino acid residues 985-1088, a region of the transporter that includes half of TM 12 and terminates just after the Walker A motif in the second nucleotide binding fold. The 7-BzDC group photoincorporates into amino acid residues 683-760, a region of the transporter that includes all of TM 7 and half of TM 8 plus the intervening extracellular loop.

Mechanisms of Taxol-induced cell death are concentration dependent.

Although the ability of Taxol to stabilize cellular microtubules is well accepted, the mechanisms by which Taxol induces growth arrest and cell death remain unclear. Recent evidence indicates that Taxol alters specific intracellular signal transduction events, such as the activation of Raf-1 kinase, that may be essential for drug-induced apoptosis. To determine whether Raf-1 kinase activation occurs at different concentrations of Taxol and in response to disruption of the normal microtubule cytoskeleton, A549 cells were treated with different concentrations of Taxol after which Raf-1 activation and the microtubule cytoskeleton were analyzed. Raf-1 activation was observed at Taxol concentrations of 9 nM and greater. However, disruption of the normal microtubule cytoskeleton was seen at lower Taxol concentrations (1-7 nM), indicating that this process begins in the absence of Raf-1 activation. Raf-1 activation correlated with the induction of a G2-M block. Depletion of Raf-1 resulted in the accumulation of cells in the G2-M phase of the cell cycle, suggesting that Raf-1 may play an important role in the passage through mitosis. Supporting this idea, Raf-1 was activated in mitotic cells. Low concentrations of Taxol induced cell death in the absence of Raf-1 activation, indicating that Taxol-induced cell death is not dependent on Raf-1 activation. At concentrations of drug lower than the critical concentration required for Raf-1 activation, p53 and p21(WAF-1) were induced independently of Raf-1. These studies suggest that Taxol-mediated cell death may result from two different mechanisms. At low Taxol concentrations (< 9 nM), cell death may occur after an aberrant mitosis by a Raf-1 independent pathway, whereas at higher Taxol concentrations (> or = 9 nM) cell death may be the result of a terminal mitotic arrest occurring by a Raf-1-dependent pathway.