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[Cancer Research 62, 3113-3119, June 1, 2002]
© 2002 American Association for Cancer Research

Experimental Therapeutics

2-Aroylindoles, a Novel Class of Potent, Orally Active Small Molecule Tubulin Inhibitors1

Thomas Beckers2,,3, Thomas Reissmann, Mathias Schmidt2, Angelika M. Burger, Heinz H. Fiebig, Udo Vanhoefer, Herwig Pongratz, Harald Hufsky, Jörg Hockemeyer, Markus Frieser and Siavosh Mahboobi

ASTA Medica Oncology, D-60314 Frankfurt/Main [T. B., T. R., M. S.]; Tumor Biology Center, D-79106 Freiburg [H. H. F., A. M. B.]; Department of Internal Medicine/German Cancer Center, University of Essen Medical School, 45112 Essen [U. V.]; and Faculty of Chemistry and Pharmacy, Institute of Pharmacy, University Regensburg, D-93040 Regensburg [S. M., H. P., J. H., M. F., H. H.] Germany


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
2-Aroylindoles with 5-methoxy-1H-2-indolyl-phenylmethanone (D-64131) as the lead structure were discovered as a new class of synthetic, small molecule tubulin inhibitors. By competitively binding with [3H]colchicine to {alpha}ß-tubulin and inhibiting microtubule formation, cycling cells were arrested in the G2-M phase of the cell division cycle. The proliferation of tumor cells from 12 of 14 different organs and tissues was inhibited with mean IC50s of 62 nM and 24 nM by D-64131 and D-68144, respectively, comparable with the potency of paclitaxel with mean IC50 of 10 nM. By measuring the cytotoxicity in a human colon carcinoma cell model with ectopic ecdysone-inducible expression of the cyclin-dependent kinase inhibitor p21WAF1, specificity toward cycling cells was demonstrated. In contrast to microtubule inhibitors from natural sources, 2-aroylindoles did not alter the polymerization-dependent GTPase activity of ß-tubulin and are not substrates of the multidrug resistance/multidrug resistance protein efflux pump. No cross-resistance toward cell lines with multidrug resistance/multidrug resistance protein independent resistance phenotypes became evident. In animal studies, no signs of systemic toxicity were observed after p.o. dosages of up to 400 mg/kg of D-64131. In xenograft experiments with the human amelanoic melanoma MEXF 989, D-64131 was highly active with treatment resulting in a growth delay of 23.4 days at 400 mg/kg. Therefore, D-64131 and analogues have the potential to be developed for cancer therapy, replacing or supplementing standard therapy regimens with tubulin-targeting drugs from natural sources.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The mitotic spindle is a complex dynamic assembly of microtubules, motor proteins (kinesin and dynein families), MAPs,4 and catastrophe factors (1) . This self-organizing machine uses energy from nucleotide hydrolysis to segregate sister chromatids accurately into daughter cells (2) . Compounds from nature targeting the mitotic spindle apparatus are potent cytotoxic drugs. Taxol (paclitaxel), the semisynthetic analogue Taxotere (docetaxel), and the Vinca alkaloids vincristine, vinblastine, and vinorelbine have significant antitumoral activity toward, breast, ovarian, and non-small cell lung cancer, among others. The discovery of new natural and semisynthetic compounds being cytotoxic by interference with tubulin has attracted much attention within the last years, and microtubules have become a compelling target for anticancer drug discovery (3 , 4) .5 Microtubules are hollow tubes consisting of {alpha}- and ß-tubulin heterodimers that polymerize parallel to a cylindrical axis (5) . Mitotic microtubules are very dynamic structures, switching between growing and shortening states, a process known as dynamic instability and driven primarily by the catastrophe rate (1 , 6) . MAPs bind to and stabilize microtubules by reducing the catastrophe rate or increasing the polymerization rate (7 , 8) .

Tubulin binding molecules interfere with the dynamic instability of microtubules and thereby arrest mitotic cells in the M-phase of the cell division cycle, finally leading to apoptotic cell death. Tubulin binding molecules from nature are colchicine and the Vinca alkaloids vinblastine and vincristine, rhizoxin, maytansine, combretastatin A4, epothilone, and paclitaxel (3) . SMTIs derived from screening of compound libraries or derivatization of natural compounds have been described recently (9, 10, 11, 12, 13, 14, 15, 16, 17) . To our current knowledge, natural and synthetic compounds bind to distinct sites within ß-tubulin and, by destabilization or stabilization of microtubules, cause mitotic catastrophe (3) . The tubulin inhibitors used for human cancer therapy have severe drawbacks, i.e., high toxicity (especially neurotoxicity), marginal oral bioavailability and poor solubility, complex synthesis or isolation procedures, and most importantly, the development of drug resistance, which is a common phenomenon. New simple and synthetic compounds with oral bioavailability and high therapeutic index for first- and second-line therapy are therefore highly attractive.

By high throughput screening for antiproliferative agents, 2-aroylindoles were identified as a novel class of antimitotic compounds (18) . In this report, we show that D-64131 as well as potent analogues are antimitotic by binding to ß-tubulin, thereby destabilizing microtubules and arresting mitotic cells in the M-phase. D-64131 and analogues displayed broad antitumoral activity against cycling cells only, which is independent from the MDR/MRP phenotype or p53 status. Finally, in nude mice D-64131 proved to be well tolerated and highly active against the human amelanoic melanoma MEXF 989 tumor xenograft.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals and Biologicals.
General chemicals, 4,6-diamidino-2-phenylindole, XTT, the {alpha}-tubulin antibody (clone 5-1-2), as well as tubulin inhibitors paclitaxel, vincristine, and colchicine were purchased from Sigma (Munich, Germany). Muristerone A was from Invitrogen (Groningen, the Netherlands). [{alpha}-32P]GTP and streptavidin-coated yttrium SPA beads were obtained from Amersham Pharmacia Biotech (Freiburg, Germany), and [3H]colchicine was from NEN (Boston, MA). Biotinylated MAP-free bovine brain tubulin was from Cytoskeleton (Denver, CO). Tubulin for the polymerization assay was purified from bovine brain according to Vallee (19) . 2-Aroylindoles were synthesized at the Institute of Pharmacy, University Regensburg (18) .

Tumor Cell Lines.
The human tumor cell lines A431 (vulva epidermoid carcinoma/CRL 1555), HeLa/KB (cervical carcinoma, CCL-17), U373 (astrocytoma, HTB-17), U87 (glioblastoma, HTB-14), SKOV3 (ovarian adenocarcinoma, HTB-77), HT29 (colon adenocarcinoma, HTB-38), MDA-MB 231 (breast adenocarcinoma, HTB-26), Hec1A (endometrial adenocarcinoma, HTB112), A549 (lung cancer, CCL 185), PC3 (prostate adenocarcinoma, CRL-1435), AsPC-1 (pancreatic adenocarcinoma, CRL1682), Cal27 (squamous cell carcinoma of the tongue, CRL2095), Saos-2 (osteogenic sarcoma, HTB-85), T24 (bladder transitional cell carcinoma, HTB-4) were from ATCC (Manassas, VA). The human colon adenocarcinoma cell line RKO and transfectant RKOp21 were described recently (20) . The murine cell line L1210 (leukemia/CCL 219) was obtained from ATCC. The L1210VCR cell line was described recently (9) . The Renca kidney carcinoma cell line (21) was kindly provided by D. Marmé (KTB, Freiburg, Germany). All cell lines were cultivated at 37°C, 5% CO2 in medium as stated on the ATCC information sheet or as published. The detailed characteristics of human parental A2780/wt (ovarian), MCF-7/wt (breast), HT1080 (fibrosarcoma), and HCT-8 (colon) cell lines as well as the multidrug-resistant, P-glycoprotein 170-overexpressing A2780/Dx5 and MCF-7/adr, the MRP-expressing HT1080/DR4, the 5-fluorouracil-resistant HT29-R1 and HT29-R24, the raltitrexed-resistant HT29/ICID, and the SN-38 resistant HCT-8/SN38 cell lines have been described (22, 23, 24, 25, 26) .

Cytotoxicity Assays.
The test compounds were dissolved in DMSO at final concentrations of 10 mM and stored at -20°C. The XTT assay (27) was used as described to determine proliferation by quantification of cellular metabolic activity. The various tumor cell lines were cultivated in microtiter plates (2–15 x 103 cells/100 µl/well) and incubated with different concentrations of cytotoxic agents for 48 h. Alternatively, drug sensitivity was assessed with the sulforhodamine B assay as described recently (9) .

Cytotoxicity in the RKO exo p21 Cell Model.
Expression of p21waf1 was induced by treatment with 3 µM muristerone A for 24 h, arresting RKO colon carcinoma cells in the G1 and G2 phases of the cell division cycle (20) . RKO cells with/without p21waf1 expression (2 x 104 cells/well induced; 6 x 103 cells/well not induced) were treated with the test compound for 48 h at 37°C. Cellular metabolic activity/proliferation was determined by the XTT assay as described above.

Indirect Immunofluorescence Microscopy.
Detection of {alpha}-tubulin in HeLa/KB cells by immunofluorescence was done as described (9) . Briefly, cells were incubated with test compound for 24 h, washed with PBS, and fixed in 50% v/v methanol:50% v/v acetone for 15 min at -20°C. After drying at room temperature and saturation with 2% w/v BSA, 10% v/v FCSi in PBS for 30 min, staining was performed with an anti-{alpha}-tubulin antibody (clone B-5-1-2; Sigma) and a Cy3-conjugated rabbit antimouse antibody (315-165-003; Dianova). Nuclear staining was performed with 1 µg/ml 4,6-diamidino-2-phenylindole as described (9) .

Flow Cytometry.
HeLa/KB cells in subconfluent, proliferating culture were exposed to the cytotoxic agents for 24 h at 37°C, detached with trypsin, and finally collected by centrifugation. After washing, fixation, and staining, cells were analyzed by FACS as described (9) . Data points were connected, and the respective IC50 calculated using a nonlinear regression program (GraphPad Prism).

Human Tumor Xenografts.
D-64131 was freshly dissolved in DMSO and subsequently diluted with PBS containing 0.05% v/v Tween 80 to obtain a final DMSO concentration of 10%. Outbred nude mice, 6–8 weeks of age, of NMRI genetic background were used for all experiments. For the experiments, the human amelanoic melanoma MEXF 989 tumor xenograft model (28) was chosen based on in vitro selectivity of D-64131 toward melanoma (data not shown) and engrafted from tumors in serial passage growing s.c. in nude mice. Fragments of ~25 mg were implanted s.c. in both flanks of the animals. Details of the animal experiments and data analysis were described by Mahboobi et al. (18) . The MEXF 989 tumor-bearing nude mice were treated p.o. with doses of 200 and 400 mg/kg/day on days 1–5, 8–9, and 15–18. In two independent experiments, the drug doses and treatment schedule used were determined as being well tolerated in non-tumor bearing nude mice before initiation of tumor experiments.

Tubulin Polymerization Assay.
The assay was basically performed according to Bollag et al. (29) . Tubulin heterodimers (0.8 mg/ml; 80 µg/assay), isolated from bovine brain by cycles of polymerization and depolymerization, were incubated with test compounds (4 µg/ml in the initial screening, different concentrations in the final IC50 determination scheme) in PEM (100 mM PIPES, 1 mM EGTA, and 1 mM MgCl2) buffer (pH 6.6) containing 1 mM GTP in a total volume of 100 µl at 37°C for 1 h. Samples (75 µl) were then transferred to a 96-well Millipore Multiscreen Durapore hydrophilic 0.22-µm pore size filtration plate. Recovered microtubules on the filters were stained with 50 µl of Amido Black solution [0.1% w/v napthol blue black (Sigma), 45% v/v methanol, and 10% v/v acetic acid] for 2 min. Vacuum was applied, and unbound dye was removed by two additions of 200 µl of destain solution (90% v/v methanol, 2% v/v acetic acid). The microtubule bound dye was eluted by incubation with 200 µl of elution solution (25 mM NaOH, 0.05 mM EDTA, and 50% v/v ethanol) for 20 min. Next, 160 µl of elution solution was transferred to a 96-well plate, and the absorbance was measured at 600 nm using the Wallac Victor Multilabel counter (Perkin-Elmer/Wallac, Freiburg, Germany).

Tubulin Binding Assay.
The tubulin binding assay was performed according to Tahit et al. (30) using biotin-labeled tubulin, streptavidin-coated yttrium SPA beads, and [3H]colchicine (1 mCi/ml; specific activity, 76.5 Ci/mmol). Briefly, the binding mixture includes 0.08 µM [3H]colchicine, 1 mM GTP, and 0.5 µg of biotin-tubulin in G-PEM buffer, pH 6.9 (80 mM PIPES, 1 mM MgCl2, 1 mM EGTA, and 5% glycerol) in 100-µl final volume. The test compound and [3H]colchicine were added before tubulin. After incubation at 37°C for 2 h, 20 µl of SPA beads (80 µg in P-GEM buffer) were added. After further incubation for 30 min under agitation at room temperature, the SPA beads were allowed to settle down for 45 min, and scintillation counting was done on a MicroBeta Trilux counting device (Perkin-Elmer Wallac).

Tubulin GTPase Assay.
The tubulin GTPase assay was performed with slight modifications according to Roychowdhury et al. (31) . Highly purified, lyophilized, MAP-free bovine brain tubulin was reconstituted in PEM buffer (pH 6.6) and stored in aliquots at -80°C. The reaction mixture used for the GTPase assay contained 1 mg/ml tubulin, 1 mM MgCl2, 100 µM {alpha}[32P]GTP (specific activity, 3000 Ci/mmol), and 1M monosodium glutamate in PEM buffer. Alternatively, 4.5% (v/v) glycerol was used for induction of tubulin polymerization. Test compounds, dissolved in 10% v/v DMSO, were added before addition of glutamate and {alpha}[32P]GTP (final concentration, 1% DMSO). Tubulin polymerization was started by incubation at 37°C for 1 h and terminated by addition of SDS at 1% final concentration. GTP hydrolysis was measured by thin-layer chromatography of the reaction mixture using polyethyleneimine cellulose plates. The chromatograms were developed with 0.35 M NH4CO3. Chromatography plates were exposed to X-ray films and quantified by phosphorimager analysis using a Fuji BAS-1800II device.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
2-Aroylindoles Are Potent Antiproliferative, Tubulin Binding Agents.
Within a robotic screening program, 2-aroylindoles were initially identified as potent, antiproliferative agents. About 160 analogues were synthesized, and D-64131 was identified as a lead structure. Structural variations were introduced in the indole and phenyl ring for analysis of structure-activity relationships. In contrast to 2-benzoyl-5-methylindole derivatives, many 5-methoxyindole derivatives exhibited high cytotoxicity, and single or multiple substitutions within the phenyl ring (e.g., OCH3, F, NO2, and NH2) were compatible with high cytotoxicity (Fig. 1Citation and Table 1Citation ; Ref. 18 ). No clear effect of electron-donating and withdrawing groups was noted and, in comparison with the natural compounds combretastatin or colchicine, multiple methoxy substitutions were no prerequisite for high biological activity, as exemplified by D-64131. This compound as well as D-68143, D-68144, D-68150, D-68172, D-70316, and D-81187, as analogues with highest antiproliferative activity against HeLa/KB cervical carcinoma and U373 glioblastoma cell lines, were selected for further characterization in vitro (Fig. 1Citation and Table 1Citation ). In mode-of-action studies, D-64131 was tested against different targets within the cell division cycle, and tubulin was found as a putative molecular target. Further studies with selected analogues subsequently showed that polymerization of bovine brain tubulin as well as binding of [3H]colchicine to biotinylated {alpha}ß-tubulin were dose-dependently inhibited by D-64131 and other analogues (Fig. 2)Citation . The IC50s from these biochemical assays correlated well with the antiproliferative activity (Table 1)Citation . Thus, we conclude that the antiproliferative activity of 2-aroylindoles is based on binding to tubulin, interfering with the function of the mitotic spindle apparatus. This conclusion was further supported by confocal microscopic analysis of mitotic spindles in HeLa/KB cervical carcinoma cells after removal of unassembled tubulin. Many cells display abnormal microtubule structures, such as fragmented mitotic spindles or multiple spindle poles after treatment with D-64131 or vincristine (data not shown). Abnormal microtubule structures are also detectable in cells treated with the tubulin-stabilizing drug paclitaxel. Nuclear blebbing and abnormal nuclear structures typical for apoptotic cells are detectable in many cells treated with D-64131, Taxol, or vincristine (data not shown).



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  		Fig. 1. Structure of selected 2-aroylindoles. Selected examples of variations of the lead structure D-64131 are shown. With the exception of D-68148, which does not have a 5-methoxy substitution within the indole moiety, all compounds depicted high cytotoxicity (see Table 1Citation ).

 

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  		Table 1 Antiproliferative activity, inhibition of tubulin polymerization, and competition binding with [3H]colchicine

The antiproliferative activity of selected 2-aroylindoles against HeLa/KB cervical carcinoma and U373 astrocytoma cells was determined by using the XTT assay as described in "Materials and Methods." Inhibition of tubulin polymerization was determined by using tubulin with MAPs, purified from bovine brain. Binding to tubulin was determined by using a competition binding assay with [3H]colchicine as the radioligand. IC50s were calculated from dose-response curves by nonlinear regression analysis. All experiments were performed at least in two replicates, and mean values of independent experiments are shown.

 


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  		Fig. 2. Inhibition of tubulin polymerization and [3H]colchicine binding. The inhibition of polymerization of bovine brain tubulin by selected 2-aroylindole analogues is shown in A, whereas inhibition of [3H]colchicine binding to biotinylated tubulin is shown in B. Mean IC50s of independent experiments are summarized in Table 1Citation .

 
Effect of D-64131 on ß-Tubulin GTPase Activity.
The dynamic instability of microtubules is driven by ß-tubulin-dependent hydrolysis of GTP (6) . Synthetic or natural compounds like paclitaxel, vincristine, or colchicine either stimulate or repress the GTPase of ß-tubulin associated with polymerization (32 , 33) . As shown in Fig. 3ACitation , only highly purified tubulin is devoid of any ß-tubulin-independent GTPase activity and was used for all additional experiments. Tubulin polymerization and ß-tubulin GTPase were activated by raising temperature to 37°C and addition of 1 M glutamate or 4.5% glycerin (Fig. 3ACitation and data not shown). In case of glycerin-dependent polymerization, the GTPase is activated by paclitaxel and colchicine, with D-64131 and vincristine having no significant effect (Fig. 3B)Citation . For glutamate-dependent polymerization, the GTPase is activated by colchicine and vincristine in concentrations <=1 µM (Fig. 3C)Citation . Paclitaxel or vincristine at higher concentrations of >1 µM are inhibitory, whereas D-64131 again showed only marginal influence (Fig. 3C)Citation . On the basis of these data, it appears that, unlike the natural tubulin inhibitors, D-64131 did not significantly effect the polymerization dependent GTPase of ß-tubulin.



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  		Fig. 3. Effect on the ß-tubulin GTPase. The GTPase activity of MAP-containing and highly purified MAP-free bovine brain tubulin is shown in A. The amount of GDP and GTP was determined under various experimental conditions as shown and described in "Materials and Methods." PEM, PIPES/EGTA/MgCl2 buffer; Glut, monosodium glutamate. MAP-free tubulin was subsequently used for the determination of the effect of colchicine, vincristine, paclitaxel, or D-64131 on the polymerization-dependent ß-tubulin GTPase. Polymerization of tubulin was induced by addition of 4.5% (v/v) glycerin (B) or 1 M glutamate (C) and incubation at 37°C for 2 h. The GTPase activity is expressed as the quotient of GDP:GTP concentrations as quantified by phosphorimager analysis (background activity at 0°C is shown as a dotted line).

 
2-Aroylindoles Are Cell Cycle Specific, Arresting Tumor Cells in G2-M.
The antiproliferative effect of 2-aroylindoles was evaluated in two independent cellular models, i.e., by flow cytometric analysis of cells in the G1, S, or G2-M phase of the cell division cycle and by determination of the cytotoxicity toward RKO colon carcinoma cells with ectopic inducible expression of p21WAF1. As shown in Fig. 4ACitation and summarized in Table 2Citation , HeLa/KB cervical carcinoma cells are dose-dependently arrested in G2-M before induction of apoptotic cell death, as evidenced by a significant number of cells in sub-G1. The RKO p21WAF1 model was chosen to prove that the cytotoxicity depends actively on cycling cells. Inducible expression of p21WAF1 renders RKO cells completely resistant toward compounds such as paclitaxel that rely on the progression through a specific phase of the cell cycle (20) . As exemplified for D-64131 and D-68144 in Fig. 4BCitation and summarized in Table 2Citation , cytotoxicity is only evident on cycling RKO cells without p21WAF1 expression. No significant cytotoxicity was detected toward RKO cells that were cell cycle arrested in G1 and G2 through inducible expression of p21Waf1. We conclude that 2-aroylindoles arrest cells in the M-phase of the cell cycle and display cytotoxicity only toward cycling cells.



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  		Fig. 4. Cell cycle specificity. The cell cycle specificity of selected compounds was determined by flow cytometry in A. The percentage of HeLa/KB cervical carcinoma cells in G2-M phase of the cell division cycle was quantified, and dose-response curves (left panel) as well as selected FACS histograms (right panel) are shown. In B, the cell cycle-dependent cytotoxicity of D-64131 and D-68144 was evaluated in the RKOp21/Waf1 cell model. RKO colon carcinoma cells were pretreated with 3 µM muristerone A to induce p21waf1, leading to cell cycle arrest in G1 and G2. Cell proliferation/metabolic activity was determined 48 h after treatment with compounds.

 

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  		Table 2 Cytotoxicity in the RKO p21waf1 model and G2-M cell cycle arrest

The antiproliferative activity of selected compounds against the RKO colon adenocarcinoma cell line with/without p21waf1 expression was determined by using the XTT assay as described in "Materials and Methods." In another experimental setting, the percentage of HeLa/KB cervical carcinoma cells arrested in G2-M after compound treatment was determined by flow cytometry. All experiments were performed at least in two replicates, and IC50s were calculated from dose-response curves by nonlinear regression analysis.

 
Antiproliferative Activity of D-64131 and D-68144 toward Different Tumor Cell Lines.
The in vitro activity spectrum toward cell lines representing different tumor entities was evaluated with D-64131 and D-68144. Paclitaxel, vincristine, and colchicine were included as reference compounds. As summarized in Table 3Citation , D-64131, D-68144, and colchicine were cytotoxic against 12 of 14 tumor types, whereas paclitaxel and vincristine were cytotoxic against 13 of 14 tumor types. The Hec1A endometrial carcinoma cell line was resistant toward all tubulin inhibitors as tested. Cytotoxicity was independent from the p53 status; cells with mutant (e.g., HT29, PC-3, MDA-MB431, and Saos-2) and wild-type p53 (e.g., A549 and RKO) were equally targeted. The mean cytotoxicity of D-68144 with 24 nM was identical to that of colchicine but 2.4- and 3.8-fold weaker compared with that of paclitaxel and vincristine, respectively. These results demonstrate that 2-aroylindoles have broad cytotoxicity toward cancer cells of different origin and an antitumor efficacy comparable with that of paclitaxel.


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  		Table 3 Cytotoxicity profile toward tumor cell lines from different tissues/organs

The antiproliferative activity of D-64131 and D-68144 in comparison to colchicine, paclitaxel, and vincristine against 13 different human tumor entities and Renca as a murine kidney carcinoma was determined by using the XTT assay as described in "Materials and Methods." All experiments were performed at least in two replicates; IC50s were calculated by nonlinear regression, and mean values of independent experiments are shown.

 
D-64131 and D-68144 Are Cytotoxic toward MDR/MRP Tumor Cell Lines.
One major mechanism of acquired drug resistance is the overexpression of efflux pumps, i.e., the p-gp170/MDR and MRP (34) . We compared the antitumoral activity of D-64131 and D-68144 to that of vincristine, paclitaxel, and colchicine in the murine L1210VCR cell line. The L1210VCR murine leukemia cell line was selected for resistance toward vincristine, overexpressing the p-gp170/MDR protein (9) . As summarized in Table 4Citation , D-64131 and D-68144 are equally potent toward the parental L1210 and L1210VCR cell lines. As expected, the resistance factor (ratio IC50 L1210VCR/IC50 L1210) for the reference compounds ranged from 70 to 109. In addition, the antitumoral efficacy of D-64131 in comparison to paclitaxel and vincristine was evaluated in resistance mediated by the MRP and in human tumor cells with resistance to cisplatin, the topoisomerase I inhibitor SN-38 (7-ethyl-10-hydroxycamptothecin) and the thymidylate synthase inhibitors 5-fluorouracil and raltitexed. Taken together, D-64131 retained unaltered cytotoxic efficacy toward all resistant sublines as tested (Table 4)Citation . In summary, the cytotoxicity of 2-aroylindoles, as represented by D-64131 and D-68144, is not altered by various resistance phenotypes of high clinical relevance.


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  		Table 4 Antitumoral efficacy toward tumor cell lines with different resistance phenotypes

The antiproliferative activity of D-64131, D-68144, vincristine, taxol, and colchicine against tumor cell lines with different resistance phenotypes was determined by using the XTT cytotoxicity assay as described in "Materials and Methods." All experiments were performed at least in two replicates, and IC50s were calculated from dose-response curves by nonlinear regression analysis; mean values of independent experiments are shown. The resistance factor (in parentheses) was calculated from the ratio of the IC50 for the resistant subline and that of their parental cell line.

 
Efficacy of D-64131 in Xenograft Experiments in Vivo.
D-68144 compared with D-64131 was less soluble, having reduced bioavailability. Thus, D-64131 was selected for evaluation of in vivo efficacy in human tumor xenografts. On the basis of the in vitro chemosensitivity profile toward 19 human xenograft-derived tumor cell lines from the Freiburg tumor panel with a mean IC70 of 0.88 µM, melanomas (2 of 2), ovarian (1 of 1) and colon (1 of 1) cancers were identified as most sensitive (data not shown). The human amelanoic melanoma MEXF 989 was selected as a potential in vivo sensitive tumor. For oral dosing, 200 and 400 mg/kg were administered on days 1–5, 7–8, and 14–18. Both dosages were well tolerated with no signs of toxicity or body weight loss. D-64131 treatment resulted in significant tumor growth inhibition during treatment (Fig. 5)Citation . We conclude that D-64131 has oral bioavailability and is well tolerated at efficacious doses.



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  		Fig. 5. Effect of D-64131 on the human melanoma MEXF 989 tumor xenograft. The treatment modalities as well as efficacy toward the melanoma MEXF 989 xenograft are shown. D-64131 was dissolved in PBS, 0.05% Tween 80, and 10% DMSO and given p.o. as indicated (group size, 12–14 tumors). Opt. T/C, day of optimal effect/%; T/C, optimum median relative tumor volume of treated/control in %; GD 200%, growth delay {cong} time until tumor doubling in days; BWC, median maximum body weight change compared with body weight at start of experiment (= 100%) in %. Arrow, stop of treatment on day 18.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
D-64131 was discovered by random screening for antiproliferative drugs, exhibiting high cytotoxicity toward human cancer cells. Mode-of-action studies revealed that D-64131 inhibited tubulin polymerization. On the basis of this finding, D-64131 was selected as a lead structure, and more than 160 analogues with the 2-aroylindole core structure were synthesized (18) . These compounds represent a new chemical entity of a synthetic SMTI. From a structural point of view, 2-aroylindoles have the indole ring as a scaffold and resemble D-24851 [N-(pyridin-4-yl)-(1-(4-chlorbenzyl)-indol-3-yl)-glyoxyl-amid], heterocombretastatins, diaryl-indoles, or 3-formyl-2-phenylindoles (9 , 13, 14, 15) , although substituents as well as the overall structures differ significantly. Other synthetic SMTIs include A-105972 (10) , the carbamate derivatives CI-980 and 1069C85 (11 , 12) , and estradiol analogues (16) , as well as T138067, a compound covalently modifying ß-tubulin (ß1, ß2, and ß4) at Cys239 (17) . Compared with taxanes, Vinca alkaloids, combretastatins, podophyllotoxins, epothilones, or dolastatins as natural compounds, synthetic SMTIs are unique concerning tubulin binding, efficacy toward resistant tumors, bioavailability, and toxicity profile.

In the present report, selected 2-aroylindoles with superior cytotoxicity were comprehensively characterized. D-64131 as well as the analogues D-68143, D-68144, D-68150, D-70316, and D-81187 (see Fig. 1Citation for chemical structures) bound to tubulin with IC50s ranking from 0.14 to 0.8 µM. In accordance with binding, tubulin polymerization was inhibited with IC50s ranking from 0.39 to 1.29 µM. For these experiments, tubulin purified by cycles of polymerization and depolymerization from bovine brain was used. Polymerization of highly purified MAP-free tubulin was inhibited equally well, excluding a direct interference of 2-aroylindoles with MAPs (data not shown). Although binding affinity and inhibition of polymerization did correlate, 10–20-fold lower compound concentrations were sufficient for cell cycle arrest, disruption of mitotic microtubules, and apoptotic cell death (Tables 1Citation and 2Citation ). This has been described for other synthetic tubulin inhibitors as well (9 , 10) . Because tubulin-independent targets are unlikely (see below), 2-aroylindoles might accumulate intracellularly, as shown for paclitaxel in HeLa cells (3) .

2-Aroylindoles compete with [3H]colchicine for binding to biotinylated tubulin, but there are exceptions such as D-68150 with partial inhibition of [3H]colchicine binding. Because bovine brain tubulin is composed of four ß-tubulin isotypes (ß1–4), isotype-specific binding is possible (35) . Another explanation might be that the binding site and/or the conformational change induced by binding of 2-aroylindoles to tubulin is distinct to that of colchicinoids, but additional experiments with radiolabeled D-64131 have to be performed to prove this hypothesis. In this regard, the fluorescence of D-64131 with {lambda}max = 512 nm might provide an assay for binding. Recent studies showed that SMTIs potently inhibiting tubulin polymerization did not compete with [3H]colchicine or [3H]vincristine for binding (Refs. 9 and 10 and data not shown), thus arguing for a new binding site or mode of action. By molecular modeling, the binding site for colchicinoids was proposed to be within ß-tubulin or, alternatively, at the {alpha}/ß interface (36) .

Microtubule inhibitors are potent antimitotic drugs, arresting dividing cells in M-phase and inducing programmed cell death. These characteristics were also evident for 2-aroylindoles. As determined by flow cytometry, HeLa/KB cervical carcinoma cells were dose-dependently arrested in M-phase with 4N chromosomes before cell death became evident by cells with <2N chromosomes (cell population in sub-G1) and a massive reduction of attached, vital cells. The superior specificity of 2-aroylindoles was shown in three independent experimental settings, i.e., the RKO p21waf1cell model, a biochemical kinase screen and, finally, signal transduction to the c-fos promoter. In the RKO p21waf1cell model, the colon adenocarcinoma cells are arrested in the G1 and G2 phase of the cell division cycle by ecdysone-inducible ectopic expression of the cdk inhibitor p21waf1 (20) . Because tubulin-targeting compounds only hit mitotic cells, RKO cells are protected by p21waf1 expression. As shown in Fig. 4BCitation and Table 2Citation , 2-aroylindoles are only cytotoxic to cycling RKO cells without p21waf1 expression. Because D-64131 is an analogue of the platelet-derived growth factor-receptor kinase inhibitor 5-methoxy-bis (1H-2-indolyl)-1-methanone (37) , specificity was evaluated against serine/threonine and tyrosine-specific kinases, i.e., platelet-derived growth factor receptor ß, Jak2, epidermal growth factor receptor/HER1, KDR/vascular endothelial growth factor-R2, protein kinase B/akt1, cyclin-dependent kinase 1/cyclin B, cyclin-dependent kinase K2/cyclin E, and protein kinase C{xi}. In concentrations up to 10 µM, all 2-aroylindoles as included in this study did not exhibit any inhibitory activity (data not shown). In the final assay for specificity, D-64131 did not affect the serum-dependent transcriptional activation of the immediate-early gene c-fos (data not shown), thus excluding inhibition of kinases like raf1, mitogen-activated protein kinase kinase, or p42/44 mitogen-activated protein kinase involved in signaling to the c-fos promoter (38) . We conclude that D-64131 and analogues are specific for tubulin, exhibiting cytotoxicity only against cycling cells.

Paclitaxel and Taxotere are stabilizing tubulin inhibitors used clinically for the treatment of breast, ovarian, and non-small cell lung cancer, whereas vincristine and vinblastine are destabilizing tubulin inhibitors indicated for acute lymphatic leukemia, Morbus Hodgkin, and advanced breast cancer (39) . Taxanes, although widely used in cancer therapy (40) , have severe drawbacks: (a) natural/semisynthetic compounds with complex structure; (b) low solubility and very limited oral bioavailability as single agents; (c) dose-limiting toxicity such as neutropenia, peripheral neuropathy, and most likely vehicle (Cremaphor EL)-related hypersensitivity reactions; and (d) development of drug resistance and tumor metastases in the brain. Clearly, there is a high medical need for tubulin inhibitors with a new mode of action and drug profile. D-64131 and analogues are very promising, displaying properties of high clinical relevance: (a) strong and broad antiproliferative activity toward tumor cells derived from different tissues/organs (Table 3)Citation ; (b) no substrate of the MDR/MRP efflux pump and no cross resistance toward tumor cells displaying various resistance phenotypes (Table 4)Citation ; and (c) oral bioavailability and efficacy at well-tolerated doses (Fig. 5)Citation .

At present, the best described mechanism of resistance to tubulin-binding agents is the MDR phenotype mediated by the P-glycoprotein 170 efflux pump (34) . Because D-64131 or D-68144 are neither substrates of the MDR/MRP efflux pump nor is there any cross-resistance, they have the potential for treatment of taxane-resistant patients. Another resistant phenotype relates to ß-tubulin structure and/or function such as described for 1A9 human ovarian carcinoma cells and paclitaxel with mutations in the class I/M40 ß-tubulin isotype (41) . Because 2-aroylindoles are structurally and mechanistically different to natural tubulin inhibitors, it is highly likely that they are active toward these cancer genotypes as well.

In animal studies in nude mice, D-64131 was well tolerated after oral dosing up to 400 mg/kg with no signs of toxicity at efficacious doses in the MEXF 989 melanoma xenograft tumor model. As such, D-64131 is superior to the synthetic SMTIs T138067 or A-105972, which were only effective after i.p. dosage (10 , 17) . Published clinical Phase II data for the carbamate CI980 are disappointing (42) , but new chemical entities such as D-24851 are headed for the clinic (9 , 43) . 2-Aroylindoles with D-64131 as a prototypic representative are new candidates for Phase I clinical trials, which are scheduled after completion of on-going preclinical development studies.


   ACKNOWLEDGMENTS
 
The technical assistance of A. Masch, E. Thoenes, S. Falk, and R. Deckenbach is gratefully acknowledged. We also thank Maria Höxter, GBF, Braunschweig for FACS analysis; J. Braunger and F. Flach, Byk Gulden GmbH, Konstanz for confocal microscopy; and U. Riemer for preparing the figures.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by Federal Ministry of Education, Science, Research and Technology (BMBF) Grant 0311334/new natural products (to S. M.) and a grant from ASTA Medica AG. Back

2 Present address: Byk Gulden GmbH, Therapeutic Area Oncology, Department RDR-B4, Byk Gulden Strasse 2, 78467 Konstanz, Germany. Back

3 To whom requests for reprints should be addressed, at Byk Gulden GmbH, Therapeutic Area Oncology, Department RDR-B4, Byk Gulden Strasse 2, 78467, Konstanz, Germany. E-mail: T.Beckers{at}vff.uni.frankfurt.de Back

4 The abbreviations used are: MAP, microtubule-associated protein; SMTI, small molecule tubulin inhibitor; D-64131, 5-methoxy-1H-2-indolyl-phenylmethanone; MDR, multidrug resistance; MRP, multidrug resistance protein; ATCC, American Type Culture Collection; XTT, 3'-[1-(phenylaminocarbonyl)-3,4-tetrazolium]-bis-(4-methoxy-6-nitro)-benzene sulfonic acid; FACS, fluorescence-activated cell sorting; SPA, scintillation proximity assay. Back

5 See the National Cancer Institute page. Compare tubulin inhibitors by checking the website http://dtp.nci.nih.gov/docs/compare/examples/antitub.html. Back

Received 8/21/01. Accepted 3/27/02.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Wittmann T., Hyman A., Desai A. The spindle: a dynamic assembly of microtubules and motors. Nat. Cell Biol., 3: E28-E34, 2001.[Medline]
  2. Nicklas R. B. How cells get the right chromosomes. Science (Wash. DC), 275: 632-637, 1997.[Abstract/Free Full Text]
  3. Jordan A., Hadfield J. A., Lawrence N. J., McGown A. T. Tubulin as a target for anticancer drugs: agents which interact with the mitotic spindle. Med. Res. Rev., 18: 259-296, 1998.[Medline]
  4. Li Q., Sham H. L., Rosenberg S. H. Antimitotic agents. Ann. Rep. Med. Chem., 34: 139-148, 1999.
  5. Amos L. A. Focusing-in on microtubules. Curr. Opin. Struct. Biol., 10: 236-241, 2000.[Medline]
  6. Mitchison T., Kirschner M. Dynamic instability of microtubule growth. Nature (Lond.), 312: 237-242, 1984.[Medline]
  7. Andersen S. S. Spindle assembly and the art of regulating microtubule dynamics by MAPs and stathmin/Op18. Trends Cell Biol., 10: 261-267, 2000.[Medline]
  8. Drewes G., Ebneth A., Mandelkow E. M. MAPs, MARKs and microtubule dynamics. Trends Biochem. Sci., 23: 307-311, 1998.[Medline]
  9. Bacher G., Nickel B., Emig P., Vanhoefer U., Seeber S., Shandra A., Klenner T., Beckers T. D-24851, a novel synthetic tubulin inhibitor, exerts curative antitumoral activity in vivo, efficacy towards multidrug resistant tumor cells, and lacks neurotoxicity. Cancer Res., 61: 392-399, 2001.[Abstract/Free Full Text]
  10. Wu-Wong J. R., Alder J. D., Alder L., Burns D. J., Han E. K., Credo B., Tahir S. K., Dayton B. D., Ewing P. J., Chiou W. J. Identification and characterization of A-105972, an antineoplastic agent. Cancer Res., 61: 1486-1492, 2001.[Abstract/Free Full Text]
  11. de Ines C., Leynadier D., Barasoain I., Peyrot V., Garcia P., Briand C., Rener G. A., Temple C., Jr. Inhibition of microtubules and cell cycle arrest by a new 1-deaza-7,8-dihydropteridine antitumor drug, CI 980, and by its chiral isomer, NSC 613863. Cancer Res., 54: 75-84, 199.[Abstract/Free Full Text]
  12. Raynaud F. I., Kelland L. R., Walton M. I., Judson I. R. Preclinical pharmacology of 1069C85, a novel tubulin binder. Cancer Chemother. Pharmacol., 35: 169-173, 1994.[Medline]
  13. Medarde M., Ramos A. C., Caballero E., Pelaez-Lamamie de Clairac R., Lopez J. L., Gravalos D. G., Feliciano A. S. Synthesis and pharmacological activity of diarylindole derivatives. Cytotoxic agents based on combretastatins. Bioorg. Med. Chem. Lett., 9: 2303-2308, 1999.[Medline]
  14. Medarde M., Ramos A. C., Caballero E., Pelaez-Lamamie de Clairac R., Lopez J. L., Gravalos D. G., Feliciano A. S. Synthesis and anti-neoplastic activity of combretastatin analogues: heterocombretastatins. Eur. J. Med. Chem., 33: 71-77, 1998.
  15. Gastpar R., Goldbrunner M., Marko D., von Angerer E. Methoxy-substituted 3-formyl-2-phenylindoles inhibit tubulin polymerization. J. Med. Chem., 41: 4965-4972, 1998.[Medline]
  16. Wang Z., Yang D., Mohanakrishnan A. K., Fanwick P. E., Nampoothiri P., Hamel E., Cushman M. Synthesis of B-ring homologated estradiol analogues that modulate tubulin polymerization and microtubule stability. J. Med. Chem., 43: 2419-2429, 2000.[Medline]
  17. Shan B., Medina J. C., Santha E., Frankmoelle W. P., Chou T. C., Learned R. M., Narbut M. R., Stott D., Wu P., Jaen J. C., Rosen T., Timmermans P. B., Beckmann H. Selective, covalent modification of ß-tubulin residue Cys-239 by T138067, an antitumor agent with in vivo efficacy against multidrug-resistant tumors. Proc. Natl. Acad. Sci. USA, 96: 5686-5691, 1999.[Abstract/Free Full Text]
  18. Mahboobi S., Pongratz Hufsky H., Hockemeyer J., Frieser M., Lyssenko A., Paper D., Bürgermeister J., Böhmer F. D., Fiebig H. H., Burger A. M., Baasner S., Beckers T. Synthetic 2-aroylindol derivatives as a new class of potent tubulin-inhibitory, antimitotic agents. J. Med. Chem., 44: 4535-4553, 2001.[Medline]
  19. Vallee R. B. Reversible assembly purification of microtubules without assembly-promoting agents and further purification of tubulin, microtubule-associated proteins, and MAP fragments. Methods Enzymol., 134: 89-190, 1986.[Medline]
  20. Schmidt M., Lu Y., Liu B., Fang M., Mendelsohn J., Fan Z. Differential modulation of paclitaxel-mediated apoptosis by p21waf1 and p27kip1. Oncogene, 19: 2423-2429, 2000.[Medline]
  21. Murphy G. P., Hrushesky W. J. A murine renal cell carcinoma. J. Natl. Cancer Inst., 50: 1013-1025, 1973.
  22. Harstrick A., Gonzales A., Schleucher N., Vanhoefer U., Lu K., Formento J. L., Milano G., Wilke H., Seeber S., Rustum Y. Comparison between short or long exposure to 5-fluorouracil in human gastric and colon cancer cell lines: biochemical mechanism of resistance. Anticancer Drugs, 9: 625-634, 1998.[Medline]
  23. Vanhoefer U., Cao S., Minderman H., Toth K., Scheper R. J., Slovak M. L., Rustum Y. M. PAK-104P, a pyridine analogue, reverses paclitaxel and doxorubicin resistance in cell lines and nude mice bearing xenografts that overexpress the multidrug resistance protein. Clin. Cancer Res., 2: 369-377, 1996.[Abstract/Free Full Text]
  24. Vanhoefer U., Muller M. R., Hilger R. A., Lindtner B., Klaassen U., Schleucher N., Rustum Y. M., Seeber S., Harstrick A. Reversal of MDR1-associated resistance to topotecan by PAK-200S, a new dihydropyridine analogue, in human cancer cell lines. Br. J. Cancer, 81: 1304-1310, 1999.[Medline]
  25. Klaassen U., Harstrick A., Schleucher N., Vanhoefer U., Schroder J., Wilke H., Seeber S. Activity- and schedule-dependent interactions of paclitaxel, etoposide and hydroperoxy-ifosfamide in cisplatin-sensitive and -refractory human ovarian carcinoma cell lines. Br. J. Cancer, 74: 224-228, 1996.[Medline]
  26. Minderman H., Vanhoefer U., Toth K., Minderman M. D., Rustum Y. M. A unique human ovarian carcinoma cell line expressing CD34 in association with selection for multidrug resistance. Cancer (Phila.), 78: 2427-2436, 1996.[Medline]
  27. Scudiero D. A., Shoemaker R. H., Paull K. D., Monks A., Tierney S., Nofziger T. H., Currens M. J., Seniff D., Boyd M. R. Evaluation of a soluble tetrazolium/formazan assay for cell growth and drug sensitivity in culture using human and other tumor cell lines. Cancer Res., 48: 4827-4833, 1988.[Abstract/Free Full Text]
  28. Roth T., Burger A. M., Dengler W., Willmann H., Fiebig H. H. Human tumor cell lines demonstrating the characteristics of patient tumors as useful models for anticancer drug screening in: relevance of tumor models for anticancer drug development. Contributions to Oncology, Vol. 54: 145-156, Karger Basel 1999.
  29. Bollag D. M., McQueney P. A., Zhu J., Hensens O., Koupal L., Liesch J., Goetz M., Lazarides E., Woods C. M. Epothilones, a new class of microtubule-stabilizing agents with a Taxol-like mechanism of action. Cancer Res., 55: 2325-2333, 1995.[Abstract/Free Full Text]
  30. Tahit S. K., Kovar P., Rosenberg S. H., Ng S. C. Rapid colchicine competition-binding scintillation proximity assay using biotin-labeled tubulin. BioTechniques, 29: 156-160, 2000.[Medline]
  31. Roychowdhury S., Panda D., Wilson L., Rasenick M. M. G protein {alpha} subunits activate tubulin GTPase and modulate microtubule polymerization dynamics. J. Biol. Chem., 274: 13485-13490, 1999.[Abstract/Free Full Text]
  32. Lin C. M., Hamel E. Effects of inhibitors of tubulin polymerization on GTP hydrolysis. J. Biol. Chem., 256: 9242-9245, 1981.[Abstract/Free Full Text]
  33. Duanmu C., Shahrik L. K., Ho H. H., Hamel E. Tubulin-dependent hydrolysis of guanosine triphosphate as a screening test to identify new antitubulin compounds with potential as antimitotic agents: application to carbamates of aromatic amines. Cancer Res., 49: 1344-1348, 1989.[Abstract/Free Full Text]
  34. Dumontet C., Sikic B. I. Mechanisms of action of and resistance to antitubulin agents: microtubule dynamics, drug transport, and cell death. J. Clin. Oncol., 17: 1061-1070, 1999.[Abstract/Free Full Text]
  35. Banerjee A., Luduena R. F. Kinetics of colchicine binding to purified ß-tubulin isotypes from bovine brain. J. Biol. Chem., 267: 13335-13339, 1992.[Abstract/Free Full Text]
  36. Bai R., Covell D. G., Pei X. F., Ewell J. B., Nguyen N. Y., Brossi A., Hamel E. Mapping the binding site of colchicinoids on ß-tubulin. 2-Chloroacetyl-2-demethylthiocolchicine covalently reacts predominantly with cysteine 239 and secondarily with cysteine 354. J. Biol. Chem., 275: 40443-40452, 2000.[Abstract/Free Full Text]
  37. Mahboobi S., Teller S., Pongratz H., Hufsky H., Sellmer A., Uecker A., Beckers T., Baasner S., Schächtele C., Truppel A., Waltenberger J., Überall F., Kassack M. U., Böhmer F. D. Bis (1H-2-indolyl)-1-methanones as a novel class of inhibitors of the platelet-derived growth factor receptor kinase. J. Med. Chem., 45: 1002-1018, 2002.[Medline]
  38. Cahill M. A., Janknecht R., Nordheim A. Signalling pathways: jack of all cascades. Curr. Biol., 6: 16-19, 1996.[Medline]
  39. Dutcher J. P., Novik Y., O’Boyle K., Marcoullis G., Secco C., Wiernik P. H. 20th-century advances in drug therapy in oncology–Part. II. J. Clin. Pharmacol., 40: 1079-1092, 2000.[Abstract]
  40. Crown J., O’Leary M. The taxanes: an update. Lancet, 355: 1176-1178, 2000.[Medline]
  41. Giannakakou P., Sackett D. L., Kang Y. K., Zhan Z., Buters J. T., Fojo T., Poruchynsky M. S. Paclitaxel-resistant human ovarian cancer cells have mutant ß-tubulins that exhibit impaired paclitaxel-driven polymerization. J. Biol. Chem., 272: 17118-17125, 1997.[Abstract/Free Full Text]
  42. Ryan C. W., Shulman K. L., Richards J. M., Kugler J. W., Sosman J. A., Ansari R. H., Vokes E. E., Vogelzang N. J. CI-980 in advanced melanoma and hormone refractory prostate cancer. Investig. New Drugs, 18: 187-191, 2000.[Medline]
  43. Nickel B., Schmidt M., Drevs J., Esser N., Marme D., Beckers T., Klenner T. Potent in vivo antitumor activity of D-24851, a novel tubulin inhibitor, in human xenografts and antimetastatic effect in the Renca model. Proc. Am. Assoc. Cancer Res., 42: 370 2001.



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