ANTICANCER RESEARCH 25: 3865-3870 (2005)

Combretastatin A-4 Resistance in H460 Human Lung Carcinoma Demonstrates Distinctive Alterations in ‚- Isotype Expression

H. WEHBE1,2, C.M. KEARNEY1 and K.G. PINNEY2

1Department of Biology and 2Department of Chemistry and Biochemistry, Institute of Biomedical Studies, Center for Drug Discovery, Baylor University, Waco, TX 76798, U.S.A.

Abstract. Tubulin isotype distribution may play a role in the site and the taxoid site (3). The and Vinca alkaloid development of anti- anti-tubulin drug resistance as well family ligands bind primarily to the unpolymerized ·,‚- as in drug efficacy and specificity. Stepwise selection was used to tubulin subunit and promote depolymerization, establish non-small cell lung carcinoma (NSCLC) H460 cells whereas the taxoid family ligands bind polymerized resistant to combretastatin A-4 (CA4), paclitaxel or vinblastine. and further stabilize polymerization. The Vinca The results demonstrated that the rate of CA4 drug resistance alkaloid site has been identified in ‚-tubulin at residues development was slower than that for paclitaxel. Western analysis 175-213 (4), and the taxoid site is near the M loop of demonstrated alterations in total ‚-tubulin and classes I, III and ‚-tubulin and the site of lateral interactions (5, 6). The IV tubulin isotypes among the resistant H460 cell lines. Class III colchicine binding site has not been conclusively identified ‚-tubulin was significantly altered in all resistant cell lines. Cells but is thought to be located at the interface of the two resistant to paclitaxel, a structural stabilizer of microtubules, monomeric subunits of the heterodimer (7, 8). exhibited an increased expression while cells resistant to CA-4 The two 50 kDa proteins of tubulin, ·- and ‚-tubulin, exist and vinblastine, structural destabilizers of tubulin, demonstrated as multiple sequence variants or isotypes. Human ‚-tubulin a reduction of the same isotype. To our knowledge, this is the first exists as six distinct isotypes designated as classes I, II, III, demonstration of resistance development and of the IVa, IVb and VI (9-11). These isotypes represent a highly corresponding tubulin isotype response for the combretastatins. homologous family of proteins that are well conserved between species and are grouped into different classes by the Microtubules participate in several cellular functions such unique carboxyl-terminal region (9). The relationship of as intracellular vesicle transport, mitosis, cell shape and ‚-tubulin and its isotypes with anti-mitotic agents suggests motility. Consequently, the basic unit of microtubules, that ‚-tubulin isotypes may have a significant regulatory tubulin, is the target of many anti-mitotic and vascular function in microtubules (12-14). Reports have also targeting agents. The potential of tubulin-binding agents as demonstrated altered isotype expression levels in cells anti-cancer drugs stems from their role in disrupting resistant to anti-mitotic anti-tubulin agents. Elevations in microtubule dynamics, which hinders mitosis and induces ‚II-, ‚III- and ‚IV-tubulin have been documented with apoptosis (1, 2). Considerable portions of the diverse group various cell lines with acquired resistance to paclitaxel of anti-mitotic drugs are natural products. (15-19). A synopsis of previous research suggests that The three small molecule tubulin-binding sites (known to significant increases in ‚III-tubulin expression levels play a date) are named according to the corresponding binding key role in a cell’s resistance to the polymerizing agent (20). ligand family and include the colchicine site, Vinca alkaloid Conversely, Kavallaris et al. (21) found a significant decrease in ‚III-tubulin in CCRF-CEM human leukemia cells resistant to the depolymerizing agents vinblastine or vincristine, which bind at the Vinca alkaloid site. Correspondence to: Christopher Kearney, Ph.D., Associate Combretastatins are tubulin-binding agents that are Professor, Department of Biology, Institute of Biomedical Studies, isolated from the South African tree Combretum caffrum (22- Center for Drug Discovery, Baylor University, One Bear Place 27). Combretastatin inhibits tubulin polymerization by binding #97388, Waco, TX 76798, U.S.A. Tel: 254-710-2131, Fax: 254-710- to the colchicine site of the ·,‚-tubulin subunit (28). Studies 2969, e-mail: [email protected] with the isolate combretastatin A-1 (CA-1) showed the drug Key Words: Microtubules, ‚-tubulin isotypes, resistance, to cause mitotic arrest in cultured cells with binding to tubulin combretastatin A-4. (26). Combretastatin A-4 (CA-4) has been found to be among

0250-7005/2005 $2.00+.40 3865 ANTICANCER RESEARCH 25: 3865-3870 (2005) the strongest anti-mitotic agents of the Combretum caffrum constituents, inhibiting tubulin polymerization and interfering with colchicine binding to tubulin (25). As shown in Figure 1, combretastatin A-4 (CA-4) has a structure which bears aspects of similarity to colchicine (24). We have established CA-4-resistant lung carcinoma lines and examined their ‚-tubulin isotype composition to determine if ‚-tubulin isotype patterns are related to cell Figure 1. Molecular structure of combretastatin A-4 and colchicine. resistance to CA-4. These results are the first to show altered ‚-tubulin expression levels in cells resistant to CA-4, an agent that binds at the colchicine site. For comparative analysis, we have also developed paclitaxel- and vinblastine-resistant lung each drug treatment, triplicate wells were seeded (5.0x103/well) and carcinoma. We were able to compare the CA-4 results to the conditioned overnight. The drugs were prepared by serial dilution with RPMI media and added to the corresponding wells. After 48 novel vinblastine-resistant cell line and a reproduced h, the MTT assay was performed. Percentage survival was paclitaxel-resistant lung carcinoma (29, 30). The results of our calculated as the OD560 of the treated sample divided by the OD560 study reveal that microtubule depolymerizers, CA-4 and of the untreated control cell line. The cell lines were tested with vinblastine induced similar isotype alterations, while the cisplatin to test for non-MDR phenotype. microtubule polymerizer, paclitaxel, induced different alterations. Our findings suggest that resistance to anti- Protein extraction and Western blotting. The cell lines were grown to tubulin agents of three unique tubulin-binding sites leads to confluency, pelleted and washed with cold PBS. The cells were lysed with 500 Ìl of ice-cold buffer (150 mM NaCl, 1% NP-40, altered ‚-tubulin isotype expression levels. 0.1% SDS, 50 mM Tris, pH 7.5, 2 mM PMSF, 5 mM Â-aminocaproic acid, 1 mM benzamide, 10 Ìg/ml apoprotein, 100 Materials and Methods Ìg/ml soybean trypsin inhibitor, 1 Ìg/Ìl leupeptin and 500 Ìl ‚-mercaptoethanol) for 30 min at 4ÆC. Samples were centrifuged Drugs. Combretastatin A-4 (CA-4) was obtained from PHARM-ECO at 10,000 xg for 10 min at 4ÆC. The supernatants containing total (Devens, MA, USA). Paclitaxel, vinblastine and verapamil were cellular protein were quantified using the BioRad protein assay purchased from ICN Biomedicals (Aurora, OH, USA), and cisplatin reagent (BioRad, Hercules, CA, USA). from Sigma (St. Louis, MO, USA). Stock solutions were prepared in Equal amounts of total protein (10 Ìg) were loaded onto 10% dimethyl sulfoxide (DMSO) and stored at –20ÆC. The working SDS-PAGE gel and stained with Coomassie blue to confirm equal solutions were diluted in culture medium to the desired concentration loading per cell line. Gels and blotters were equilibrated in before use; the highest concentration of DMSO was 0.1% (v/v). modified Towbin transfer buffer (25 mM Tris, 192 mM glycine, 20% methanol) for 15 min prior to transfer. PVDF membrane was Cell culture. A panel of cells lines obtained from the American Type pre-wet with methanol for 15 sec and then with transfer buffer for Culture Collection (Rockville, MD, USA) was initially assayed 15 min. Protein transfer was performed at 10 V for 20 min per against CA-4 to screen for drug sensitivity. The human non-small cell 1 mini-gel with BioRad Trans-Blot® Semi-Dry Transfer Cell. The lung carcinoma (NSCLC), NCI-H460, was selected for resistance gel was stained to confirm complete transfer of protein. Equal based on significant cytotoxicity. H460 cells were grown and loading and efficient transfer of proteins were confirmed by maintained in RPMI-1640 supplemented with penicillin-streptomycin, staining the membrane with Ponceau S red before fungizone® and 10% heat-inactivated fetal bovine serum (Invitrogen, immunodetection. The membrane was blocked for 1 h at RT with Carlsbad, CA, USA) and maintained at 37ÆC in a humidified 5% 5% dried non-fat milk in TBS-Tween buffer (20 mM Tris-HCl, pH CO2 atmosphere. The H460 parental cell line was selected for 7.5, 500 mM NaCl, 0.05% (v/v) Tween 20) and then incubated for resistance to CA-4, paclitaxel or vinblastine by incubation with 1 h at RT with monoclonal mouse antibodies against P-gp, ‚-, ‚I-, stepwise increments of each drug, beginning at 2 nM. Cells were ‚II-, ‚III-, or ‚IV-tubulin (1:500, Sigma). The membrane was treated with the drugs for 24 h, once a week, and every 2-3 weeks, the incubated for 1 h with horseradish peroxidase linked rabbit anti- drug dose was increased by 2 nM increments until 30 nM drug mouse secondary antibody (1:10,000, Sigma), washed, and resistance was reached. The cells were washed frequently with PBS developed with enhanced chemiluminescence (ECL) reagent to ensure removal of the drug. To select non-MDR-resistant cell (Amersham, Arlington Heights, IL, USA). Protein expression was lines, cells were evolved in the presence of 10 ÌM verapamil, an detected using a Fluor-S Max Imager (BioRad) and quantified with inhibitor of P-glycoprotein (P-gp) drug efflux pump that produces a Quantity One densitometry software (BioRad). The results were MDR phenotype. Resistance was maintained by acute 24-h exposure reported as a normalized ratio of average volume total pixel to the corresponding drug and verapamil once a month. All cells used number of the resistant cell to the parental H460 cell. Western for the experiments were grown logarithmically in drug-free medium blots were obtained from at least three independent experiments. at least a week before experiments. Statistical analysis. Data are expressed as the mean ± the standard In vitro cytotoxicity assay. Cytoxicity assays was assessed using a deviation from at least three independent experiements (performed modified MTT assay (31). MTT (3-(4,5-dimethylthiazol-2-yl)-2,5- in triplicate for MTT assays), unless otherwise noted. For diphenyl tetrazolium bromide) was purchased from Sigma. For quantitative Western analysis, the differences between the groups

3866 Wehbe et al: Combretastatin A-4 Resistance and ‚-Tubulin were analyzed using an Anova Single Factor test comparing the Table I. In vitro cytotoxicity assays: panel of cell lines with combretastatin resistant cell lines to the parental cell line data. Statistical A-4. significance was considered as p<0.05. Cell line IC50 (nM) Results CEM 0.25±0.03 MOLT-4 4.1±0.61 Cytotoxicity of CA-4. To select a moderately sensitive cell line HT29 5.51x104±0.88 5 for drug resistance, a panel of cell lines was screened for CA-4 MCF-7 1.65x10 ±0.31 H460 7.3±1.37 cytotoxicity (Table I). The leukemia cell lines CEM and BXPC-3 5.7x104±0.00 MOLT-4 showed high sensitivity with IC50 values of 0.25 nM DU-145 7.7±1.96 and 4.1 nM, respectively. Prostate (DU-145) and NSCLC-H460 aData represents the average ± STDEV of three independent carcinomas displayed high sensitivity with IC50 values of 7.7 nM and 7.3 nM. Pancreatic (BXPC-3), mammary (MCF-7) and experiments with each experiment run in triplicate wells. colorectal (HT29) cancer cell lines had much lower sensitivity to 4 5 CA-4 with IC50 values from 5.5x10 nM to 1.65x10 nM. From this data, the H460 cell line was selected for drug resistance Western analysis. ‚-tubulin and isotypes I, II, III and IV were against CA-4 as it showed significant cytotoxicity and a similar analyzed by Western blotting and compared to the parental IC50 value to paclitaxel and vinblastine (Table II). cell line (Figure 2). Total ‚-tubulin was increased in all resistant cell lines. This increase in total tubulin has been Resistance and cross-resistance of cell lines. Using step-wise previously reported for both depolymerizing and selection, drug resistance to CA-4, paclitaxel or vinblastine was polymerizing agents (17, 32). C30 cells showed a significant established in the NSCLC-H460 cell line in the presence of increase in ‚I-tubulin, with a 1.4-fold increase (p=0.002). verapamil, a P-gp drug efflux pump inhibitor. The C30, P30 No significant change in class II was detected. Class III and V30 cell lines represent H460 parental cells selected at 30 tubulin was significantly altered in resistant cell lines, with nM CA-4, paclitaxel and vinblastine, respectively. Resistance P30 cells showing an increase of 1.6-fold (p=0.0004) and was non-MDR-1-mediated as levels of P-gp protein were C30 and V30 showing a reduction by 1.6- and 1.3-fold, undetectable by Western analysis (data not shown). As shown respectively (p=0.0002, p=0.045 respectively). V30 in Table II, C30 cells were 3-fold resistant to CA-4, and cross- demonstrated a significant decrease in ‚IV-tubulin with resistance studies showed that the cells were 10-fold resistant reductions of 1.4-fold (p=0.015). to paclitaxel and negligibly resistant to vinblastine. P30 cells were 10-fold resistant to paclitaxel and 3-fold cross-resistant to Discussion vinblastine; the cells showed negligible resistance to CA-4. V30 cells were 5-fold resistant to vinblastine, 11-fold resistant to To our knowledge, this is the first report of the establishment paclitaxel and negligibly resistant to CA-4. Cisplatin, an anti- of CA-4 resistance and characterization of ‚-tubulin isotype cancer agent with a non-tubulin-binding mode of action, was alterations. In addition, the rate of resistance to CA-4 is assayed against the cell lines to test for the MDR phenotype. considerably lower than the rate of development of paclitaxel Compared to the parental cell line, C30, P30 and V30 resistance. The results of this study demonstrate that cytotoxicity to cisplatin was not altered (Table II). resistance to CA-4, paclitaxel and vinblastine, three agents

Table II. In vitro cytotoxicity assay: resistance and cross-resistance studies.

IC50 values (nM) and Resistance factor

H460wild C30* Resistance factor P30* Resistance factor V30* Resistance factor

CA-4 7.0±0.6 19.0±1.7 2.7±0.4 8.5±0.2 1.2±0.1 9.2±0.6 1.3±0.1 Paclitaxel 8.1±0.4 80.0±2.0 9.9±0.6 77.0±4.9 9.6±1.0 87.0±3.6 10.8±1.0 Vinblastine 5.9±0.1 6.0±0.3 1.0±0.0 15.7±4.2 2.7±0.7 26.3±2.1 4.5±0.4 Cisplatin 20.0±2.6 16.7±3.2 0.8±0.1 25.3±3.1 1.3±0.0 21.7±0.6 1.1±0.2 a Resistance factor is the IC50 value of the resistant cell divided by the IC50 value of the parental cell, which represents the fold-increase in resistance. bData represents the average ± STDEV of three independent experiments with each experiment run in triplicate wells. *p<0.0001

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agents have been limited compared to studies with paclitaxel and Vinca alkaloids. Furthermore, the severe toxicity of colchicine has limited its chemotherapeutic value and chemoresistance study. Hence, the combretastatins, which demonstrate clinical efficacy (37), are model agents to study the chemoresistance response to agents that bind at the colchicine site of tubulin. After five months of similar treatment, the level of resistance to the polymerizing stabilizer, paclitaxel, evolved faster than the depolymerizing agents, CA-4 and vinblastine. The P30 cells had a 10-fold resistance whereas the C30 and V30 cells were 3- and 5-fold resistant at 30 nM of drug. It is interesting that at 30 nM of CA-4 resistance, cells demonstrated an IC50 of 19 nM. Previous studies with other drugs have shown IC50 values of resistant cell lines at the maximum treated dose, but not at a lower concentration (17, 29, 30). This trait is a unique characterization of the CA-4-resistant cell line and is not true for previous resistant studies found in the literature. The molecular mechanism for this phenotype is the subject of further investigation, with the possibilities of altered microtubule dynamics or resistance stability. Our findings (Table II) demonstrate that alterations of total ‚-tubulin and isotypes I and III for cell lines resistant to the depolymerizing agent vinblastine are consistent with the data of Kavallaris et al. (19), who found an increase in Figure 2. Western analysis of the cellular protein extracts of parental and total tubulin, no change in ‚I- and a significant decrease in resistant H460 cells with antibodies to ‚-tubulin and ‚-tubulin isotypes I, II, ‚III-tubulin in CCRF-CEM cells resistant to vinblastine. In III and IV. Total protein was extracted in the presence of protease inhibitors our study, CA-4 induced changes similar to vinblastine for and quantified by the BioRad protein assay reagent. (A) Coommassie blue- ‚III- and total tubulin and a significant increase in stained polyacrylamide gel (10%) demonstrating equal loading of total ‚ -tubulin. In addition, our results indicated a decrease in protein (10 Ìg) onto 10% SDS-PAGE gel. (B) Representative Western blots I of samples incubated with antibodies to ‚-tubulin and ‚-tubulin isotypes I, ‚IV- tubulin for V30 cells. In contrast, Sirotnak et al. (39), II, III and IV. Ponceau S red was used to demonstrate equal transfer of found an increased expression of ‚I- and ‚IV-tubulin in protein. Blots were developed with enhanced chemiluminescence (ECL) Vinca alkaloid resistance of Chinese hamster ovary cells; reagent and protein expression was detected. The figure represents Western these results did not indicate any alteration of ‚III-tubulin. blots of three independent experiments. Western analysis results suggest that multidrug resistance did not play a role in establishing resistance as an increased expression of the drug efflux pump P-gp was not detected in the resistant cell lines. Because an increase of isotypes III and binding distinct tubulin sites, confers altered ‚-tubulin isotype IV is demonstrated in P30 cells, a decreased expression of the expression levels in NSCLC-H460. Our results demonstrate isotypes should increase sensitivity to paclitaxel. Interestingly, that ‚III-tubulin, an isotype linked to H460 paclitaxel cross-resistance studies of C30 and V30 cells with paclitaxel chemoresistance (33), is altered in the lung carcinoma cells (Table II) did not demonstrate this expected increase of resistant to combretastatin, paclitaxel, or vinblastine chemosensitivity. In fact, the cells showed a 10-fold resistance compared to the parental cell line. to paclitaxel. Cross-resistance only develops to paclitaxel and The relationship between the level of sensitivity or does not develop in the other drugs. A previous drug-resistant resistance to a given tubulin-binding agent and the cell line study in which the multidrug-resistant phenotype was expression of a given tubulin isotype has yet to be fully not present also revealed a similar cross-resistance to tubulin- understood. Studies have shown various mechanisms by binding agents (38). The mechanism for this cross-resistance which anti-tubulin drug resistance could be facilitated has not been identified, but this phenomenon indicates that including genetic mutations, altered isotype expression, cross-talk mechanisms are involved in chemoresistance. increased tubulin synthesis and tubulin isotype composition The tubulin analysis of paclitaxel-resistant lung carcinoma (19, 20, 32, 34-36). Studies on altered ‚-tubulin isotypes in cells was comparable to previous work with prostate human cancer cell lines resistant to colchicine-binding carcinoma by the Ranganathan group (17); total ‚-tubulin

3868 Wehbe et al: Combretastatin A-4 Resistance and ‚-Tubulin

Figure 3. Quantitative results of Western analysis levels of tubulin and tubulin isotypes in parental and resistant H460 cells. Bars representing ■H460 ■ C30 ■ P30 ■ V30. Protein expression was detected and quantified with BioRad’s Quantity One densitometry software. Bars represent the normalized ratio of the average volume pixel number and STDEV of resistant cell lines to parental H460 cell line per ‚-tubulin blotted. Western blots were obtained from three independent experiments. *p<0.05 and **p<0.005

levels and ‚III-tubulin were significantly increased. This distribution and specificity of certain drugs to individual study additionally revealed a slight increase in class IV tumor cell lines, may lead to improved therapeutic tubulin. The paclitaxel-resistant H460 cells of Han et al. (30) intervention in the treatment of solid core tumors. overexpress P-glycoprotein, and they also demonstrated an increase in total ‚-tubulin. Chu et al. (29) also showed an Acknowledgements increase in total ‚-tubulin with non-MDR-mediated paclitaxel-resistant H460 cells. Tubulin isotypes were not The authors are most appreciative of the generous financial analyzed in either of these latter studies. support provided by The Welch Foundation (Grant No. AA-1278 In conclusion, our results raise the possibility that the to KGP) and Oxigene, Inc., Waltham, MA, USA. sensitivity and resistance of tumors to anti-mitotic drugs can be modulated by the tubulin isotype composition of References microtubules in the tumor cell and further ascertain the importance of tubulin isotypes as determinants of 1 Jordan MA, Toso RJ, Thrower D and Wilson L: Mechanism of chemoresponse. In addition, the rate of development of mitotic block and inhibition of cell proliferation by taxol at low concentrations. Proc Natl Acad Sci USA 90: 9552-9556, 1993. resistance is drug-specific and probably tissue-specific. 2 Jordan MA, Wendell K, Gardiner S, Derry WB, Copp H and Further studies comparing the tubulin expression, in the Wilson L: Mitotic block induced in HeLa cells by low panel of cell lines originally assayed against CA-4, may concentrations of paclitaxel (taxol) results in abnormal mitotic reveal a correlation between drug sensitivity/resistance, exit and apoptotic cell death. Cancer Res 56: 816-825, 1996. tumor type and ‚-tubulin isotype levels. This study suggests 3 Nogales E: Structural insights into microtubule function. Ann that resistance to tubulin-binding agents with similar modes Rev Biochem 69: 277-302, 2000. of action, i.e. microtubule depolymerizers or polymerizer, 4 Rai SS and Wolff J: Localization of the vinblastine-binding site on ‚-tubulin. J Biol Chem 271: 14707-14711, 1996. confer similar alterations in ‚-tubulin isotypes, but further 5 Nogales E, Whittaker M, Milligan RA and Downing KH: High- studies on a panel of variable tissue cell lines are needed to resolution model of the microtubule. Cell 96: 79-88, 1999. satisfy this hypothesis. A clearer understanding of the 6 Nogales E, Wolf SG and Downing KH: Structure of the ·‚ tubulin seemingly complex tubulin system, including isotype dimer by electron crystallography. Nature 391: 199-202, 1998.

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J Med Chem Received August 11, 2005 38: 1666-1672, 1995. Accepted September 7, 2005

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