Comparison of the Effects of Vinblastine, Vincristine, Vindesine, and Vinepidine on Microtubule Dynamics and Cell Proliferation in Vitro

Home , Vinblastine, Vincristine, Vindesine

[CANCER RESEARCH 45, 2741-2747, June 1985]

Mary Ann Jordan,1 Richard H. Hirnes,2 and Leslie Wilson3

Department of Biological Sciences, University of California, Santa Barbara, California 93106 [M. A. J., L W.], and Department of Biochemistry, University of Kansas, Lawrence, Kansas 66045 [R. H. H.]

ABSTRACT ing RNA and DNA synthesis (11-13), lipid biosynthesis (13), cyclic nucleotide metabolism (14, 15), glutathione metabolism Vinepidine, a new derivative of vincristine, and three clinically (16), and calmodulin-dependent Ca2+-transport ATPase (17). In used Catharanthus derivatives, vinblastine, vincristine, and vin- fact, it is not clear to what extent the cell-killing and antitumor desine, were examined for their abilities to inhibit net tubulin properties of the Catharanthus alkaloids are due to their inter addition at the assembly ends of bovine brain microtubules at actions with tubulin and microtubules. In a number of cases, the steady state. Although all four derivatives were generally similar order of efficacy of the drugs against tumors does not correlate in potency, their relative abilities to inhibit tubulin addition were with reported binding strengths to tubulin (3). However, in vivo distinguishable. Vinepidine and vincristine were the most potent experiments are complicated by considerations of differences in derivatives (KÂ¡,0.079 Â±0.018 (SD) ^M and 0.085 Â±0.013 Â¿iM- delivery to the tumors, uptake of the drugs by the tumors, and respectively), followed by vindesine (KÂ¡,0.110 Â±0.007 fiM) and metabolic conversions. vinblastine (KÂ¡,0.178 Â±0.025 J/M). In contrast to their relative In the present study, we have evaluated the ability of a new abilities to inhibit microtubule assembly In vitro, vinblastine and vincristine derivative, vinepidine (4'-epi,4'-deoxyvincristine) its derivative, vindesine, were generally more potent than vincris (Chart 1), to inhibit the initial rate and final extent of microtubule tine and vinepidine in inhibiting cell proliferation in culture. Vin polymerization in vitro and have investigated the relative abilities blastine was nine times more potent than the weakest derivative, of vinepidine and 3 clinically used Catharanthus derivatives, vinepidine, in B16 melanoma cells. In L-cells, vinblastine com vincristine, vinblastine, and vindesine to inhibit net tubulin addi pletely inhibited growth at 40 nw, whereas vincristine and vin tion to the assembly ends of microtubules at steady state. The desine caused about 25% inhibition, and vinepidine was inactive. abilities of the 4 derivatives to inhibit tubulin addition to steady- When B16 melanoma cells were treated with drug before being state microtubules in vitro were compared with the abilities of injected into mice, retardation of tumor growth was best achieved the derivatives to inhibit proliferation of B16 melanoma and L- with vindesine, one of the weaker of the four derivatives In vitro. cells in culture. We found that the relative abilities of the 4 The results demonstrate that chemical differences among the derivatives to inhibit net tubulin addition to microtubule assembly Catharanthus derivatives, which affect to small extents the abili ends correlated poorly with the relative abilities of the derivatives ties of the derivatives to inhibit microtubule assembly In vitro, to inhibit cell growth. result in significant differences in the order and the magnitude of the abilities of the drugs to inhibit cell growth. MATERIALS AND METHODS

Purification of Microtubule Protein. Bovine brain microtubule protein INTRODUCTION was isolated by 3 cycles of depolymerization and polymerization in vitro in the absence of glycerol by the procedure of Asnes and Wilson (18) as The dimeric Catharanthus roseus alkaloids (commonly called modified by Farrell eÃal. (19). The preparation was stored frozen as Vinca alkaloids) are a group of antimitotic drugs used in cancer pellets at -70Â°C. Before use, pellets were thawed, suspended in cold chemotherapy. Individual C. roseus derivatives show different polymerization buffer (0.1 M 2-(W-morpholino)ethanesulfonic acid: 1 rnw degrees of efficacy toward a variety of tumors and also exhibit MgSCvl mM ethyleneglycol bis(/3-aminoethyl ether)-/V,A/,/v",/V'-tetraa- different toxic side effects (1-3). Thus, there has been interest cetic acid, pH 6.75) and centrifuged at 40,000 x g (5Â°C)for 20 min. The in developing new analogues of the natural compounds in order supernatant, consisting of approximately 70% tubulin and 30% MAPs,4 to improve effectiveness and to decrease toxicity. was used in polymerization and flux experiments. The 4 drugs were The actions of the Catharanthus alkaloids in cells have been either provided by Eli Lilly & Co. or were purchased. Microtubule Polymerization. Polymerization experiments were con ascribed, at least in part, to inhibition of microtubule function. ducted at 30Â°Cin 0.5-ml solutions of microtubule protein (1.6 to 2.5 mg/ They are known to disrupt microtubules in cells (4), and in vitro ml) in polymerization buffer containing 0.1 mw GTP and a GTP-regener- at relatively low concentrations, they inhibit assembly of tubulin ating system consisting of acetate kinase and acetyl phosphate (20). into microtubules (5-8), and at higher concentrations, they inter Solutions, initially at 0Â°C,were warmed to 30Â°Cto begin polymerization. act directly with microtubules to cause peeling of protofilaments Polymerization was quantitated by measurement of turbidity at 350 nm from microtubule walls (5, 8-10). However, the Catharanthus using a Gilford Model 2400 recording spectrophotometer equipped with alkaloids can affect a number of different cellular systems includ- a constant-temperature cuvet chamber. The rates of polymerization were determined from the slopes of the absorbance traces, measured between 1To whom requests for reprints should be addressed. 2 Recipient of American Cancer Society Grant CH-98. 20 and 80% of the maximum absorbance attained for each drug concen 3 Recipient of USPHS Grant CA36389 awarded by the National Cancer Institute, tration. The final extents of polymerization were determined from the Department of Health and Human Services, and by a grant-in-aid from Eli Lilly & absorbance at 60 min. The drug concentrations which produced 50% Co. Received 11/15/84; revised 3/5/85; accepted 3/7/85. 4 The abbreviation used is: MAP, microtubule-associated protein.

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dioactivity (26). Background adsorption to filters, which constituted 12 to 15% of the incorporation obtained in the absence of any drug, was determined by a 40-min [3H]GTP pulse in the presence of 50 ^M podophyllotoxin and was subtracted from all other sample values. Electron Microscopy of Drug-induced Polymers. The effect of the Catharanthus alkaloids on the structure of the polymers formed was also determined. After addition of the drugs either to solutions of microtubule protein followed by warming to 30Â°Cor to steady-state microtubules at CHjO 30Â°C, samples of the suspensions diluted and stabilized with a 5-fold excess of 30Â°C buffered 50% sucrose, were applied to pariodion and carbon-coated electron microscope grids for approximately 30 sec, drawn off by a torn edge of filter paper, then followed sequentially by cytochrome c (1 mg/ml, 15 sec), water (3 drops, drawn off), and 1% VLB CH3 COOCH3 OCOCHj CH2CH3 OH aqueous uranyl acetate (20 sec, drawn off). All electron microscopic YDS CH3 CONH2 OH CH2CH3 OH preparations were viewed in a Philips 300 electron microscope at 80 kV. Effects on Cell Proliferation. 816 melanoma tumor tissue provided VCR CHO COOCH3 OCOCHj CHjCH, OH by Eli Lilly & Co., was passed serially by bilateral s.c. implantation in the VPD CHO COOCH3 OCOCH3 H CH2CH3 axillary region of adult female C57BL/6 mice. Expiants were prepared in Dulbecco's modified Eagle's minimal essential medium from Grand Island Chart 1. Structures of vinblastine (VLB),vindesine(VOS),vincristine (VCR),and vinepidine (VPD). Vindoline moiety is the "lower" portion of the molecule, and Biological Co., containing 10% calf serum and passed about 20 times velbanamineis the "upper" portion of the molecule(3). before freezing in 10% dimethyl sulfoxide in liquid nitrogen. Frozen cells were thawed and grown for several passages before an experiment. inhibition of rate and final extent of polymerization were determined from Cells were plated in 2 ml of medium in 35-mm plates at a concentration log-concentration dependence plots. of about 5x10* cells/ml and grown for 24 h at 37Â°Cin an atmosphere Steady-State Flux Assay and Properties of Steady-State Bovine of 5% CO2 and 95% air. At this time, experiments were conducted to Brain Microtubule System. When assembled to steady state in vitro in determine (a) the acute or (b) the chronic effects of the drugs. To test the presence of a GTP-regenerating system, the critical subunit concen the effects of chronic treatment, the medium was replaced with fresh trations for growth are different for the 2 ends of the MAP-containing medium lacking or containing low concentrations of the drugs, and bovine brain microtubules used in this study. This results in growth at proliferation was continued for 3 days. Cell proliferation was measured one microtubule end (the net assembly end) and a precisely balanced in the presence of 4 nw drugs using B16 melanoma cells and 40 nw shortening at the opposite end (the net disassembly end) (21, 22). The drugs using L-cells (the latter provided by Dr. P. A. Kitos) in the same net addition of tubulin at assembly ends and balanced net loss at medium. Cell counts were done each day in a Coulter Counter after disassembly ends produce a flux or "treadmilling" of tubulin from one detaching the cells with trypsin and EDTA. To test the effects of acute end of the microtubule to the other in vitro. At steady state, a constant treatment, cells were treated with a 1 ^M concentration of the drugs for mean microtubule length and a constant number of microtubule ends are 2 h. The medium was removed, and the plates were washed 3 times maintained in suspension. Thus, the microtubules do not show evidence with phosphate-buffered saline and then incubated in fresh medium of the dynamic instability behavior reported recently for MAP-free micro lacking drugs. The medium was changed at Days 1, 3, 5, and 7. In all tubules by Mitchison and Kirschner (23) (discussed in further detail in cell proliferation experiments, duplicate plates were counted for each Ref. 24). time point. The SD in 90% of the measurements was usually less than Inhibition of the net addition of tubulin to the assembly ends of steady- 5% and never exceeded 10%. state bovine brain microtubules (flux inhibition) by drugs is measured The effect of treatment of B16 melanoma cells in culture with a high using [3H]GTP as a probe. Once steady state is attained, a trace of [3H]- concentration of drugs on the subsequent growth of tumors in mice was GTP is added which exchanges rapidly with unlabeled GTP bound at the also determined. Cells were grown on 100-mm dishes, treated for 2 h exchangeable guanine nucleotide site of soluble tubulin. Upon incorpo with 5 /Â¿Mdrug,washed 3 times, released with trypsin, and collected by centrifugation. The cell mass was suspended in Eagle's minimal essential ration of subunits at microtubule assembly ends, the labeled GTP be comes hydrolyzed to GDP and nonexchangeably bound in the microtu medium containing 8% calf serum at an approximate concentration of bule. In control microtubule suspensions, the [3H]guanine nucleotide is 2.5 to 3.0 x 107 cells/ml. A volume containing 2.5 x 106 cells was loaded incorporated in a time-dependent linear manner at a rate equivalent to onto a trocar, and the cells were injected into the axillary regions of adult approximately a 1.5-^m length of added subunits per microtubule per h female C57BL/6 mice. Both axillary regions received an injection. Tumors (24, 25). were removed and weighed after 17 days. For steady-state experiments, microtubule protein (2.1 to 2.2 mg/ml) in polymerization buffer containing 0.1 rriM GTP and the GTP regenerating system was polymerized to steady state at 30Â°C.All drugs were assayed RESULTS simultaneously using the same microtubule suspension. Aliquots (400 /Â¿I) of the steady-state microtubule suspension were added to prewarmed Effects of Vinepidine on Microtubule Assembly in Vitro. tubes containing desired concentrations of drugs. After 15 min of equili Vinepidine, like other Catharanthus alkaloids that have been bration, [3H]GTP (125 Ci/mol; ICN) was added to each sample tube. studied, inhibits microtubule assembly in a concentration-de Duplicate determinations of the flux incorporation of [3H]GTP for all drug concentrations were made 40 min after addition of the [3H]GTP pulse. pendent manner. Vinepidine inhibited the rate of microtubule polymerization by 50% at approximately 0.27 ^M (Chart 2), and The time used was in the linear portion of the flux-incorporation curve (26). After the [3H]GTP pulse, 100-jil samples diluted into 4 ml microtu- it inhibited the final extent of microtubule polymerization by 50% bule-stabilizing solution (30% glycerol: 10% dimethyl sulfoxide:0.1 M 2- at approximately 0.84 /IM (Charts 2 and 3). These K,s are similar (A/-morpholino)ethanesulfonic acid: 1 rrtM MgSO4: 1 mw ethyleneglycol to those reported previously for vinblastine, vincristine, and bisffi-aminoethyl ether)-A/,A/,N',W'-tetraacetic acid: 5 mw ATP, pH 6.75), vindesine (5, 6, 8). the microtubules were collected by filtering through glass fiber filters, At concentrations approximately 10-100-fold higher than that and the filters were assayed for trapped (microtubule-incorporated) ra required to cause 50% inhibition of polymerization, vinepidine

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Ends by Catharanthus Alkaloids. Interpretation of the inhibition of polymerization of microtubules from solutions of tubulin and microtubule-associated proteins is complicated because 2 proc esses are involved, nucleation (formation of nucleating centers) and elongation (growth of the microtubules). A simpler approach and perhaps one that is more relevant to the effects of the Catharanthus alkaloids on the dynamics of microtubules in pro liferating cells is to examine the net incorporation of tubulin at microtubule assembly ends after adding the drugs to microtu bules already at steady state. A single action is measured in this kind of assay, which depends solely upon the concentration of microtubule assembly ends in suspension. The abilities of vinepidine, vincristine, vinblastine, and vinde sine to inhibit net addition of tubulin dimers at assembly ends of steady-state microtubules were determined simultaneously on aliquots of single steady-state microtubule suspensions (Chart 4). Vinepidine and vincristine were the most potent inhibitors of assembly-end addition with K,s that were approximately half that 20 60 found for vinblastine and two-thirds that of vindesine (KÂ¡:vinepi TIME (minutes) dine, 0.079 Â±0.018 (SD) /Â¿M;vincristine, 0.085 Â±0.013 pu; Chart 2. Time course of inhibition of microtubule polymerization by vinepidine vindesine, 0.110 Â±0.007 Â¿Â¿M;vinblastine,0.178 Â±0.025 ^M. as measured by turbidity (absorbance) at 350 nm. Polymerization studies were conducted as described in 'Materials and Methods" at a microtubule protein Effects on Cell Proliferation in Vitro. The 4 drugs when present continuously ("chronic"-treatment regimen), inhibited concentration of 1.62 mg/ml (tubulin plus microtubule-associated proteins) and at the indicated concentrations of vinepidine. Absorbancy at 350 nm is presented as 816 melanoma cell proliferation in the 10~9 to 10~8 M concentra the percentage of maximum absorbance of the control. tion range. The order of effectiveness when compared at a single concentration, 4 x 10~9 M, was vinblastine > vindesine > vin cristine > vinepidine (Chart 5a). By comparing the concentrations of the drugs required to produce 50% inhibition of cell prolifera 100 tion, we found that the relative effectiveness was vinblastine, 1.0, vindesine, 0.77, vincristine, 0.45, and vinepidine, 0.11, ex actly the reverse of the order of potency for inhibition of steady- 80 Om state tubulin addition in vitro. To test the relative effectiveness Â¡I IS 60

I 0.2 0.5 1.0 2.0 VINEPIDINE CONCENTRATION, juM Chart 3. Inhibition of the extent of microtubule assembly at 60 min by vinepidine. Data were taken from the same experiment as partially shown in Chart 2.

produced a concentration-dependent depolymerization of microtubules and concomitant peeling and fraying of their ends with formation of filamentous spirals (Fig. 1, a and b). In addition to causing spiral formation from intact microtubules, vinepidine also 0.02 0.05 0.10 0.20 0.50 1.00 caused microtubule protein to polymerize directly into rings (at 5 CONCENTRATION Â¿IM)orspirals (at 50 UM) when added at the time the solutions Chart 4. Inhibition of steady-state tubulin flux by vinepidine (VPD). vincristine were warmed (Fig. 1, c to e). These ultrastructural effects are (VCR), vindesine (VOS), and vinblastine (VLB). Flux incorporation during a 40-min pulse with |3H|GTP was assayed as described under "Materials and Methods" at properties also shared by vinblastine, vincristine, and vindesine a microtubule protein concentration of 2.2 mg/ml and at the indicated drug (5,8-10,27,28). concentrations. Results are presented as percentage inhibition of incorporation into Inhibition of Steady-State Tubulin Addition to Microtubule a control nondrug-treated sample.

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1000 -

Days Charte. Effect of Catharanthus alkaloids on L-cell proliferation. Cells were grown as described under "Materials and Methods" in the absence and presence of 40 nw drugs. At Days 1,2, and 3 after addition of the drugs, cells were released from duplicate plates and counted in a Coulter Counter. VPD, vinepidine; VCR, vincristine; VOS, vindesine; VLB, vinblastine.

100 Table 1 8 10 Effect on tumor growth after treatment of cells with drugs Days Experimental conditions are given in "Materials and Methods." Chart 5. Effect of Catharanthus alkaloids on B16 melanoma cell proliferation, a, cells plated and grown as described under "Materials and Methods" in the absence DrugNone (mg)1.31of tumor and presence of 4 x 10~9 M drugs. At Days 1, 2, and 3 after addition of the drugs, Â±0.26s (12)" cells were released from duplicate plates and counted in a Coulter Counter; b, cells Vindesine 0.21 Â±0.065(12) treated for 2 h with a 1 JIM concentration of each drug, and after removal of the Vincristine 0.56 Â±0.14 (10) drugs, were allowed to proliferate. VPD, vinepidine; VCR, vincristine; VOS, vindesine; Vinepidine 0.64 Â±0.16 (12) VLB, vinblastine. VinblastineWt 0.67 Â±0.18 (10) Â°Mean Â±SD. 6 Numbers in parentheses, number of tumors. during acute treatment, cells were treated for 2 h with much higher concentrations (1 pM) of each drug and allowed to grow after removal of the compounds (Chart 50). Again the order of abilities of the derivatives to inhibit growth of cultured cells. effectiveness was vinblastine > vindesine > vincristine > vine The mechanism of microtubule assembly inhibition by vinblas tine has been studied extensively in recent years (see Refs. 5-8 pidine, with vinepidine being significantly less effective than the and 10). Microtubule assembly inhibition by low vinblastine con other 3 alkaloids, although as the time of proliferation was centrations occurs at very low vinblastine to tubulin ratios ("sub- extended, the differences among the 4 derivatives diminished. A stoichiometric poisoning"). While the mechanism is not under 10-fold higher concentration of drugs was required to inhibit proliferation of L-cells as compared with B16 melanoma cells in stood in detail, it seems clear that at concentrations that inhibit chronic treatment (Chart 6). With this cell line, vinblastine was polymerization but do not produce formation of filamentous again the most effective alkaloid (producing complete inhibition spirals, vinblastine acts by binding rapidly and reversibly to at 40 nw), and vinepidine was completely inactive, with little microtubule ends and inhibiting the rate of tubulin addition (8). It difference between vincristine and vindesine. In another type of is reasonable to expect that other vinblastine congeners will experiment, cultures of B16 melanoma cells were treated with inhibit microtubule polymerization in vitro in a manner similar to high concentrations of each alkaloid prior to injection into mice that of vinblastine. (see "Materials and Methods" and Table 1). All drugs retarded Vinepidine is a new vincristine derivative prepared at the Lilly research laboratories by modification of the parent compound. It the subsequent growth of tumors in the animals, with vindesine is identical to vincristine in the vindoline ("lower") moiety but being the most potent. Differences among vinepidine, vincristine, differs in the velbanamine ("upper") moiety in the R4 and R5 and vinblastine were not detected. substituents in the piperidine ring (see Chart 1). Vinepidine inhibited the initial rate and final extent of microtubule polymeri DISCUSSION zation in vitro by 50% at concentrations of 0.27 and 0.84 UM, respectively. It also caused depolymerization of microtubules One purpose of this study was to examine the action of and concomitant formation of filamentous spirals at microtubule vinepidine, a new derivative of vincristine, on microtubule polym ends at 10- to 100-fold higher concentrations than those required erization in vitro and on the proliferation of cells in culture. A for assembly inhibition. By these criteria, vinepidine appears second purpose was to determine the relative abilities of 4 active similar in action and relative potency to the 3 clinically used Catharanthus derivatives to inhibit the net addition of tubulin at Catharanthus derivatives, vinblastine, vincristine, and vindesine. the assembly ends of steady-state microtubules in vitro and to Using the flux assay, we found that like vinblastine, vinepidine, evaluate how well these relative abilities correlated with the vindesine, and vincristine inhibited net tubulin addition at micro-

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. Table 2 noma cell proliferation. At the concentration tested, vinblastine Relativeinhibitory effects of Catharanthusderivatives completely inhibited L-cell proliferation, vincristine and vindesine Inhibition of Order of potency inhibited weakly (~25%), and vinepidine was inactive. Vindesine VPD" = VCR > VDS > VLB Steady-state tubulin addition treatment of cultured B16 melanoma cells was 3 times as B16 melanomacell proliferation VLB > VDS > VCR > VPD L-cell proliferation VLB Â»VDS s VCR > VPD effective in preventing the subsequent tumor production by these Tumor production by B16 melanomacells VDS > VCR = VPD = VLB cells in mice as was treatment by the other 3 drugs. [This 8VPD, vinepidine;VCR, vincristine; VDS, vindesine;VLB, vinblastine. corroborates and extends a previous study (35) in which vinde sine was found to be more effective than vincristine using this tubule assembly ends in concentration-dependent manner. Tub experimental protocol.] ulin addition was exquisitely sensitive to drug inhibition, with half- The results of other studies in which the effects of the Cathar maximal inhibition occurring at much lower concentrations than anthus derivatives on cell proliferation were examined also do those required to inhibit microtubule assembly beginning with not correlate positively with the results of the tubulin-microtubule unassembled microtubule protein. Furthermore, use of the flux assay or the tubulin-binding affinities. Howard ef al. (14) steady-state assay in which the effect of the 4 derivatives was found the relative inhibition of L1210 cell proliferation to be measured simultaneously with a fixed concentration of microtu vindesine > vinblastine > vincristine, and Brade (1) and Sweeney bule ends permitted us to distinguish the relative potencies of ef al. (36) report vindesine to be more effective than vinblastine the 4 derivatives, which has been difficult to accomplish by other and vincristine in prolonging the life of mice bearing B16 mela in vitro polymerization assays. Vinepidine and vincristine were noma tumors. Reviews by Brade (1) and Gerzon (3) point out the most potent of the four Catharanthus alkaloids when tested that there is not a single order of effectiveness of these drugs in the flux assay (Table 2), although it is clear that the potencies when tested on varying tumor types nor is there a single order of the 4 derivatives differed by little more than a factor of 2. of toxicity. The abilities of the Catharanthus derivatives to inhibit tubulin The in vivo (whole-animal) variability undoubtedly reflects the flux in vitro might be expected to be related to the association many factors involved in getting drugs to and into tumor cells in constants for the binding of the Catharanthus derivatives to vivo. The lack of correlation between in vitro microtubule assays tubulin. Evaluation of the binding constants, however, is compli and the results we and others have obtained using cultured cells cated by the very large discrepancies in values reported in the may be the result of (a) differential rates and extents of cellular literature. For example, association constants for vinblastine as uptake and perhaps release of these 4 alkaloids and/or (b) high as 6 x 106/w (29, 30) and as low as 2.3 x 10"/M (31) have inhibitory effects of these alkaloids on cell processes not involv been reported. Similarly, reported constants for vincristine bind ing microtubules or tubulin. The differences we found between ing to tubulin range from 8 x 106/w (30) to 3.5 x 104/w (31, 32). the potency of vinblastine when (a) applied continuously to Interestingly, the wide variations that have been observed in the cultured cells or (b) applied briefly (2 h) and then washed out binding constants may be real and perhaps are reflecting suggest that differential uptake and release, even in cultured changes in the binding site determined by solution conditions or cells, may well be a significant factor. Lengsfeld ef al. (37) found different states of tubulin association. that vinblastine was released faster than vincristine from HeLa Despite the wide discrepancies in the absolute magnitudes of cells, although Donoso and Hirnes (35) found that differential the binding constants, there does seem to be internal consist effects of vindesine and vincristine on B16 melanoma cell prolif ency in the relative affinities for vinblastine, vincristine, and eration could not be explained by differential uptake or release. vindesine for tubulin (there have been no determinations for In summary, the data demonstrate that small chemical differ vinepidine). Investigators who observed the lowest binding affin ences among the Catharanthus derivatives which affect to small ities found that the affinity for vincristine was approximately 46% extents the abilities of the derivatives to inhibit microtubule higher than for vinblastine (31, 32). Those reporting the highest assembly in vitro result in significant differences in the order and affinities observed a binding constant for vincristine that was magnitude of the abilities of the drugs to inhibit cell growth. 33% higher than for vinblastine. In 2 studies, it was determined Thus, important differences in a pharmacological parameter such that vindesine bound to tubulin with an affinity between that of as cellular uptake may exist among the 4 drugs, and/or one or vincristine and vinblastine (33, 34). Although different tubulins all of the derivatives may inhibit cell growth by other mecha- with different degrees of purity and different methods were used nism(s) in addition to inhibition of microtubule polymerization. for determining the binding constants, it seems clear that the relative order of binding to tubulin is vincristine > vindesine > ACKNOWLEDGMENTS vinblastine which is identical to the order of potency for inhibiting The excellent technical assistance of Jeanne Ellermeier, Mary Barron, Dr. tubulin addition to microtubule assembly ends. Indeed, the rela Timothy O'Brien, and PeterStaats is gratefully acknowledged.We thank Dr. Gerald tive binding affinities observed in one study in which all 3 com E. Gutonski and GeorgeBoder for supplyingthe Catharanthusalkaloids, Dr. Gerald pounds were compared (34) was 1:0.7:0.5 (vincristine:vin- A. Poorefor providing the B16 melanomatumor line, and Herb Miller for preparing the microtubule protein. We also appreciate the help provided by Dr. J. Alejandro desine:vinblastine) which correlates very well with their relative Donoso. abilities to inhibit steady-state tubulin flux (1:0.8:0.5, vincristine:vindesine:vinblastine). We found, however, that the relative flux inhibitory abilities of REFERENCES these drugs did not correlate with the relative abilities of the 1. Brade, W. P. Critical review of pharmacology,toxicology, pharmacokineticsof drugs to inhibit cell proliferation (Table 2). Vinblastine, which was vincristine, vindesine,vinblastine.Contrib. Oncol., 6: 95-123,1980. 2-fold less potent than vinepidine in blocking tubulin flux in vitro, 2. Creasey, W. A. The Vinca alkaloids and similar compounds. Cancer Chemo- ther., 3: 79-96, 1981. was 9 times more effective than vinepidine against B16 mela 3. Gerzon, K. DimericCatharanthusalkaloids.In: J. M. Cassadyand J. D. Douros

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(eds.). Anticancer Agents Based on Natural Product Models, pp. 271-317. substoichiometric nucleotide levels using a GTP regenerating system. New York: Academic Press, Inc., 1980. Biochem. Biophys. Res. Commun., 74: 440-447, 1977. 4. George, P., Journey, L. J., and Goldstein, M. N. Effect of vincristine on the fine 21. Margolis, R. L., and Wilson, L. Opposite end assembly and disassembly of structure of HeLa cells during mitosis. J. Nati. Cancer Inst., 35.' 355-375, microtubules at steady state in vitro. Cell, 73:1-8,1978. 1965. 22. Farrell, K. W., and Jordan, M. A. A kinetic analysis of assembly-disassembly 5. Himes. R. H., Kersey. R. N., Heller-Bettinger, I., and Samson, F. E. Action of at opposite microtubule ends. J. Biol. Chem., 257: 3131-3138, 1982. the Vinca alkaloids vincristine, vinblastine, and desacetyl vinblastine amide on 23. Mitchison, T., and Kirschner, M. Dynamic instability of microtubule growth. microtubules in vitro. Cancer Res., 36. 3798-3802,1976. 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Fig. 1. Electron micrographs of microtubules and microtubule protein treated with vinepidine. In a, microtubule protein (1.7 mg/ml) was assembled to steady state. Vinepidine (final concentration, 5 UM)was added, and incubation was continued for 70 min before negative staining. Microtubule ends are clearly frayed. Rings and a few spirals (arrows) are present, x 86,000; oar, 0.2 Â»m.b, same as a, except that vinepidine concentration was 50 UM. Long spirals are seen both attached to microtubule ends and free in suspension. Rings are also present, x 86,000; oar, 0.2 ^m. In c to e, vinepidine was added before initiation of assembly. Samples were taken for negative staining after 90 min of incubation. In c and d, vinepidine (5 Â¡M)produces spirals and rings, x 100,000; bar, 0.1 pm.

CANCER RESEARCH VOL. 45 JUNE 1985 2746

CANCER RESEARCH VOL. 45 JUNE 1985 2747

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1985 American Association for Cancer Research. Comparison of the Effects of Vinblastine, Vincristine, Vindesine, and Vinepidine on Microtubule Dynamics and Cell Proliferation in Vitro

Mary Ann Jordan, Richard H. Himes and Leslie Wilson

Cancer Res 1985;45:2741-2747.

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