REVIEW ARTICLE
`Mechanisms of Action of and Resistance to Antitubulin Agents:
`Microtubule Dynamics, Drug Transport,
`and Cell Death
`
`By Charles Dumontet and Branimir I. Sikic
`
`Purpose: To analyze the available data concerning
`mechanisms of action of and mechanisms of resistance
`to the antitubulin agents, vinca alkaloids and taxanes,
`and more recently described compounds.
`Design: We conducted a review of the literature on
`classic and recent antitubulin agents, focusing particu-
`larly on the relationships between antitubulin agents
`and their intracellular target, the soluble tubulin/
`microtubule complex.
`Results and Conclusion: Although it is widely ac-
`cepted that antitubulin agents block cell division by
`inhibition of the mitotic spindle, the mechanism of action
`of antitubulin agents on microtubules remains to be
`determined. The classic approach is that vinca alkaloids
`depolymerize microtubules, thereby increasing the
`soluble tubulin pool, whereas taxanes stabilize microtu-
`bules and increase the microtubular mass. More recent
`
`data suggest that both classes of agents have a similar
`mechanism of action, involving the inhibition of microtu-
`bule dynamics. These data suggest that vinca alkaloids
`and taxanes may act synergistically as antitumor agents
`and may be administered as combination chemother-
`apy in the clinic. However, enhanced myeloid and neu-
`rologic toxicity, as well as a strong dependence on the
`sequence of administration, presently exclude these
`combinations outside the context of clinical trials. Al-
`though the multidrug resistance phenotype mediated
`by Pgp appears to be an important mechanism of
`resistance to these agents, alterations of microtubule
`structure resulting in altered microtubule dynamics
`and/or altered binding of antitubulin agents may consti-
`tute a significant mechanism of drug resistance.
`J Clin Oncol 17:1061-1070. r 1999 by American
`SocietyofClinicalOncology.
`
`TUBULIN-BINDING AGENTS constitute a large fam-
`
`ily of compounds that have been used in a wide
`variety of ways, including as herbicides and antiparasitics
`and in human therapeutics. The first tubulin-binding agent to
`be used in humans was colchicine, extracted from Colchi-
`cum autumnale, which has been administered to patients
`with gout since sixth century AD.1 The ability of colchicine
`to block cells in metaphase made it a powerful tool in the
`study of mitosis.2 Tubulin, the building block of microtu-
`bules, was first identified as the ‘‘colchicine-binding pro-
`tein.’’3 The ability of some compounds to act electively on
`nonhuman cells, such as yeast, has been shown to be due to
`differences in these compounds’ abilities to bind to human
`versus nonhuman tubulins. Despite structural constraints,
`significant variations in the primary structure of tubulin, as
`well as the emergence of various isotypes, have occurred
`during evolution.4
`tubulin-
`In the field of antineoplastic chemotherapy,
`binding agents constitute an important class of compounds,
`with broad activity both in solid and in hematologic
`neoplasias.5-11 These agents are believed to block cell
`division by interfering with the function of the mitotic
`spindle, blocking the cells at the metaphase/anaphase junc-
`tion of mitosis.12,13 Vinca alkaloids, the earliest tubulin-
`binding agents to be used in the clinic as antimitotics, have
`been described as ‘‘microtubule depolymerizing agents.’’ At
`high concentrations,
`these agents reduce or abolish the
`
`microtubule content of cells in culture and prevent polymer-
`ization of purified tubulin in vitro. Conversely, the taxanes
`paclitaxel and docetaxel promote the polymerization of
`purified tubulin in vitro and, at high concentrations, enhance
`the fraction of polymerized tubulin in cells and they have
`thus been referred to as ‘‘microtubule stabilizing agents.’’
`
`MICROTUBULE STRUCTURE AND FUNCTION
`Microtubules are composed of a backbone of tubulin
`dimers and microtubule-associated proteins (MAPs).14 Al-
`pha- and beta-tubulin peptides, both of which have molecu-
`lar masses close to 50 kd, combine stoichiometrically to
`form tubulin dimers. Gamma-tubulin, which is less abun-
`dant, appears to be localized in the centrosomes.15 Chaper-
`
`From the Service d’He´matologie, Centre Hospitalier Lyon Sud,
`Pierre Be´nite, France; and Oncology Division, Department of Medi-
`cine, Stanford University School of Medicine, Stanford, CA.
`Submitted July 7, 1998; accepted November 4, 1998.
`Supported in part by National Institutes of Health grant nos. RO1 CA
`52168 and RO1 CA 68217 (B.I.S.), Department of the Army grant no.
`DAMD 17-94-J-4352 (B.I.S.), the Ligue Contre le Cancer de Saoˆne et
`Loire (C.D.), and the Association Pour la Recherche Contre le Cancer
`(C.D.).
`Address reprint requests to Dr Charles Dumontet, Service
`d’He´matologie, Centre Hospitalier Lyon Sud, 69495 Pierre Be´nite
`Cedex, France; email cd@hematologie.univ-lyon1.fr.
`r 1999 by American Society of Clinical Oncology.
`0732-183X/99/1703-1061
`
`JournalofClinicalOncology, Vol 17, No 3 (March), 1999: pp 1061-1070
`
`1061
`
`Information downloaded from jco.ascopubs.org and provided by at INFOTRIEVE on June 20, 2014 from 216.33.62.192
`Copyright © 1999 American Society of Clinical Oncology. All rights reserved.
`
`002009
`
`AVENTIS EXHIBIT 2016
`Mylan v. Aventis, IPR2016-00712
`
`

`
`1062
`
`onins as well as proteins involved in tubulin folding appear
`to play an essential role in the synthesis of functional tubulin
`subunits.16 Alpha- and beta-tubulins have been studied in
`many species, gamma-tubulin has been studied in a few, and
`sequence analyses have demonstrated strong conservation
`throughout evolution from yeast to human.4 Alpha- and
`beta-tubulins exist under the form of isotypes, which are
`distinguished by slightly different amino acid sequences.17,18
`Thanks to the work of Cowan et al19-21 and Dobner et al,22
`six alpha- and six beta-tubulin isotypes have been described
`in mammals. The analysis of human tubulin genes has been
`complicated by the fact that many of the genes of the tubulin
`multigene family, identified by screening of genomic librar-
`ies, are in fact pseudogenes, which do not code for intact
`proteins.23 The six mammalian beta-tubulin isotypes may be
`grouped into six classes, according to their C-terminal
`amino acid composition, which is the most highly divergent
`portion between isotypes, although they are highly con-
`served between species (Table 1). Posttranslational modifica-
`tions have been reported, including phosphorylation and
`glutamylation (reviewed in Luduena18).
`The strong intraspecies conservation of beta-tubulin iso-
`types has prompted a number of investigators to search for
`functional differences specific to the various isotypes. Analy-
`sis of tubulin isotype expression in various tissues has
`demonstrated a complex pattern of distribution, suggesting
`functional specificity. In neurons,
`there is evidence of
`isotype segregation within cells, as well as differential
`synthesis and phosphorylation during neurite outgrowth.24
`Conversely, immunohistochemical analyses of various micro-
`tubules (spindle, interphase, midbody, manchette, flagella)
`have failed to show segregation of isotypes into specialized
`microtubular structures, as have experiments with trans-
`fected tubulin isotypes.25,26 The nature and degree of the
`functional specificities of beta-tubulin isotypes remain con-
`troversial.18
`
`DYNAMICS AND FUNCTION
`Microtubules are highly dynamic structures that are in
`unstable equilibrium with the pool of soluble tubulin dimers
`
`DUMONTET AND SIKIC
`
`present in the cell. There is constant incorporation of free
`dimers into the polymerized structures and release of dimers
`into the soluble tubulin pool. Polymerization of tubulin
`dimers may be influenced by a number of factors, such as
`guanosine triphosphate, which binds to one exchangeable
`site on beta-tubulin and one nonexchangeable site on
`alpha-tubulin; the ionic environment; and MAPs. MAPs
`constitute a complex family of proteins, including MAP2,
`MAP4, Mip-90, tau, and STOP, many of which have been
`shown to regulate tubulin polymerization and function.27-31
`Many results have been reported on tubulin polymerization,
`with studies using highly purified tubulin, usually obtained
`from bovine brain, an abundant source. The development of
`real-time contrast videomicroscopy has allowed direct visu-
`alization of the behavior of individual microtubules.
`Microtubule ends have the ability to switch stochastically
`between growing and shortening states, both in cells and in
`vitro. This phenomenon, called dynamic instability, is an
`essential property that makes microtubules some of the most
`plastic protein polymers in the cell.32 Microtubules have a
`plus end, which is kinetically more dynamic than the other
`(the minus end). Although both ends alternately grow or
`shorten, net growing occurs at
`the plus end and net
`shortening at the minus end. When both of these actions
`occur simultaneously, the microtubule is said to be treadmill-
`ing, a phenomenon that is believed to be critical in the polar
`movement of chromosomes during anaphase.33
`Microtubules are complex polymeric structures that are
`involved in a number of cellular functions.3,14 They play a
`critical role not only in mitosis but also in intracellular
`transport, axonemal motility, and constitution of the cytoskel-
`eton. The abundant amount of tubulin in neurons and the role
`of microtubules in axonal transport are thought to contribute
`to the neurologic toxicity of tubulin-binding agents in the
`clinic.34 It is widely accepted that the antimitotic effect of the
`tubulin-binding agents used as anticancer agents is due to
`their effect on the mitotic spindle. However, these com-
`pounds also affect microtubules in interphase cells, altering
`neurite morphogenesis, as well as adhesion and locomotion
`properties.35-37 Other antitumor effects of taxanes have been
`
`Table 1. Beta-Tubulin Isotypes in Vertebrates
`
`Class
`
`Human
`
`I
`II
`III
`IVa
`IVb
`V
`VI
`
`M40
`hb
`hb
`h5b
`hb
`ND
`hb
`
`Isotype
`
`Chicken
`
`cb
`b1/cb
`b
`
`9
`4
`
`—
`
`b
`cb
`b
`
`2
`
`1
`
`c
`c
`
`c
`
`c
`
`Mouse
`
`b5
`7
`b2
`2
`4
`b
`mb4
`3
`b3
`5
`D
`6
`b
`
`% Homology
`(mouse/ human)
`
`m
`
`m
`
`m
`
`m
`N
`m
`
`100
`100
`6
`9
`100
`100
`—
`1
`1
`
`Abbreviation: ND, not described in this species.
`
`C-Terminal Sequence
`
`Expression
`
`EEEEDFGEEAEEEA
`DEQGEFEEEGEEDEA
`9
`EEEGEMYEDDEEESESQGPK
`EEGEFEEEAEEEVA
`EEGEFEEEAEEEVA
`NDGEEEAFEDDEEEINE
`9
`EEDEEVTEEAEMEPEDKGH
`
`All tissues
`Major: neuronal, many tissues
`Minor: neuronal
`Major: neuronal
`Major: testis, many tissues
`All tissues except in neurons
`Hematopoietic specific
`
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`Copyright © 1999 American Society of Clinical Oncology. All rights reserved.
`
`

`
`TUBULIN-BINDING AGENTS AND CANCER
`
`described that appear to be independent of the antimitotic
`activity. Paclitaxel modifies the motility of paclitaxel-
`resistant ovarian carcinoma cells in vitro and displays
`antiangiogenic activity in vivo.38,39 The specific action of
`tubulin-binding agents on the mitotic spindle may be
`attributed to the fact that mitotic microtubules are consider-
`ably more dynamic than interphase microtubules, with a
`much shorter half-life.40 Conversely, the absolute require-
`ment of a functional spindle for the proper migration of
`chromosomes during anaphase may explain why this stage
`of the cell cycle is particularly vulnerable to tubulin active
`agents, even though these compounds act on other cellular
`microtubules as well.41
`
`HOW TUBULIN-BINDING AGENTS WORK
`Despite considerable efforts, the exact binding sites of
`tubulin-binding agents on microtubules have not been
`identified. However, Nogales et al42 recently presented the
`results of a crystallographic analysis that defined the pacli-
`taxel binding site more precisely. Although it has been
`shown that tubulin dimers are the targets of these com-
`pounds, whether the beta-tubulin subunit is the exclusive
`binding site for these compounds has not been clearly
`determined.1,43,44 Although evaluation of total accumulation
`of labeled compounds in cells is technically straightforward,
`quantification of drug binding to microtubules is more
`difficult. Cells displaying the multidrug resistance (MDR)
`phenotype have a reduced amount of total drug, because of
`increased drug efflux. However, to date, there are no reports
`describing a specific association between resistance to
`tubulin-binding agents and reduced drug binding to microtu-
`bules.
`their effects on
`Colchicine and vinca alkaloids exert
`microtubules under different conditions. Unlike vinca alka-
`loids, colchicine must first bind to soluble tubulin before
`acting on microtubule dynamics. At substoichiometric con-
`centrations (, one molecule of drug for each molecule of
`tubulin), these compounds dramatically affect microtubule
`dynamics, without causing depolymerization.32 It is believed
`that
`tubulin-colchicine and tubulin–vinca alkaloid com-
`plexes, and unbound vincas, bind to and ‘‘poison’’ microtu-
`bule ends, changing both on- and off-rate constants, thereby
`considerably reducing their ability to grow or shorten.45 At
`higher concentrations, these compounds bind stoichiometri-
`cally to tubulin subunits and can induce rapid polymer
`disassembly, giving rise to nonmicrotubular structures such
`as vincristine-induced spiral protofilaments. The net effect
`of these high concentrations is a reduction or a disappear-
`ance of the normal microtubule network of the cell.
`Taxanes, on the other hand, bind to polymerized tubulin
`only.46 There is a binding site for paclitaxel on each tubulin
`
`1063
`
`dimer in microtubules, and the ability of paclitaxel to induce
`polymerization is associated with stoichiometric binding of
`paclitaxel to microtubules. However, at lower, substoichio-
`metric, concentrations (one molecule of paclitaxel for 200 to
`600 molecules of tubulin), paclitaxel suppresses microtubule
`dynamics without significantly altering the microtubule
`polymer mass.47,48 Paclitaxel also modifies the rigidity of
`microtubules, an effect that may contribute significantly to
`its effect on mitosis.49 Thus, at very low concentrations, all
`of these compounds share the ability to reduce microtubule
`dynamics while not significantly affecting the amount of
`polymerized tubulin.
`Attempts have been made to correlate the isotype compo-
`sition of microtubules with their dynamic properties and/or
`their different abilities to bind tubulin-binding agents. Lu-
`duena et al50 reported that colchicine binding was biphasic in
`preparations of bovine brain tubulin, which is a mix of
`classes I, II, III, and IV, but monophasic in the case of renal
`tubulin, which does not contain class III beta-tubulin.
`Falconer et al51 showed that colchicine-stable microtubules
`preferentially incorporate class II beta-tubulin. Lobert et al52
`reported that the interaction of vinblastine with tubulin is
`identical for all beta-tubulin isotypes but
`that class III
`beta-tubulin differs from unfractionated tubulin in its ability
`to associate into paclitaxel-stabilized microtubules. Laferri-
`ere and Brown53 found that paclitaxel promoted the polymer-
`ization and posttranslational modifications of class III beta-
`tubulin in an embryonal carcinoma cell line. Panda et al54
`reported that immunopurified isotypes of tubulin display
`different assembly properties in vitro. Derry et al55 showed
`that paclitaxel differentially modulates the dynamics of
`microtubules assembled from unfractionated and purified
`beta-tubulin isotypes.
`Taken together, these data suggest that tubulin isotypes
`may be important determinants of microtubule dynamics.
`These results, as well as those showing altered tubulin
`isotype content in resistant cell lines, suggest that the isotype
`composition of microtubules may influence sensitivity to
`tubulin-active agents. However, the tubulin isotype profile
`of mammalian cells is complex and is variable from one
`tissue to another. At present, no simple relationship has been
`established between the level of expression of a given
`tubulin isotype and the degree of sensitivity or resistance to a
`given tubulin-binding agent.
`
`MECHANISMS OF RESISTANCE TO ANTITUBULIN
`DRUG TRANSPORT
`At present, the best described mechanism of resistance to
`tubulin-binding agents is the MDR phenotype, mediated by
`the 170-kd Pgp efflux pump, encoded by the mdr1 gene.56,57
`
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`Copyright © 1999 American Society of Clinical Oncology. All rights reserved.
`
`

`
`1064
`
`Both the vinca alkaloids and the taxanes are good substrates
`for this pump.58,59 In a number of cases, development of cell
`lines resistant to vincristine or paclitaxel has been shown to
`be associated with the expression of mdr1.57,60 The multi-
`drug resistance protein has also been shown to be an efficient
`transporter of vinca alkaloids, but not taxanes.61,62 Presently,
`little is known concerning the significance of the MDR
`phenotype in the emergence of resistant tumors in patients
`treated with tubulin-binding agents. Clinical trials aiming to
`sensitize MDR-positive tumors to agents such as vinblastine
`with Pgp modulators have been disappointing.63
`Altered metabolism and/or subcellular distribution, alter-
`ations of the interaction between drugs and their target
`(microtubules), and altered response to cell cycle arrest
`induced by mitotic blockage are among the possible non-
`MDR mechanisms of resistance to tubulin-binding agents
`(Fig 1). To date, there have been no reports of cell lines that
`are resistant to tubulin-binding agents because of altered
`metabolism of these compounds. Regulation of glutathione
`levels by buthionine sulfoximine has been reported to
`influence paclitaxel-induced cytotoxicity, but it is not clear
`whether this is due to an effect on drug metabolism or to a
`direct interaction between glutathione and tubulin.64,65
`
`MICROTUBULE DYNAMICS AND RESISTANCE
`TO TUBULIN-BINDING AGENTS
`Cabral et al66-68 described a model in which resistance to
`tubulin-binding agents is associated with the presence of
`alterations in microtubule stability. According to these
`authors, some cells contain ‘‘hypostable’’ microtubules, with
`a spontaneous tendency toward depolymerization, and ‘‘hy-
`
`DUMONTET AND SIKIC
`
`perstable’’ microtubules, with a relative resistance to depoly-
`merization. In this model, cells with hypostable microtu-
`bules are particularly susceptible to the depolymerizing
`agents and display hypersensitivity to vinca alkaloids while
`displaying resistance to the stabilizing agents (Fig 2).
`Conversely, cells containing hyperstable microtubules are
`resistant to the vinca alkaloids but relatively sensitive to the
`taxanes. This model offers an explanation for the phenom-
`enon of paclitaxel-dependent cell lines, in which cells do not
`grow in the absence of paclitaxel.69 According to this model,
`the dependence on paclitaxel is due to the presence of
`extremely hypostable microtubules that, in the absence of a
`stabilizing agent, disassemble spontaneously and are incom-
`patible with normal cell function.
`Using clinically relevant concentrations of vinblastine
`and paclitaxel, Jordan et al12,13,70 showed that both depoly-
`merizing and stabilizing agents exert antimitotic effects by
`reducing spindle microtubule dynamics, with no significant
`alteration in the distribution of tubulin between the soluble
`and the polymerized forms. Using real-time differential-
`interference contrast videomicroscopy, these authors ana-
`lyzed the dynamic behavior of individual microtubules and
`found that vinblastine strongly reduces microtubule dynam-
`ics, without significantly modifying the length of the micro-
`tubules (or absolute microtubular mass). Analyzing the
`effects of paclitaxel at low concentrations, these authors
`found the same effect on microtubule dynamics, with no
`significant alteration in microtubule length. In terms of the
`interactions of tubulin-binding agents with microtubules, the
`most meaningful equilibrium to consider may therefore be
`between highly dynamic microtubules and less dynamic
`
`Fig 1. Potential mechanisms of
`resistance to tubulin-binding agents
`(TBA). 1: Efflux of drug by a mem-
`brane pump. 2: Altered metabolism
`or distribution of agent. 3: Altered
`interaction of agent with microtu-
`bules. 4:
`Inadequate induction of
`apoptotic signal.
`
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`Copyright © 1999 American Society of Clinical Oncology. All rights reserved.
`
`

`
`TUBULIN-BINDING AGENTS AND CANCER
`
`1065
`
`Fig 2. Models describing effects
`of tubulin-binding agents on soluble
`tubulin/microtubule complex. (A) Ca-
`bral model: Equilibrium between
`soluble tubulin dimers and polymer-
`ized tubulin (microtubules). Hy-
`postable microtubules are sensitive
`to vinca alkaloids, hyperstable mi-
`crotubules to taxanes.
`(B) Jordan
`and Wilson model: Equilibrium be-
`tween highly dynamic microtubules
`and weakly dynamic microtubules.
`Binding of a drug to microtubules
`reduces or suppresses dynamics of
`highly dynamic microtubules, form-
`ing stabilized microtubules.
`
`microtubules, rather than between polymerized and soluble
`tubulins (Fig 2).
`These two models differ significantly in their prediction of
`cross-resistance to the vinca alkaloids and the taxanes. The
`Cabral model suggests that cells resistant to depolymerizing
`agents may be sensitive to stabilizing agents and vice versa.
`Conversely, in the Jordan and Wilson model, these two types
`of compounds exert the same suppressive effects on microtu-
`bule dynamics, and cells resistant to one class of compounds
`may thus be cross-resistant to the other, at least in terms of
`interaction with the intracellular target. However, the concen-
`trations involved in the two models differ greatly, and the net
`effect on microtubule polymerization, a critical parameter in
`the Cabral model, probably occurs only at high concentra-
`tions of drugs. These high concentrations, which may allow
`stoichiometric interaction between the tubulin-binding agents
`and tubulin, may be difficult or impossible to achieve
`clinically.
`There is a growing body of evidence suggesting that some
`combinations of vinca alkaloids and taxanes may be benefi-
`cial in terms of antitumor activity. Aoe et al71 reported
`synergy between vinorelbine and docetaxel on a human lung
`cancer cell line in vitro, and Photiou et al72 showed synergy
`between paclitaxel and vinorelbine against human mela-
`noma lines. In the P388 murine model, Knick et al73 reported
`not only a significant percentage of long-term cures with the
`combination of vinorelbine and paclitaxel, but also a re-
`duced toxicity of these agents when they were used in
`combination. Of note is the importance of the delay between
`the administration of these two agents: the same doses were
`lethal to 80% of the animals when administered 24 hours
`
`apart but well tolerated when administered less than 6 hours
`apart. Preliminary reports of combinations of vinorelbine
`with paclitaxel or docetaxel in patients with advanced breast
`cancer or lung cancer suggest promising activity with no
`substantial increase in toxicity.74-76 Conversely, Monnier et
`al,77 who studied the effects of the combination of docetaxel
`and vinorelbine in 26 chemotherapy-naive patients with
`non–small-cell lung carcinoma, reported substantial hemato-
`logic and mucosal
`toxicity, with two toxic deaths, and
`studies of paclitaxel-vinorelbine combinations showed se-
`vere and/or frequent neurotoxicity.78,79 Additional clinical
`data are clearly required to evaluate the benefit of the
`combination of vinca alkaloids and taxanes, and such
`combinations should not be administered outside prospec-
`tive clinical trials.
`
`TUBULIN GENES AND DRUG RESISTANCE
`The available data suggest that alterations in microtubule
`structure and/or function represent an important, and poten-
`tially complex, mechanism of resistance to tubulin-binding
`agents. A number of cell lines resistant to tubulin-binding
`agents in vitro have been shown to contain tubulin alter-
`ations, in terms of total tubulin content, tubulin polymeriza-
`tion, or tubulin isotype content.80-82 We reported that the
`KPTA5 cell line, which is exclusively resistant to taxanes,
`displays increased expression of the class IVa tubulin
`isotype.82 Conversely, the KCVB2 cell line, which does not
`express mdr1, is cross-resistant to vinca alkaloids and to
`taxanes and has a reduced amount of total tubulin, a higher
`percentage of polymerized tubulin, and a higher content of
`class III tubulin isotype.83 Various investigators have re-
`
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`Copyright © 1999 American Society of Clinical Oncology. All rights reserved.
`
`

`
`1066
`
`ported altered expression of tubulin isotypes in resistant cell
`lines.81,84,85 Haber et al86 reported that in the murine cell line
`J774, resistance to paclitaxel is associated with a 21-fold
`increase in class II beta-tubulin isotype. In paclitaxel-
`resistant human prostate cancer cells, on the other hand,
`class III beta-tubulin appears to be overexpressed.87 Muta-
`tions of tubulin isotype genes have also been reported in
`lines.88 Reproducing resistant pheno-
`paclitaxel-resistant
`types by modifying the tubulin isotype composition of cells
`has proven to be difficult89 and has been impeded due to the
`fact that there are often multiple alterations of the soluble
`tubulin/microtubule complex in resistant lines.
`
`PROGRAMMED CELL DEATH (APOPTOSIS)
`Tubulin-binding agents induce apoptosis in tumor cells in
`vitro, as do a great number of other chemotherapeutic
`agents.90 The mechanism by which mitotic blockage induces
`apoptosis remains to be determined, although it is increas-
`ingly clear that a number of regulatory molecules,91,92 as
`well as oncogenes,93 bind to the mitotic apparatus. It is
`highly probable, although the mechanism is poorly under-
`stood, that genes that protect cells against apoptosis, such as
`mutant p53, bcl-2, and bcl-x, may induce resistance to
`tubulin-binding agents.94,95 MAPs are also likely to be
`involved in mechanisms of resistance to drug-induced
`apoptosis. MAP4, the expression of which is negatively
`regulated by wild-type p53, has been shown to increase
`sensitivity to paclitaxel.96,97 Tau overexpression has been
`described in estramustine-resistant human prostatic carci-
`noma cells.98
`The relationship between p53 alterations and sensitivity
`to antitubulin agents is complex. Functional p53 causes cell
`cycle arrest in the G1 phase in case of DNA damage, thereby
`allowing DNA repair and enhanced survival in normal cells.
`It was thus expected that abnormal p53 would sensitize
`tumor cells to DNA-damaging agents. In most cases,
`however, abnormal p53 was associated with drug resistance.
`These unexpected findings were attributed to the fact that
`tumor cells that did not express functional p53 were unable
`
`DUMONTET AND SIKIC
`
`to initiate apoptosis because of the DNA damage they had
`sustained. The temporary inactivation of p53 by acute
`human papillomavirus or the permanent inactivation ob-
`tained in p53-null mice is associated with increased sensitiv-
`ity to paclitaxel.99,100 Woods et al101 suggested that paclitaxel
`induces apoptosis through two different pathways: a p53-
`independent pathway occurring in cells blocked in prophase,
`which is observed both in p53-expressing and in p53-null
`mouse embryo fibroblasts; and a p53-dependent mechanism,
`which occurs in cells that accumulate in G1 and requires
`functional p53. The observation by various authors that
`vinca alkaloids and paclitaxel induce p53 may thus be
`interpreted as a resistance mechanism of the cell against the
`cytotoxic effect of paclitaxel.102,103 Paclitaxel has been
`shown to modulate the level of expression of genes involved
`104 The ability to
`in apoptotic regulation, such as bcl-xL.
`regulate gene expression appears to be an important property
`of paclitaxel but not of docetaxel.105
`
`NEW TUBULIN-BINDING AGENTS
`New antitubulin agents are currently being evaluated
`(Table 2). Spurred by the encouraging results obtained with
`taxanes, research has continued, yielding alkylating paclitax-
`els that bind irreversibly to tubulin and are active at lower
`concentrations on tumor cell lines.106 Nontaxane stabilizing
`agents have also been described. Estramustine suppresses
`microtubule dynamics and displays synergism with vinblas-
`tine.107,108 Discodermolide, extracted from the Caribbean
`sponge Discodermia dissoluta, stabilizes microtubules more
`potently than paclitaxel and inhibits the growth of breast
`cancer cell lines in vitro.109,110 The macrolides epothilones A
`and B also share the ability to arrest cells in mitosis and
`promote the formation of microtubular bundles in nonmi-
`totic cells.111,112 A number of peptide agents have been
`shown to block cell division by interfering with microtubule
`function. These include dolastatin and cryptophycin, which
`behave as depolymerizing agents and inhibit the binding of
`vinblastine to tubulin.113-115 Cryptophycin induced more
`prolonged depletion of microtubules in vitro than did
`
`Compound
`
`Origin
`
`Discodermolide
`
`Sponge (Discodermiadissoluta)
`
`Table 2. New Antitubulin Agents
`
`Competes
`With
`
`Taxanes
`
`Range of
`Activity
`
`, nM
`
`Epothilones A and B
`
`Myxobacterium (Sorangiumcellulosum)
`
`Taxanes
`
`Dolastatin
`Cryptophycins
`Curacin A
`
`Mollusk (Dolabellaauricularia)
`Cyanobacterium
`Cyanobacterium (Lyngbyamajuscula)
`
`Vincas
`Vincas
`Colchicine
`
`Abbreviation: NA, not available.
`
`nM
`
`mM
`pM
`nM
`
`Sensitivity
`to MDR
`
`Low
`
`Low
`
`High
`Low
`NA
`
`Comments
`
`Possibly immunosuppressive, more potent
`than paclitaxel
`No endotoxin-like effect; equipotent with
`paclitaxel
`Peptide
`Peptide; active in murine models
`Thiazoline ring–containing lipid
`
`Information downloaded from jco.ascopubs.org and provided by at INFOTRIEVE on June 20, 2014 from 216.33.62.192
`Copyright © 1999 American Society of Clinical Oncology. All rights reserved.
`
`

`
`TUBULIN-BINDING AGENTS AND CANCER
`
`1067
`
`vinblastine.116 Many of these new compounds, with the
`exception of dolastatin, are weakly transported in Pgp-
`expressing cells and thus retain activity in cells expressing
`the MDR phenotype. Although the first
`tubulin-binding
`agents have been extracted from plants and trees, most of the
`recent and promising compounds have been found in marine
`organisms.
`In conclusion, tubulin-binding agents constitute a diverse
`group of compounds with many applications in medicine.
`Cytotoxic tubulin-binding agents are unique among antican-
`cer drugs in that they target the mitotic spindle rather than
`DNA. Although vincas and taxanes may differ in their gross
`
`effect on cellular cytoskeleton in culture, these compounds
`seem to share a common mechanism of action—namely, the
`inhibition of microtubule dynamics. An important conse-
`quence is that the understanding, and possibly the therapeu-
`tic modulation, of factors influencing microtubular dynam-
`ics will be essential to improve the therapeutic efficacy of
`these compounds. Because of the high tubulin content in
`neuronal tissues, these agents also share a common side
`effect: neurotoxicity. The discovery of new marine com-
`pounds that are not MDR substrates offers great hope for the
`expansion of the role of this family of agents in the treatment
`of cancer.
`
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