Microtubules: a dynamic target in cancer therapy

The tubulin/microtubule system is an important target for anticancer therapy. Two of the most clinically valuable groups of these agents are the vinca alkaloids and taxanes. In recent years, new tubulin-binding agents have been under preclinical or clinical development. One of these classes of agents, epothilones, has shown great promise in phase III clinical trials. What all these agents share in common, is that they bind to beta-tubulin and disrupt microtubule function during mitosis which in turn leads to mitotic arrest and cell death. In addition, these agents can inhibit angiogenesis. Not withstanding their effectiveness, drug resistance can pose a major clinical problem. This review provides an overview of the mechanisms mediating resistance to tubulin-binding agents related to the cellular target and discusses strategies to overcome this important clinical problem.     

Synthetic lethal hubs associated with vincristine resistant neuroblastoma

Chemotherapy of cancer experiences a number of shortcomings including development of drug resistance. This fact also holds true for neuroblastoma utilizing chemotherapeutics as vincristine. We performed a comparative analysis of molecular and cellular mechanisms associated with vincristine resistance utilizing cell line as well as human tissue data. Differential gene expression analysis revealed molecular features, processes and pathways afflicted with drug resistance mechanisms in general, and specifically with vincristine significantly involving actin associated features. However, specific mode of resistance as well as underlying genotype of parental, vincristine sensitive cells apparently exhibited significant heterogeneity. No consensus profile for vincristine resistance could be derived, but resistance-associated changes on the level of individual neuroblastoma cell lines as well as individual patient profiles became clearly evident. Based on these prerequisites we utilized the concept of synthetic lethality aimed at identifying hub proteins which when inhibited promise to induce cell death due to a synthetic lethal interaction with down-regulated, chemoresistance associated features. Our screening procedure identified synthetic lethal hub proteins afflicted with actin associated processes holding synthetic lethal interactions to down-regulated features individually found in all chemoresistant cell lines tested, therefore promising an improved therapeutic window. Verification of such synthetic lethal hub candidates in human neuroblastoma tissue expression profiles indicated the feasibility of this screening approach for addressing vincristine resistance in neuroblastoma.     

Mechanisms of cross-resistance between nucleoside analogues and vincristine or daunorubicin in leukemic cells

The aim of this study was to clarify the biochemical and molecular mechanisms behind the cross-resistance to nucleoside analogues (NAs) in four erythroleukemic cell lines with acquired resistance to the anthracycline daunorubicin and to the vinca alkaloid vincristine, expressing high levels of p-glycoprotein (P-gp, MDR1). All resistant strains exhibited cross-resistance to NA (cladribine and cytosine arabinoside)-induced apoptosis, assessed by caspase-3-like activation and were less sensitive to NA cytotoxicity in MTT assay. Real-time PCR and enzyme activity analysis showed reduced amounts of deoxycytidine kinase (35-80%) and elevated levels of 5'-nucleotidases (50-100%). The ratio 5'-nucleotidase to deoxycytidine kinase increased between 2.5- and 7.5-folds in resistant cells. This is in agreement with the observation that 5'-nucleotidase/deoxycytidine kinase ratio might be an important factor in predicting resistance to NAs. Implications of this finding for combining anthracyclines or vinca alkaloids with NAs toward leukemic cells are discussed.     

Aging of tubulin at neutral pH: the destabilizing effect of vinca alkaloids.

The effect of the vinca alkaloid drugs, vincristine, vinblastine, catharanthine, and vindoline, on the aging process of tubulin has been examined. It was found that addition of vincristine or vinblastine accelerated by a factor of 3-3.5 the transformation of tubulin from the 5.8 S alpha-beta-tubulin dimer to paucidisperse polymers, with an average sedimentation coefficient of 9 S, previously observed in the absence of drugs (V. Prakash and S. N. Timasheff, 1982, J. Mol. Biol. 160, 499-515). This transformation of tubulin from 5.8 S to "9 S" followed pseudo-first-order kinetics whether the starting protein was predominantly dimeric (i.e., at low drug concentration) or self-associated into the reversible linear polymers induced by the vinca alkaloid drugs at high drug concentration (G. C. Na and S. N. Timasheff, 1980, Biochemistry 19, 1355-1365; V. Prakash and S. N. Timasheff, 1985, Biochemistry 24, 5004-5010). Identical kinetics were found in a fluorescence examination of the loss by tubulin of its ability to bind colchicine specifically, indicating that the rate determining step is a protein conformational change that induces a major change in the far uv circular dichroism spectrum of tubulin. The found lack of an effect of dithiothreitol on the aging and aggregation processes is consistent with the irreversible aggregation being due to the intermolecular coalescence of nonpolar patches on the protein. The observations that vincristine binds to aged tubulin and that the aging of tubulin is accompanied by quenching of the tryptophan fluorescence similar to that which occurs on the binding of the vinca drugs has led to the proposal that the vinca alkaloids stabilize the aged conformation of the protein by interacting with nonpolar regions that may be related to the aggregation sites.     

Vincristine attenuates doxorubicin cardiotoxicity

Our aim was to test the hypothesis that the vinca alkaloid vincristine could prevent doxorubicin-induced cardiomyocyte death and to identify the mechanisms involved. Adult mouse cardiac myocytes were incubated for 24 h with doxorubicin, with and without concurrent vincristine. Trypan blue exclusion showed that 50–60% of myocytes treated with doxorubicin alone survived. Concurrent vincristine treatment increased survival to 85%. Treatment with doxorubicin + vincristine activated the prosurvival signal Akt and diminished cytochrome C release. The PI3K/Akt inhibitor LY294002 and the MEK/ERK inhibitor PD98059 augmented doxorubicin cardiotoxicity and attenuated salvage during concurrent vincristine treatment, indicating that the mechanism of vincristine cardioprotection involves activation of specific survival signals. Vincristine retarded the onset of apoptosis in association with a delay in poly(ADP) ribose polymerase activation. Vincristine also exhibited greater protection than the antioxidant MPG. These novel findings may have clinical implications for the prevention of doxorubicin cardiomyopathy.     

Identification of glycan structure alterations on cell membrane proteins in desoxyepothilone B resistant leukemia cells

Resistance to tubulin-binding agents used in cancer is often multifactorial and can include changes in drug accumulation and modified expression of tubulin isotypes. Glycans on cell membrane proteins play important roles in many cellular processes such as recognition and apoptosis, and this study investigated whether changes to the glycan structures on cell membrane proteins occur when cells become resistant to drugs. Specifically, we investigated the alteration of glycan structures on the cell membrane proteins of human T-cell acute lymphoblastic leukemia (CEM) cells that were selected for resistance to desoxyepothilone B (CEM/dEpoB). The glycan profile of the cell membrane glycoproteins was obtained by sequential release of N- and O-glycans from cell membrane fraction dotted onto polyvinylidene difluoride membrane with PNGase F and β-elimination respectively. The released glycan alditols were analyzed by liquid chromatography (graphitized carbon)-electrospray ionization tandem MS. The major N-glycan on CEM cell was the core fucosylated α2-6 monosialo-biantennary structure. Resistant CEM/dEpoB cells had a significant decrease of α2-6 linked sialic acid on N-glycans. The lower α2-6 sialylation was caused by a decrease in activity of β-galactoside α2-6 sialyltransferase (ST6Gal), and decreased expression of the mRNA. It is clear that the membrane glycosylation of leukemia cells changes during acquired resistance to dEpoB drugs and that this change occurs globally on all cell membrane glycoproteins. This is the first identification of a specific glycan modification on the surface of drug resistant cells and the mechanism of this downstream effect on microtubule targeting drugs may offer a route to new interventions to overcome drug resistance.     

A polymer-dependent increase in phosphorylation of beta-tubulin accompanies differentiation of a mouse neuroblastoma cell line

We have examined the phosphorylation of cellular microtubule proteins during differentiation and neurite outgrowth in N115 mouse neuroblastoma cells. N115 differentiation, induced by serum withdrawal, is accompanied by a fourfold increase in phosphorylation of a 54,000-mol-wt protein identified as a specific isoform of beta-tubulin by SDS PAGE, two-dimensional isoelectric focusing/SDS PAGE, and immunoprecipitation with a specific monoclonal antiserum. Isoelectric focusing/SDS PAGE of [35S]methionine-labeled cell extracts revealed that the phosphorylated isoform of beta-tubulin, termed beta 2, is one of three isoforms detected in differentiated N115 cells, and is diminished in amounts in the undifferentiated cells. Taxol, a drug which promotes microtubule assembly, stimulates phosphorylation of beta-tubulin in both differentiated and undifferentiated N115 cells. In contrast, treatment of differentiated cells with either colcemid or nocodazole causes a rapid decrease in beta-tubulin phosphorylation. Thus, the phosphorylation of beta-tubulin in N115 cells is coupled to the levels of cellular microtubules. The observed increase in beta-tubulin phosphorylation during differentiation then reflects developmental regulation of microtubule assembly during neurite outgrowth, rather than developmental regulation of a tubulin kinase activity.     

The effect of 5'-(N,N-dimethyl)-amiloride on cytotoxic activity of doxorubicin and vincristine in CEM cell lines

Intracellular pH (pHi) plays an important role in anticancer drug accumulation in cancer cells. Resistant cells often express membrane P-glycoprotein responsible for active drug extrusion and participating in increased pHi. In the present paper, we report on the influence of Na+/H+-exchanger inhibitor, 5'-(N,N-dimethyl)-amiloride (AMI), on the cytotoxic effects of doxorubicin (DOXO) and vincristine (VCR) in the parental CEM, and resistant CEM/DNR and CEM/VCR cell lines. The obtained results revealed a potentiating effect of AMI to both anticancer drugs in parental CEM line. However, AMI did not significantly potentiate the effect of DOXO or VCR in resistant CEM cell lines. We conclude, that inhibition of Na+/H+-exchanger by AMI is not sufficient for reversal of drug resistance in the tested CEM/DNR and CEM/VCR cell lines and the possible change in pHi does not affect the mechanisms of cell resistance.     

Structural insight into the inhibition of tubulin by vinca domain peptide ligands

The tubulin vinca domain is the target of widely different microtubule inhibitors that interfere with the binding of vinblastine. Although all these ligands inhibit the hydrolysis of GTP, they affect nucleotide exchange to variable extents. The structures of two vinca domain antimitotic peptides--phomopsin A and soblidotin (a dolastatin 10 analogue)--bound to tubulin in a complex with a stathmin-like domain show that their sites partly overlap with that of vinblastine and extend the definition of the vinca domain. The structural data, together with the biochemical results from the ligands we studied, highlight two main contributors in nucleotide exchange: the flexibility of the tubulin subunits' arrangement at their interfaces and the residues in the carboxy-terminal part of the beta-tubulin H6-H7 loop. The structures also highlight common features of the mechanisms by which vinca domain ligands favour curved tubulin assemblies and destabilize microtubules.     

Markedly altered membrane transport and intracellular binding of vincristine in multidrug-resistant Chinese hamster cells selected for resistance to vinca alkaloids

Studies of a multidrug-resistant variant (DC-3F/VCRd-5L) of Chinese hamster lung cells selected for resistance to vinca alkaloids revealed marked alterations in transport and intracellular binding of [3H]vincristine compared to parental DC-3F cells. Influx of [3H]vincristine in DC-3F cells appears to be an equilibrating, but mediated, process. Although saturation kinetics for [3H]vincristine influx were not demonstrated, an extremely high temperature-dependence (Q10 27-37 degrees C = 5-6) and trans-inhibition of influx following preloading of cells with nonradioactive vincristine argue in favor of a carrier-mediated process. Efflux of [3H]vincristine from parental cells conformed to first-order kinetics (t1/2 37 degrees = 3.6 +/- 0.4) and exhibited a lower temperature-dependence (Q10 27-37 degrees C = 3-3.5) than influx. In variant vs. parental cells, influx of [3H]vincristine was reduced 24-fold and efflux was increased two-fold, accounting for the large (approximately 48-fold) reduction in steady-state level of exchangeable drug accumulating in variant cells. Otherwise, transport of [3H]vincristine in these cells showed characteristics similar to parental DC-3F cells. Also, the rate and amount of intracellular binding of [3H]vincristine in variant cells was almost 40-fold lower than in parental cells. These alterations in influx and efflux of [3H]vincristine and its intracellular binding appear to account, at least to a major extent, for the high level of resistance (2,750-fold) of this variant to vinca alkaloids. In contrast, cross-resistance of this variant to daunomycin (178-fold) could be explained only minimally by a transport alteration. Only a two-fold increase in efflux of [3H]daunomycin was demonstrated in variant vs. parental cells along with some decrease in intracellular binding. Influx of [3H]daunomycin was unaltered. In view of these results, we conclude that these two agents most likely do not share the same route for entry in these cells but might share the same efflux route.     

A ubiquitous beta-tubulin disrupts microtubule assembly and inhibits cell proliferation

Vertebrate tubulin is encoded by a multigene family that produces distinct gene products, or isotypes, of both the alpha- and beta-tubulin subunits. The isotype sequences are conserved across species supporting the hypothesis that different isotypes subserve different functions. To date, however, most studies have demonstrated that tubulin isotypes are freely interchangeable and coassemble into all classes of microtubules. We now report that, in contrast to other isotypes, overexpression of a mouse class V beta-tubulin cDNA in mammalian cells produces a strong, dose-dependent disruption of microtubule organization, increased microtubule fragmentation, and a concomitant reduction in cellular microtubule polymer levels. These changes also disrupt mitotic spindle assembly and block cell proliferation. Consistent with diminished microtubule assembly, there is an increased tolerance for the microtubule stabilizing drug, paclitaxel, which is able to reverse many of the effects of class V beta-tubulin overexpression. Moreover, transfected cells selected in paclitaxel exhibit increased expression of class V beta-tubulin, indicating that this isotype is responsible for the drug resistance. The results show that class V beta-tubulin is functionally distinct from other tubulin isotypes and imparts unique properties on the microtubules into which it incorporates.     

Review: postchaperonin tubulin folding cofactors and their role in microtubule dynamics

The microtubule cytoskeleton consists of a highly organized network of microtubule polymers bound to their accessory proteins: microtubule-associated proteins, molecular motors, and microtubule-organizing proteins. The microtubule subunits are heterodimers composed of one alpha-tubulin polypeptide and one beta-tubulin polypeptide that should undergo a complex folding processing before they achieve a quaternary structure that will allow their incorporation into the polymer. Due to the extremely high protein concentration that exists at the cell cytoplasm, there are alpha- and beta-tubulin interacting proteins that prevent the unwanted interaction of these polypeptides with the surrounding protein pool during folding, thus allowing microtubule dynamics. Several years ago, the development of a nondenaturing electrophoretic technique made it possible to identify different tubulin intermediate complexes during tubulin biogenesis in vitro. By these means, the cytosolic chaperonin containing TCP-1 (CCT or TriC) and prefoldin have been demonstrated to intervene through tubulin and actin folding. Various other cofactors also identified along the alpha- and beta-tubulin postchaperonin folding route are now known to have additional roles in tubulin biogenesis such as participating in the synthesis, transport, and storage of alpha- and beta-tubulin. The future characterization of the tubulin-binding sites to these proteins, and perhaps other still unknown proteins, will help in the development of chemicals that could interfere with tubulin folding and thus modulating microtubule dynamics. In this paper, current knowledge of the above postchaperonin folding cofactors, which are in fact chaperones involved in tubulin heterodimer quaternary structure achievement, will be reviewed.     

Cancer Cells Acquire Mitotic Drug Resistance Properties Through Beta I-Tubulin Mutations and Alterations in the Expression of Beta-Tubulin Isotypes

Background

Anti-mitotic compounds (microtubule de-stabilizers) such as vincristine and vinblastine have been shown clinically successful in treating various cancers. However, development of drug-resistance cells limits their efficacies in clinical situations. Therefore, experiments were performed to determine possible drug resistance mechanisms related to the application of anti-mitotic cancer therapy.

Principal Findings

A KB-derived microtubule de-stabilizer-resistant KB-L30 cancer cell line was generated for this study. KB-L30 cells showed cross-resistance to various microtubule de-stabilizers including BPR0L075, vincristine and colchicine through multiple-drug resistant (MDR)-independent mechanisms. Surprisingly, KB-L30 cells showed hyper-sensitivity to the microtubule-stabilizer, paclitaxel. Results of the RT-PCR analysis revealed that expression of both class II and III β-tubulin was down-regulated in KB-L30 cells as compared to its parental KB cancer cells. In addition, DNA sequencing analysis revealed six novel mutation sites present in exon four of the βI-tubulin gene. Computational modeling indicated that a direct relationship exists between βI-tubulin mutations and alteration in the microtubule assembly and dynamic instability in KB-L30 cells and this predicted model was supported by an increased microtubule assembly and reduced microtubule dynamic instability in KB-L30 cells, as shown by Western blot analysis.

Conclusions and Significance

Our study demonstrated that these novel mutations in exon four of the βI-tubulin induced resistance to microtubule de-stabilizers and hyper-sensitivity to microtubule stabilizer through an alteration in the microtubule assembly and dynamics in cancer cells. Importantly, the current study reveals that cancer cells may acquire drug resistance ability to anti-mitotic compounds through multiple changes in the microtubule networks. This study further provided molecular information in drug selection for patients with specific tubulin mutations.     

Recurrent laryngeal nerve paralysis in patients receiving vincristine and vinblastine.

Three patients receiving vincristine or binblastine developed recurrent laryngeal nerve paralysis, which resolved when the drug was withdrawn. The presence of mediastinal lymphadenopathy in patients with lymphoma may obscure the correct diagnosis, with the danger that the condition will produce respiratory distress if vinca alkaloid treatment is continued.     

Calmodulin, activated cyclic nucleotide phosphodiesterase, microtubules, and vinca alkaloids

Calmodulin 1.8 and 10.6 microM, inhibited the polymerization of bovine brain microtubules by 30 and 50%, respectively. Two 55,000- to 68,000-dalton calmodulin-binding protein as well as calmodulin-dependent and -independent phosphodiesterases (PDE) were found associated with microtubule proteins. Among the antimicrotubule drugs, such as colchicine, podophyllotoxin, griseofulvin, and vinca alkaloids (vinblastine, desacetylvinblastine amide, and vincristine), the vinca alkaloids were selective inhibitors of calmodulin-activated PDE activity. This action of vinca alkaloids resides in the catharanthine moiety of vinblastine molecule. An alpha 2 inhibitor, yohimbine, affects the microtubules, and a series of alpha-adrenoceptor blocking agents were examined for their effects on calmodulin-dependent PDE. The relative order of the potency is phenoxybenzamine = dibenamine greater than phentolamine greater than yohimbine greater than prazosin greater than tolazoline, and the first four drugs in this series were selective inhibitors of calmodulin action. Inasmuch as phenoxybenzamine and dibenamine are alkylating agents, the effects of antineoplastic alkylating agents on the calmodulin action were also examined. Busulphan, melphalan, 1-(2-chloroethyl)-3-cyclohexyl-l-nitrosourea, and streptozotocin (up to 4 mM) were not selective inhibitors of calmodulin action. Maytansine, a vinca alkaloid-type antimicrotubule agents as well as an alkylating agent, selectively inhibited the calmodulin with a potency similar to vincristine. In addition, phenoxybenzamine affected Ca2+-dependent fluorescence induced by the interaction between calmodulin and hydrophobic fluorescent probes, whereas vinblastine was ineffective. However, the binding of vinblastine to calmodulin is calcium dependent. Studies such as these suggest the importance of physical and structural considerations in drugs binding to calmodulin as well as at least two different binding sites for drugs on calmodulin.     

Cancer drug-resistance and a look at specific proteins: Rho GDP-dissociation inhibitor 2, Y-box binding protein 1, and HSP70/90 organizing protein in proteomics clinical application

Resistance to anti-cancer drugs is a well recognized problem and very often it is responsible for failure of the cancer treatment. In this study, the proteome alterations associated with the development of acquired resistance to cyclin-depedent kinases inhibitor bohemine, a promising anti-cancer drug, were analyzed with the primary aim of identifying potential targets of resistance within the cell that could pave a way to selective elimination of specific resistant cell types. A model of parental susceptible CEM T-lymphoblastic leukemia cells and its resistant counterpart CEM-BOH was used and advanced 2-D liquid chromatography was applied to fractionate cellular proteins. Differentially expressed identified proteins were further verified using immunoblotting and immunohistochemistry. Our study has revealed that Rho GDP-dissociation inhibitor 2, Y-box binding protein 1, and the HSP70/90 organizing protein have a critical role to play in resistance to cyclin-depedent kinases inhibitor. The results indicated not only that quantitative protein changes play an important role in drug-resistance, but also that there are various other parameters such as truncation, post-translational modification(s), and subcellular localization of selected proteins. Furthermore, these proteins were validated for their roles in drug resistance using different cell lines resistant to diverse representatives of anti-cancer drugs such as vincristine and daunorubicin.     

Cellular functions of the Spir actin-nucleation factors

The initiation of actin polymerization from free monomers requires actin-nucleation factors. Spir proteins nucleate actin polymerization by a novel mechanism that is distinct from actin nucleation by the Arp2/3 complex or by formins. In vitro actin polymerization assays and electron microscopic data show that Spire nucleates actin polymerization by binding four actin monomers to a cluster of four Wiskott-Aldrich syndrome protein-homology domain 2 (WH2) domains in the central region of the proteins. Although the exact cell biological function and regulation of Spir proteins is still unknown, data from genetic studies in Drosophila, cell biological studies and protein interaction experiments have provided insight into the biology of these interesting and novel actin-nucleation factors and suggest a role in vesicle transport processes and in the coordination of cortical microtubule and actin filaments. Phosphorylation by mitogen-activated protein kinases and interaction with Rho GTPases have been proposed as regulatory mechanisms.     

Functional cooperation between the microtubule and actin cytoskeletons

In diverse cell types, microtubule (MT) and actin filament networks cooperate functionally during a wide variety of processes, including vesicle and organelle transport, cleavage furrow placement, directed cell migration, spindle rotation, and nuclear migration. The mechanisms by which MTs and actin filaments cooperate to mediate these different processes can be grouped into two broad categories: coordinated MT- and actin-based transport to move vesicles, organelles, and cell fate determinants; and targeting and capture of MT ends at cortical actin sites. Over the past several years, a growing number of cellular factors that bridge these cytoskeletal systems have been identified. These include 'hetero-motor' complexes (physically associated myosin and kinesin), myosin-CLIP170 complexes, formin homology (FH) proteins, dynein and the dynactin complex, Kar9p, coronin, Kelch repeat-containing proteins, and ERM proteins.