Microtubule drugs: action,selectivity, and resistance across the kingdoms of life

Microtubule drugs such as paclitaxel, colchicine, vinblastine, trifluralin, or oryzalin form a chemically diverse group that has been reinforced by a large number of novel compounds over time. They all share the ability to change microtubule properties. The profound effects of disrupted microtubule systems on cell physiology can be used in research as well as anticancer treatment and agricultural weed control. The activity of microtubule drugs generally depends on their binding to α- and β-tubulin subunits. The microtubule drugs are often effective only in certain taxonomic groups, while other organisms remain resistant. Available information on the molecular basis of this selectivity is summarized. In addition to reviewing published data, we performed sequence data mining, searching for kingdom-specific signatures in plant, animal, fungal, and protozoan tubulin sequences. Our findings clearly correlate with known microtubule drug resistance determinants and add more amino acid positions with a putative effect on drug-tubulin interaction. The issue of microtubule network properties in plant cells producing microtubule drugs is also addressed.     

Design, synthesis, and anticancer activity of phosphonic acid diphosphate derivative of adenine-containing butenolide and its water-soluble derivatives of paclitaxel with high antitumor activity

Synthesis of adenine derivative of triphosphono-gamma-(Z)-ethylidene-2,3-dimethoxybutenolide 4 was accomplished by treatment of phosphonate 3 with 5-phosphoribosyl 1-pyrophosphate in the presence of 5-phosphoribosyl 1-pyrophosphate synthetase. It was found that triphosphonate 4 functions as an irreversible stoichiometric inactivator of the Escherichia coli ribonucleoside diphosphate reductase (RDPR). Triphosphonate 4 exhibited potent inhibitory activity against murine leukemias (L1210 and P388), breast carcinoma (MCF7), and human T-lymphoblasts (Molt4/C8 and CEM/0) cell lines. Paclitaxel ester derivatives of adenine-containing triphosphono-gamma-(Z)-ethylidene-2,3-dimethoxybutenolide 8-10 were also synthesized. Like triphosphonate 4, compound 8 exhibited inhibitory property toward RDPR. It also induced microtubule assembly similar to paclitaxel (5). The structure of the chlorodiester linker in 8 was found to account for this dual property. After treatment of MCF7 cells with compounds 4, 5, and 8, fluorescence microscope examination demonstrated the presence of nucleus shrinkage or segmentation. Bifunctional prodrug 8 exhibited higher lipophilicity than 4 and higher water-solubility than 5. Pro-dual-drug 8 exhibited more pronounced anticancer activity relative to that of the triphosphonate 4 and paclitaxel (5). In contrast, compound 9, resulting from the linkage of triphosphonate 4 and paclitaxel (5) through a diester unit, was only found to function as a highly water-soluble prodrug for paclitaxel (5). It induced microtubule assembly in vitro, but did not show inhibitory property toward RDPR. On the other hand, compound 10, an aggregate of triphosphonate 4 and paclitaxel (5), neither functioned as an inhibitor of RDPR nor exhibited microtubule assembly stimulating activity in vitro.     

Cellular studies reveal mechanistic differences between taccalonolide A and paclitaxel

Taccalonolide A is a microtubule stabilizer that has cellular effects almost identical to paclitaxel. However, biochemical studies show that, unlike paclitaxel, taccalonolide A does not enhance purified tubulin polymerization or bind tubulin/microtubules. Mechanistic studies aimed at understanding the nature of the differences between taccalonolide A and paclitaxel were conducted. Our results show that taccalonolide A causes bundling of interphase microtubules at concentrations that cause antiproliferative effects. In contrast, the concentration of paclitaxel that initiates microtubule bundling is 31-fold higher than its IC₅₀. Taccalonolide A''s effects are further differentiated from paclitaxel in that it is unable to enhance the polymerization of tubulin in cellular extracts. This finding extends previous biochemical results with purified brain tubulin to demonstrate that taccalonolide A requires more than tubulin and a full complement of cytosolic proteins to cause microtubule stabilization. Reversibility studies were conducted and show that the cellular effects of taccalonolide A persist after drug washout. In contrast, other microtubule stabilizers, including paclitaxel and laulimalide, demonstrate a much higher degree of cellular reversibility in both short-term proliferation and long-term clonogenic assays. The propensity of taccalonolide A to alter interphase microtubules at antiproliferative concentrations as well as its high degree of cellular persistence may explain why taccalonolide A is more potent in vivo than would be expected from cellular studies. The close linkage between the microtubule bundling and antiproliferative effects of taccalonolide A is of interest given the recent hypothesis that the effects of microtubule targeting agents on interphase microtubules might play a prominent role in their clinical anticancer efficacy.Key words: taccalonolide, paclitaxel, microtubule stabilizer, microtubule targeted agent, tubulin, microtubule, laulimalide, antimitotic agent, drug persistence     

FTIR spectral signature of the effect of cardiotonic steroids with antitumoral properties on a prostate cancer cell line

We show in the present work that the infrared (IR) spectrum of human PC-3 prostate cancer cells exposed to anticancer drugs could offer a unique opportunity to get a fingerprint of all the major biochemical components (DNA, RNA, proteins, lipids, etc.) present in the cells and to identify with high sensitivity the signature of the metabolic changes induced by anticancer drugs.We investigated here the FTIR-related signatures of the effect of 4 structurally-related cardiotonic steroids (CS), i.e. ouabain, 19-hydroxy-2″-oxovoruscharin, hellebrin and 19-hydroxy-hellebrin on PC-3 cancer cells incubated between 0 and 36 h in the absence (control) or the presence of the CS. For each molecule a single spectral signature described the largest part of the time dependent modifications with a possible very minor second component. The spectral signatures characterizing the effects of each of the four CS were unique but very similar when compared to the signature of the effect of an intercalating anticancer drug, i.e. doxorubicin, selected as a positive reference compound in our study, suggesting a fully distinct set of cellular perturbations. The current study thus illustrates that Fourier Transform Infrared (FTIR) analyses can be used to identify, among the perturbations induced on a given cancer cell line, the features common to a group of anticancer compounds as well as features specific to every single drug.     

Cellular studies reveal mechanistic differences between taccalonolide A and paclitaxel

Taccalonolide A is a microtubule stabilizer that has cellular effects almost identical to paclitaxel. However, biochemical studies show that, unlike paclitaxel, taccalonolide A does not enhance purified tubulin polymerization or bind tubulin/microtubules. Mechanistic studies aimed at understanding the nature of the differences between taccalonolide A and paclitaxel were conducted. Our results show that taccalonolide A causes bundling of interphase microtubules at concentrations that cause antiproliferative effects. In contrast, the concentration of paclitaxel that initiates microtubule bundling is 31-fold higher than its IC₅₀. Taccalonolide A’s effects are further differentiated from paclitaxel in that it is unable to enhance the polymerization of tubulin in cellular extracts. This finding extends previous biochemical results with purified brain tubulin to demonstrate that taccalonolide A requires more than tubulin and a full complement of cytosolic proteins to cause microtubule stabilization. Reversibility studies were conducted and show that the cellular effects of taccalonolide A persist after drug washout. In contrast, other microtubule stabilizers, including paclitaxel and laulimalide, demonstrate a much higher degree of cellular reversibility in both short-term proliferation and long-term clonogenic assays. The propensity of taccalonolide A to alter interphase microtubules at antiproliferative concentrations as well as its high degree of cellular persistence may explain why taccalonolide A is more potent in vivo than would be expected from cellular studies. The close linkage between the microtubule bundling and antiproliferative effects of taccalonolide A is of interest given the recent hypothesis that the effects of microtubule targeting agents on interphase microtubules might play a prominent role in their clinical anticancer efficacy.     

Microtubule-interfering activity of parthenolide

Parthenolide is an active sesquiterpene lactone present in a variety of medicinal herbs, well known as anti-inflammatory drug. It has recently been proposed as a chemotherapeutic drug, but the pharmacological pathways of its action have not yet been fully elucidated. Firstly, we explored whether the anticancer properties of parthenolide may be related to a tubulin/microtubule-interfering activity. We additionally compared bioactivities of parthenolide with those checked after combined treatments with paclitaxel in human breast cancer MCF-7 cells. Parthenolide exerted in vitro stimulatory activity on tubulin assembly, by inducing the formation of well-organized microtubule polymers. Light microscopy detections showed that parthenolide-induced alterations of either microtubule network and nuclear morphology happened only after combined exposures to paclitaxel. In addition, the growth of MCF-7 cells was significantly inhibited by parthenolide, which enhanced paclitaxel effectiveness. In conclusion, the antimicrotubular and antiproliferative effects of parthenolide, well known microtubule-stabilizing anticancer agent, may influence paclitaxel activity. The tubulin/microtubule system may represent a novel molecular target for parthenolide, to be utilized in developing new combinational anticancer strategies.     

Mutations in the β-tubulin binding site for peloruside A confer resistance by targeting a cleft significant in side chain binding

Peloruside A is a microtubule-stabilizing macrolide that binds to β-tubulin at a site distinct from the taxol site. The site was previously identified by H-D exchange mapping and molecular docking as a region close to the outer surface of the microtubule and confined in a cavity surrounded by a continuous loop of protein folded so as to center on Y340. We have isolated a series of peloruside A-resistant lines of the human ovarian carcinoma cell line A2780(1A9) to better characterize this binding site and the consequences of altering residues in it. Four resistant lines (Pel A-D) are described with single-base mutations in class I β-tubulin that result in the following substitutions: R306H, Y340S, N337D and A296S in various combinations. The mutations are localized to peptides previously identified by Hydrogen-Deuterium exchange mapping, and center on a cleft in which the drug side chain appears to dock. The Pel lines are 10–15-fold resistant to peloruside A and show cross resistance to laulimalide but not to any other microtubule stabilizers. They show no cross-sensitivity to any microtubule destabilizers, nor to two drugs with targets unrelated to microtubules. Peloruside A induces G₂/M arrest in the Pel cell lines at concentrations 10–15 times that required in the parental line. The cells show notable changes in morphology compared with the parental line.Key words: Apeloruside A, laulimalide, paclitaxel, drug resistance, mitotic arrest, binding site, β-tubulin     

Role of gamma-synuclein in microtubule regulation

Gamma-synuclein is a neuronal protein found in peripheral and motor nerve systems. It becomes highly expressed in metastatic but not in primary tumor or normal tissues. The close association between gamma-synuclein expression and cancer spreading has been demonstrated in a broad range of malignancies. Our previous study showed that exogenous expression of gamma-synuclein in ovarian and breast cancer cells significantly enhanced cell migration and resistance to paclitaxel-induced apoptotic death. In our current research, we found that gamma-synuclein can affect microtubule properties and act as a functional microtubule associated protein. In vitro assays revealed that gamma-synuclein can bind and promote tubulin polymerization, induce the microtubule bundling and alter microtubule morphology developed in the presence of microtubule associated protein 2 (MAP2). Using cancer cell lysate, gamma-synuclein protein was found to be localized in both cytosolic compartment and extracted cytoskeleton portion. Immunofluorescence staining demonstrated that gamma-synuclein can colocalize with microtubule in HeLa cells and decrease rigidity of microtubule bundles caused by paclitaxel. In human ovarian cancer epithelial A2780 cells, gamma-synuclein overexpression improved cell adhesion and microtubule structure upon paclitaxel treatment. Importantly, it led to microtubule-dependent mitochondria clustering at perinuclear area. These observations suggest that overexpression of gamma-synuclein may reduce cell chemo-sensitivity of tumor cells through decreasing microtubule rigidity. In summary, our studies suggested that gamma-synuclein can directly participate in microtubule regulation.     

End-binding protein 1 stimulates paclitaxel sensitivity in breast cancer by promoting its actions toward microtubule assembly and stability

Paclitaxel is a microtubule-targeting agent widely used for the treatment of many solid tumors. However, patients show variable sensitivity to this drug, and effective diagnostic tests predicting drug sensitivity remain to be investigated. Herein, we show that the expression of end-binding protein 1 (EB1), a regulator of microtubule dynamics involved in multiple cellular activities, in breast tumor tissues correlates with the pathological response of tumors to paclitaxel-based chemotherapy. In vitro cell proliferation assays reveal that EB1 stimulates paclitaxel sensitivity in breast cancer cell lines. Our data further demonstrate that EB1 increases the activity of paclitaxel to cause mitotic arrest and apoptosis in cancer cells. In addition, microtubule binding affinity analysis and polymerization/depolymerization assays show that EB1 enhances paclitaxel binding to microtubules and stimulates the ability of paclitaxel to promote microtubule assembly and stabilization. These findings thus reveal EB1 as a critical regulator of paclitaxel sensitivity and have important implications in breast cancer chemotherapy.     

Heightened sensitivity to paclitaxel in Class IVa beta-tubulin-transfected cells is lost as expression increases

Stably transfected Chinese hamster ovary cell lines expressing increasing levels of beta4a, a class IV neuronal-specific beta-tubulin, were compared for effects on microtubule organization, assembly, and sensitivity to antimitotic drugs. It was found that beta4a reduced microtubule assembly in proportion to its abundance and thereby caused supersensitivity to microtubule disruptive drugs such as colcemid, vinblastine, and nocodazole. However, the response to paclitaxel was more complex. Low expression of beta4a caused supersensitivity to paclitaxel, whereas higher expression resulted in the loss of supersensitivity. The results suggest that beta4a may possess an enhanced ability to bind paclitaxel that increases sensitivity to the drug and acts substoichiometrically. At high levels of beta4a expression, however, microtubule disruptive effects counteract the assembly promoting pressure exerted by paclitaxel binding, and drug supersensitivity is lost. beta4a-Tubulin differs from the more ubiquitous beta4b isotype at relatively few amino acid residues, yet beta4b expression has little effect on microtubule assembly or drug response. To determine which amino acids mediate the effects of beta4a expression, beta4a and beta4b were altered by site-directed mutagenesis and expressed in Chinese hamster ovary cells. The introduction of N332S or N335S mutations into beta4b-tubulin was sufficient to confer microtubule disruption and increased colcemid sensitivity. On the other hand, mutation of Ala(115) to serine in beta4a-tubulin almost completely reversed heightened sensitivity to paclitaxel, but introduction of an S115A mutation into beta4b had no effect, suggesting that a complex interaction of multiple amino acids are necessary to produce this phenotype.     

Antimicrotubular and cytotoxic activity of geiparvarin analogues, alone and in combination with paclitaxel

Geiparvarin is an antiproliferative compound isolated from the leaves of Geijera parviflora, and may represent a new drug which targets tubulin. To better explore the potential use of this agent, we investigated the antimicrotubular and cytotoxic effects of new synthetic aromatic derivatives of geiparvarin. These drugs inhibited polymerization of microtubular protein, particularly when the assembly was induced by paclitaxel. The microtubular network organization of fibroblasts was altered more effectively by some drugs. Normal microtubule architecture completely disappeared when the cells were treated simultaneously with drugs and paclitaxel: microtubules depolymerized or were reorganized into bundles, in a similar but more disarrayed fashion than that observed after treatment with paclitaxel alone. Cytotoxicity studies showed a dose-dependent effect, whereas combined administration of drugs and paclitaxel increased cytotoxicity, more effectively in paclitaxel versus derivatives administration alone.     

ATPase activity of nucleotide binding domains of human MDR3 in the context of MDR1

Although human MDR1 and MDR3 share 86% similarity in their amino acid sequences and are predicted to share conserved domains for drug recognition, their physiological transport substrates are quite different: MDR1 transports xenobiotics and confers multidrug resistance, while MDR3 exports phosphatidylcholine into bile. Although MDR1 shows high ATPase activity, attempts to demonstrate the ATPase activity of human MDR3 have not succeeded. Therefore, it is possible that the difference in the functions of these proteins is caused by their different ATPase activities. To test this hypothesis, a chimera protein containing the transmembrane domains (TMDs) of MDR1 and the nucleotide binding domains (NBDs) of MDR3 was constructed and analyzed. The chimera protein was expressed on the plasma membrane and conferred resistance against vinblastine and paclitaxel, indicating that MDR3 NBDs can support drug transport. Vanadate-induced ADP trapping of MDR3 NBDs in the chimera protein was stimulated by verapamil as was MDR1 NBDs. The purified chimera protein showed drug-stimulated ATPase activity like MDR1, while its V_max was more than 10-times lower than MDR1. These results demonstrate that the low ATPase activity of human MDR3 cannot account for the difference in the functions of these proteins, and furthermore, that TMDs determine the features of NBDs. To our knowledge, this is the first study analyzing the features of human MDR3 NBDs.