Rotenone inhibits mammalian cell proliferation by inhibiting microtubule assembly through tubulin binding

Rotenone, a widely used insecticide, has been shown to inhibit mammalian cell proliferation and to depolymerize cellular microtubules. In the present study, the effects of rotenone on the assembly of microtubules in relation to its ability to inhibit cell proliferation and mitosis were analyzed. We found that rotenone inhibited the proliferation of HeLa and MCF-7 cells with half maximal inhibitory concentrations of 0.2 +/- 0.1 microm and 0.4 +/- 0.1 microm, respectively. At its effective inhibitory concentration range, rotenone depolymerized spindle microtubules of both cell types. However, it had a much stronger effect on the interphase microtubules of MCF-7 cells compared to that of the HeLa cells. Rotenone suppressed the reassembly of microtubules in living HeLa cells, suggesting that it can suppress microtubule growth rates. Furthermore, it reduced the intercentrosomal distance in HeLa cells at its lower effective concentration range and induced multipolar-spindle formation at a relatively higher concentration range. It also increased the level of checkpoint protein BubR1 at the kinetochore region. Rotenone inhibited both the assembly and the GTP hydrolysis rate of microtubules in vitro. It also inhibited the binding of colchicine to tubulin, perturbed the secondary structure of tubulin, and reduced the intrinsic tryptophan fluorescence of tubulin and the extrinsic fluorescence of tubulin-1-anilinonaphthalene-8-sulfonic acid complex, suggesting that it binds to tubulin. A dissociation constant of 3 +/- 0.6 microm was estimated for tubulin-rotenone complex. The data presented suggest that rotenone blocks mitosis and inhibits cell proliferation by perturbing microtubule assembly dynamics.     

The benzophenanthridine alkaloid sanguinarine perturbs microtubule assembly dynamics through tubulin binding. A possible mechanism for its antiproliferative activity

Sanguinarine has been shown to inhibit proliferation of several types of human cancer cell including multidrug-resistant cells, whereas it has minimal cytotoxicity against normal cells such as neutrophils and keratinocytes. By analyzing the antiproliferative activity of sanguinarine in relation to its effects on mitosis and microtubule assembly, we found that it inhibits cancer cell proliferation by a novel mechanism. It inhibited HeLa cell proliferation with a half-maximal inhibitory concentration of 1.6 +/- 0.1 microM. In its lower effective inhibitory concentration range, sanguinarine depolymerized microtubules of both interphase and mitotic cells and perturbed chromosome organization in mitotic HeLa cells. At concentrations of 2 microM, it induced bundling of interphase microtubules and formation of granular tubulin aggregates. A brief exposure of HeLa cells to sanguinarine caused irreversible depolymerization of the microtubules, inhibited cell proliferation, and induced cell death. However, in contrast with several other microtubule-depolymerizing agents, sanguinarine did not arrest cell cycle progression at mitosis. In vitro, low concentrations of sanguinarine inhibited microtubule assembly. At higher concentrations (> 40 microM), it altered polymer morphology. Further, it induced aggregation of tubulin in the presence of microtubule-associated proteins. The binding of sanguinarine to tubulin induces conformational changes in tubulin. Together, the results suggest that sanguinarine inhibits cell proliferation at least in part by perturbing microtubule assembly dynamics.     

Mechanism of mitotic arrest induced by dolastatin 15 involves loss of tension across kinetochore pairs

Dolastatin 15 (DL15) is a potent, tubulin-targeted, vinca-site binding, anticancer agent that induces mitotic arrest and inhibit cell proliferation in a variety of cell types. Several analogs of DL15, including LU 103793 and tasidotin, have been progressed to clinical trials for different types of cancer. DL15 has been known to interfere with cellular microtubules and purified tubulin in vitro. However, the molecular mechanism with which the peptide arrests cells in mitosis is poorly understood. This study reports a possible antimitotic mechanism of action of DL15. DL15 inhibited HeLa cell proliferation in a concentration-dependent manner with a half-maximal inhibitory concentration (IC₅₀) of 2.8 ± 0.3 nM, induced mitotic arrest, disrupted cellular microtubules near its IC₅₀ for cell proliferation, and inhibited the re-polymerization of cellular microtubules. By staining the centrosomes of DL15-treated cells with anti-γ tubulin antibodies, the study found a significant reduction in interpolar distances in mitotic HeLa cells, indicating a disruption in the normal assembly dynamics of the microtubules. The study further found that DL15 induced a loss of tension across the kinetochore pairs as indicated by a reduction in interkinetochore distance. In response to this loss of tension, the tension-sensing checkpoint protein BuBR1 accumulated at the kinetochores, promoting mitotic arrest. In vitro, DL15 promoted formation of curved and fragmented polymers of microtubule proteins and inhibited tubulin decay in a manner similar to vinca-site binding agents such as phomopsin A. Together, the data indicate that the mitotic arrest induced by DL15 involves a loss of tension across the kinetochore pairs due to disruption of normal assembly dynamics of microtubules.     

Cellular Effects of Curcumin on Plasmodium falciparum Include Disruption of Microtubules

Curcumin has been widely investigated for its myriad cellular effects resulting in reduced proliferation of various eukaryotic cells including cancer cells and the human malaria parasite Plasmodium falciparum. Studies with human cancer cell lines HT-29, Caco-2, and MCF-7 suggest that curcumin can bind to tubulin and induce alterations in microtubule structure. Based on this finding, we investigated whether curcumin has any effect on P. falciparum microtubules, considering that mammalian and parasite tubulin are 83% identical. IC₅₀ of curcumin was found to be 5 µM as compared to 20 µM reported before. Immunofluorescence images of parasites treated with 5 or 20 µM curcumin showed a concentration-dependent effect on parasite microtubules resulting in diffuse staining contrasting with the discrete hemispindles and subpellicular microtubules observed in untreated parasites. The effect on P. falciparum microtubules was evident only in the second cycle for both concentrations tested. This diffuse pattern of tubulin fluorescence in curcumin treated parasites was similar to the effect of a microtubule destabilizing drug vinblastine on P. falciparum. Molecular docking predicted the binding site of curcumin at the interface of alpha and beta tubulin, similar to another destabilizing drug colchicine. Data from predicted drug binding is supported by results from drug combination assays showing antagonistic interactions between curcumin and colchicine, sharing a similar binding site, and additive/synergistic interactions of curcumin with paclitaxel and vinblastine, having different binding sites. This evidence suggests that cellular effects of curcumin are at least, in part, due to its perturbing effect on P. falciparum microtubules. The action of curcumin, both direct and indirect, on P. falciparum microtubules is discussed.     

An Antitubulin Agent BCFMT Inhibits Proliferation of Cancer Cells and Induces Cell Death by Inhibiting Microtubule Dynamics

Using cell based screening assay, we identified a novel anti-tubulin agent (Z)-5-((5-(4-bromo-3-chlorophenyl)furan-2-yl)methylene)-2-thioxothiazolidin-4-one (BCFMT) that inhibited proliferation of human cervical carcinoma (HeLa) (IC(50), 7.2±1.8 μM), human breast adenocarcinoma (MCF-7) (IC(50), 10.0±0.5 μM), highly metastatic breast adenocarcinoma (MDA-MB-231) (IC(50), 6.0±1 μM), cisplatin-resistant human ovarian carcinoma (A2780-cis) (IC(50), 5.8±0.3 μM) and multi-drug resistant mouse mammary tumor (EMT6/AR1) (IC(50), 6.5±1μM) cells. Using several complimentary strategies, BCFMT was found to inhibit cancer cell proliferation at G2/M phase of the cell cycle apparently by targeting microtubules. In addition, BCFMT strongly suppressed the dynamics of individual microtubules in live MCF-7 cells. At its half maximal proliferation inhibitory concentration (10 μM), BCFMT reduced the rates of growing and shortening phases of microtubules in MCF-7 cells by 37 and 40%, respectively. Further, it increased the time microtubules spent in the pause (neither growing nor shortening detectably) state by 135% and reduced the dynamicity (dimer exchange per unit time) of microtubules by 70%. In vitro, BCFMT bound to tubulin with a dissociation constant of 8.3±1.8 μM, inhibited tubulin assembly and suppressed GTPase activity of microtubules. BCFMT competitively inhibited the binding of BODIPY FL-vinblastine to tubulin with an inhibitory concentration (K(i)) of 5.2±1.5 μM suggesting that it binds to tubulin at the vinblastine site. In cultured cells, BCFMT-treatment depolymerized interphase microtubules, perturbed the spindle organization and accumulated checkpoint proteins (BubR1 and Mad2) at the kinetochores. BCFMT-treated MCF-7 cells showed enhanced nuclear accumulation of p53 and its downstream p21, which consequently activated apoptosis in these cells. The results suggested that BCFMT inhibits proliferation of several types of cancer cells including drug resistance cells by suppressing microtubule dynamics and indicated that the compound may have chemotherapeutic potential.     

Perturbation of microtubule polymerization by quercetin through tubulin binding: a novel mechanism of its antiproliferative activity

The dietary flavonoid quercetin has a broad range of biological activities, including potent antitumor activity against several types of tumors. Recently, it has been shown that quercetin inhibits cancer cells proliferation by depleting cellular microtubules and perturbing cellular microtubule functions. However, the direct interactions of quercetin with tubulin and microtubules have not been examined so far. Here, we found that quercetin inhibited polymerization of microtubules and depolymerized microtubules made from purified tubulin in vitro. The binding of quercetin with tubulin was studied using quercetin fluorescence and intrinsic tryptophan fluorescence of tubulin. Quercetin bound to tubulin at a single site with a dissociation constant of 5-7 microM, and it specifically inhibited colchicine binding to tubulin but did not bind at the vinblastine site. In addition, quercetin perturbed the secondary structure of tubulin, and the binding of quercetin stimulated the intrinsic GTPase activity of soluble tubulin. Further, quercetin stabilized tubulin against decay and protected two cysteine residues of tubulin toward chemical modification by 5,5'-dithiobis-2-nitrobenzoic acid. Our data demonstrated that the binding of quercetin to tubulin induces conformational changes in tubulin and a mechanism through which quercetin could perturb microtubule polymerization dynamics has been proposed. The data suggest that quercetin inhibits cancer cells proliferation at least in part by perturbing microtubule functions through tubulin binding.     

Antimitotic antifungal compound benomyl inhibits brain microtubule polymerization and dynamics and cancer cell proliferation at mitosis, by binding to a novel site in tubulin

The antifungal agent benomyl [methyl-1-(butylcarbamoyl)-2-benzimidazolecarbamate] is used throughout the world against a wide range of agricultural fungal diseases. In this paper, we investigated the interaction of benomyl with mammalian brain tubulin and microtubules. Using the hydrophobic fluorescent probe 1-anilinonaphthalene-8-sulfonic acid, benomyl was found to bind to brain tubulin with a dissociation constant of 11.9 +/- 1.2 microM. Further, benomyl bound to at a novel site, distinct from the well-characterized colchicine and vinblastine binding sites. Benomyl altered the far-UV circular dichroism spectrum of tubulin and reduced the accessibility of its cysteine residues to modification by 5,5'-dithiobis-2-nitrobenzoic acid, indicating that benomyl binding to tubulin induces a conformational change in the tubulin. Benomyl inhibited the polymerization of brain tubulin into microtubules, with 50% inhibition occurring at a concentration of 70-75 microM. Furthermore, it strongly suppressed the dynamic instability behavior of individual brain microtubules in vitro as determined by video microscopy. It reduced the growing and shortening rates of the microtubules but did not alter the catastrophe or rescue frequencies. The unexpected potency of benomyl against mammalian microtubule polymerization and dynamics prompted us to investigate the effects of benomyl on HeLa cell proliferation and mitosis. Benomyl inhibited proliferation of the cells with an IC(50) of 5 microM, and it blocked mitotic spindle function by perturbing microtubule and chromosome organization. The greater than expected actions of benomyl on mammalian microtubules and mitosis together with its relatively low toxicity suggest that it might be useful as an adjuvant in cancer chemotherapy.     

NMK-TD-100, a Novel Microtubule Modulating Agent,Blocks Mitosis and Induces Apoptosis in HeLa Cells by Binding to Tubulin

Thiadiazoles are one of the most widely utilized agents in medicinal chemistry, having a wide range of pharmacologic activity. Microtubules (MTs) have always remained a sought-after target in rapidly proliferating cancer cells. We screened for the growth inhibitory effect of synthetic 5-(3-indolyl)-2-substituted-1,3,4-thiadiazoles on cancer cells and identified NMK-TD-100, as the most potent agent. Cell viability experiments using human cervical carcinoma cell line (HeLa cells) indicated that the IC₅₀ value was 1.42±0.11 µM for NMK-TD-100 for 48 h treatment. In further study, we examined the mode of interaction of NMK-TD-100 with tubulin and unraveled the cellular mechanism responsible for its anti-tumor activity. NMK-TD-100 induced arrest in mitotic phase of cell cycle, caused decline in mitochondrial membrane potential and induced apoptosis in HeLa cells. Immunofluorescence studies using an anti-α-tubulin antibody showed a significant depolymerization of the interphase microtubule network and spindle microtubule in HeLa cells in a concentration-dependent manner. However, the cytotoxicity of NMK-TD-100 towards human peripheral blood mononuclear cells (PBMC) was lower compared to that in cancer cells. Polymerization of tissue purified tubulin into microtubules was inhibited by NMK-TD-100 with an IC₅₀ value of 17.5±0.35 µM. The binding of NMK-TD-100 with tubulin was studied using NMK-TD-100 fluorescence enhancement and intrinsic tryptophan fluorescence of tubulin. The stoichiometry of NMK-TD-100 binding to tubulin is 1:1 (molar ratio) with a dissociation constant of ~1 µM. Fluorescence spectroscopic and molecular modeling data showed that NMK-TD-100 binds to tubulin at a site which is very near to the colchicine binding site. The binding of NMK-TD-100 to tubulin was estimated to be ~10 times faster than that of colchicine. The results indicated that NMK-TD-100 exerted anti-proliferative activity by disrupting microtubule functions through tubulin binding and provided insights into its potential of being a chemotherapeutic agent.     

4',6-Diamidino-2-phenylindole, a fluorescent probe for tubulin and microtubules

A new fluorophor for tubulin which has permitted the monitoring of microtubule assembly in vitro is reported. DAPI (4',6-diamidino-2-phenylindole), a fluorophor already known as a DNA intercalator, was shown to bind specifically to a unique tubulin site as a dimer (KD(app) = 43 +/- 5 microM at 37 degrees C) or to tubulin associated in microtubules (KD(app) = 6 +/- 2 microM at 37 degrees C) with the same maximum enhancement in fluorescence. When tubulin polymerization was induced with GTP, the change in DAPI affinity for tubulin resulted in an enhancement of DAPI binding and, consequently, of fluorescence intensity. DAPI, whose binding site is different from that of colchicine, vinblastine, or taxol, did not interfere greatly with microtubule polymerization. It induced a slight diminution of the critical concentration for tubulin assembly due to a decrease in the depolymerizing rate constant. Moreover, DAPI did not interfere with GTP hydrolysis correlated with tubulin polymerization, but it decreased the GTPase activity at the steady state of tubulin assembly. Even at substoichiometric levels DAPI can be used to follow the kinetics of microtubule assembly.     

Cytoplasmic dynein-mediated assembly of pericentrin and gamma tubulin onto centrosomes

Centrosome assembly is important for mitotic spindle formation and if defective may contribute to genomic instability in cancer. Here we show that in somatic cells centrosome assembly of two proteins involved in microtubule nucleation, pericentrin and gamma tubulin, is inhibited in the absence of microtubules. A more potent inhibitory effect on centrosome assembly of these proteins is observed after specific disruption of the microtubule motor cytoplasmic dynein by microinjection of dynein antibodies or by overexpression of the dynamitin subunit of the dynein binding complex dynactin. Consistent with these observations is the ability of pericentrin to cosediment with taxol-stabilized microtubules in a dynein- and dynactin-dependent manner. Centrosomes in cells with reduced levels of pericentrin and gamma tubulin have a diminished capacity to nucleate microtubules. In living cells expressing a green fluorescent protein-pericentrin fusion protein, green fluorescent protein particles containing endogenous pericentrin and gamma tubulin move along microtubules at speeds of dynein and dock at centrosomes. In Xenopus extracts where gamma tubulin assembly onto centrioles can occur without microtubules, we find that assembly is enhanced in the presence of microtubules and inhibited by dynein antibodies. From these studies we conclude that pericentrin and gamma tubulin are novel dynein cargoes that can be transported to centrosomes on microtubules and whose assembly contributes to microtubule nucleation.     

Synthesis and evaluation of alpha-hydroxymethylated conjugated nitroalkenes for their anticancer activity: inhibition of cell proliferation by targeting microtubules

The Morita-Baylis-Hillman (MBH) type reaction of a variety of aromatic and heteroaromatic conjugated nitroalkenes with formaldehyde in the presence of stoichiometric amounts of imidazole and catalytic amounts (10 mol %) of anthranilic acid at room temperature provided the corresponding hydroxymethylated derivatives in moderate to good yield. The parent nitroalkenes and their MBH adducts were subsequently screened for their anticancer activity. Some of the MBH adducts were found to inhibit cervical cancer (HeLa) cell proliferation at low micromolar concentrations with half-maximal inhibitory concentrations in the range of 1-2 microM. The antiproliferative activity of 3-((E)-2-nitrovinyl)furan and three potent MBH adducts, namely, hydroxymethylated derivatives of 3-((E)-2-nitrovinyl)thiophene, 1-methoxy-4-((E)-2-nitrovinyl)benzene, and 1,2-dimethoxy-4-((E)-2-nitrovinyl)benzene was correlated well with their antimicrotubule activity. At their effective concentration range, the tested compounds perturbed the organization of mitotic spindle microtubules and chromosomes. In the presence of hydroxymethylated nitroalkenes, abnormal bipolar or multipolar mitotic spindles were apparent. Interphase microtubules were found to be significantly depolymerized at relatively higher concentrations of the tested compounds. These compounds inhibited tubulin assembly into microtubules in vitro by binding to tubulin at a site distinct from the vinblastine and colchicine binding sites. The compounds reduced the intrinsic tryptophan fluorescence of tubulin and the fluorescence of tubulin-1-anilinonaphthalene-8-sulfonic acid (ANS) complex indicating that they induced conformational changes in the tubulin. The results suggest that hydroxymethylated nitroalkenes exert their antiproliferative activity at least in part by depolymerizing cellular microtubules through tubulin binding and indicate that hydroxymethylated nitroalkenes are promising lead compounds for cancer therapy.     

Curcumin inhibits FtsZ assembly: an attractive mechanism for its antibacterial activity

The assembly and stability of FtsZ protofilaments have been shown to play critical roles in bacterial cytokinesis. Recent evidence suggests that FtsZ may be considered as an important antibacterial drug target. Curcumin, a dietary polyphenolic compound, has been shown to have a potent antibacterial activity against a number of pathogenic bacteria including Staphylococcus aureus, Staphylococcus epidermidis and Enterococcus. We found that curcumin induced filamentation in the Bacillus subtilis 168, suggesting that it inhibits bacterial cytokinesis. Further, curcumin strongly inhibited the formation of the cytokinetic Z-ring in B. subtilis 168 without detectably affecting the segregation and organization of the nucleoids. Since the assembly dynamics of FtsZ protofilaments plays a major role in the formation and functioning of the Z-ring, we analysed the effects of curcumin on the assembly of FtsZ protofilaments. Curcumin inhibited the assembly of FtsZ protofilaments and also increased the GTPase activity of FtsZ. Electron microscopic analysis showed that curcumin reduced the bundling of FtsZ protofilaments in vitro. Further, curcumin was found to bind to FtsZ in vitro with a dissociation constant of 7.3+/-1.8 microM and the agent also perturbed the secondary structure of FtsZ. The results indicate that the perturbation of the GTPase activity of FtsZ assembly is lethal to bacteria and suggest that curcumin inhibits bacterial cell proliferation by inhibiting the assembly dynamics of FtsZ in the Z-ring.     

Interaction of tubulin and cellular microtubules with the new antitumor drug MDL 27048. A powerful and reversible microtubule inhibitor

We have characterized the binding of trans-1-(2,5-dimethoxyphenyl)-3-[4-(dimethylamino)phenyl]-2-methyl-2- propen- 1-one (MDL 27048) to purified procine brain tubulin, and the inhibition of microtubule assembly by this compound in vitro and using cultured cells. Binding measurements were performed by difference absorption and fluorescence spectroscopy. MDL 27048 binds to one site/tubulin heterodimer with an apparent equilibrium constant Kb = (2.8 +/- 0.8) X 10(6) M-1 (50 mM 2-(N-morpholino)ethanesulfonic acid, 1 mM [ethylenebis(oxyethylenenitrilo)]tetraacetic acid, 0.5 mM MgCl2, 0.1 mM GTP buffer, pH 6.7, at 25 degrees C). Podophyllotoxin displaced the binding of MDL 27048, suggesting an overlap with the colchicine-binding site. Assembly of purified tubulin into microtubules was inhibited by substoichiometric concentrations of MDL 27048, which also induced a slow depolymerization of preassembled microtubules. The cytoplasmic microtubules of PtK2 cells were disrupted in a concentration and time-dependent manner by MDL 27048, as observed by indirect immunofluorescence microscopy. Maximal depolymerization took place with 2 X 10(-6) M MDL 27048 in 3 h. When the inhibitor was washed off from the cells, fast microtubule assembly (approximately 8 min) and complete reorganization of the cytoplasmic microtubule network (15-30 min) were observed. MDL 27048 also induced mitotic arrest in SV40-3T3 cell cultures. Due to all these properties, this anti-tumor drug constitutes a new and potent microtubule inhibitor, characterized by its specificity and reversibility.     

Dynein Light Chain 1 (LC8) Association Enhances Microtubule Stability and Promotes Microtubule Bundling

Dynein light chain 1 (LC8), a highly conserved protein, is known to bind to a variety of different polypeptides. It functions as a dimer, which is inactivated through phosphorylation at the Ser-88 residue. A loss of LC8 function causes apoptosis in Drosophila embryos, and its overexpression induces malignant transformation of breast cancer cells. Here we show that LC8 binds to tubulin, promotes microtubule assembly, and induces the bundling of reconstituted microtubules in vitro. Furthermore, LC8 decorates microtubules both in Drosophila embryos and in HeLa cells, increases the microtubule stability, and promotes microtubule bundling in these cells. Microtubule stability influences a number of different cellular functions including mitosis and cell differentiation. The LC8 overexpression reduces the susceptibility of microtubules to cold and nocodazole-induced depolymerization in tissue-cultured cells and increases microtubule acetylation, suggesting that LC8 stabilizes microtubules. We also show that LC8 knockdown or transfection with inhibitory peptides destabilizes microtubules and inhibits bipolar spindle assembly in HeLa cells. In addition, LC8 knockdown leads to the mitotic block in HeLa cells. Furthermore, molecular docking analysis using the crystal structures of tubulin and LC8 dimer indicated that the latter may bind at α-β tubulin junction in a protofilament at sites distinct from the kinesin and dynein binding sites. Together, we provide the first evidence of a novel microtubule-associated protein-like function of LC8 that could explain its reported roles in cellular metastasis and differentiation.     

Curcumin inhibits metastatic progression of breast cancer cell through suppression of urokinase-type plasminogen activator by NF-kappa B signaling pathways

Curcumin (1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione), is extracted from the plant Curcuma longa. It was recently reported for its anticancer effect on several types of cancer cells in vitro however, the molecular mechanisms of this anticancer effect are not fully understood. In the present study, we evaluated the effects of curcumin on human mammary epithelial carcinoma MCF-7 cells. Cells were treated with curcumin and examined for cell viability by MTT assay. The cells invasion was demonstrated by transwell assay. The binding activity of NF-κB to DNA was examined in nuclear extracts using Trans-AM NF-κB ELISA kit. Western blot was performed to detect the effect of curcumin on the expression of uPA. Our results showed that curcumin dose-dependently inhibited (P < 0.05) the proliferation of MCF-7 cells. Meanwhile, the adhesion and invasion ability of MCF-7 cells were sharply inhibited when treated with different concentrations of curcumin. Curcumin also significantly decreased (P < 0.05) the expression of uPA and NF-κB DNA binding activity, respectively. It is concluded that curcumin inhibits the adhesion and invasion of MCF-7 cells through down-regulating the protein expression of uPA via of NF-κB activation. Accordingly, the therapeutic potential of curcumin for breast cancer deserves further study.     

Possible regulation of the in vitro assembly of bovine brain tubulin by the bovine thioredoxin system

Microtubule assembly in vitro and in vivo is highly sensitive to a variety of sulfhydryl-reactive reagents, raising the question of the possible existence of a physiological sulfhydryl-mediated system for regulating microtubule assembly. However, the specific reagents which have previously been used to inhibit microtubule assembly in vitro are either nonphysiological or, if physiological, effective only at concentrations much higher than their physiological ones. Because of reports of association in vivo between microtubules and the sulfhydryl-reactive proteins thioredoxin and thioredoxin reductase, we decided to examine the interaction in vitro between microtubules and the thioredoxin system, comprising thioredoxin, thioredoxin reductase and NADPH. At pH 6.8, both the mammalian and the Escherichia coli thioredoxin systems inhibited microtubule assembly by 4-35% (19 +/- 9%) by reducing one intra-subunit disulfide bond in the tubulin dimer. The thioredoxin-reducible disulfide of the tubulin dimer remains protected from thioredoxin in the assembled microtubules. Thioredoxin or thioredoxin reductase alone, or together in the absence of NADPH, were incapable of either reducing tubulin or inhibiting microtubule assembly. Microtubules formed from reduced tubulin were found to be stable and morphologically identical to those obtained from native tubulin dimers. Since the components of the thioredoxin system were used at concentrations similar to their physiological ones, our results suggest a potential role of the thioredoxin system in regulation of microtubule assembly in vivo.     

The natural naphthoquinone plumbagin exhibits antiproliferative activity and disrupts the microtubule network through tubulin binding

Plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone), a naphthoquinone isolated from the roots of Plumbaginaceae plants, has potential antiproliferative activity against several tumor types. We have examined the effects of plumbagin on cellular microtubules ex vivo as well as its binding with purified tubulin and microtubules in vitro. Cell viability experiments using human non-small lung epithelium carcinoma cells (A549) indicated that the IC 50 value for plumbagin is 14.6 microM. Immunofluorescence studies using an antitubulin FITC conjugated antibody showed a significant perturbation of the interphase microtubule network in a dose dependent manner. In vitro polymerization of purified tubulin into microtubules is inhibited by plumbagin with an IC 50 value of 38 +/- 0.5 microM. Its binding to tubulin quenches protein tryptophan fluorescence in a time and concentration dependent manner. Binding of plumbagin to tubulin is slow, taking 60 min for equilibration at 25 degrees C. The association reaction kinetics is biphasic in nature, and the association rate constants for fast and slow phases are 235.12 +/- 36 M (-1) s (-1) and 11.63 +/- 11 M (-1) s (-1) at 25 degrees C respectively. The stoichiometry of plumbagin binding to tubulin is 1:1 (mole:mole) with a dissociation constant of 0.936 +/- 0.71 microM at 25 degrees C. Plumbagin competes for the colchicine binding site with a K i of 7.5 microM as determined from a modified Dixon plot. Based on these data we conclude that plumbagin recognizes the colchicine binding site to tubulin. Further study is necessary to locate the pharmacophoric point of attachment of the inhibitor to the colchicine binding site of tubulin.     

Characterization and in vitro polymerization of Tetrahymena tubulin

Tetrahymena tubulin was purified from the cell extract using DEAE-Sephadex A-50 ion-exchanger and ammonium sulfate precipitation. About 2.2% of the total protein in the 20,000 X g supernatant was recovered as DEAE-Sephadex-purified tubulin fraction. Applying the temperature-dependent polymerization-depolymerization method to this fraction in the presence of Tetrahymena outer fibers as a seed, almost pure tubulin was obtained. Tetrahymena tubulin dimer showed different behavior on SDS-polyacrylamide gels from porcine brain tubulin, and showed very low affinity for colchicine, amounting to about one-twentieth of the binding to porcine brain tubulin. The tubulin fraction failed to polymerize into microtubules by itself. Addition of a small amount of the ciliary outer fiber fragment induced polymerization as demonstrated by viscometric measurements, but the reconstituted microtubules were very unstable in the absence of glycerol. Microtubule-depolymerizing agents such as Ca2+ ions, low temperature, or colchicine all inhibited in vitro polymerization. Although Tetrahymena tubulin purified by the polymerization-depolymerization method could copolymerize with porcine brain microtubules, the DEAE-Sephadex-purified tubulin fraction suppressed the initial rate of porcine brain microtubule assembly in vitro. There seemed to be no differences between cytoplasmic tubulin and outer fiber tubulin in colchicine binding activity or SDS-gel electrophoretic behavior, or between the fine structure of both reconstituted microtubules observed by electron microscopy.     

Human chromosomes and centrioles as nucleating sites for the in vitro assembly of microtubules from bovine brain tubulin

Treatment of HeLa cells with Colcemid at concentrations of 0.06-0.10 mug/ml leads to irreversible arrest in mitosis. Colcemid-arrested cells contained few microtubules, and many kinetochores and centrioles were free of microtubule association. When these cells were exposed to microtubule reassembly buffer containing Triton X-100 and bovine brain tubulin at 37 degrees C, numerous microtubules were reassembled at all kinetochores of metaphase chromosomes and in association with centriole pairs. When bovine brain tubulin was eliminated from the reassembly system, microtubules failed to assemble at these sites. Similarly, when EGTA was eliminated from the reassembly system, microtubules failed to polymerize. These results are consistent with other investigations of in vitro microtubule assembly and indicate that HeLa chromosomes and centrioles can serve as nucleating sites for the assembly of microtubules from brain tubulin. Both chromosomes and centrioles became displaced from their C-metaphase configurations during tubulin reassembly, indicating that their movements were a direct result of microtubule formation. Although both kinetochore- and centriole- associated microtubules were assembled and movement occurred, we did not observe direct extension of microtubules from kinetochores to centrioles. This system should prove useful for experimental studies of spindle microtubule formation and chromosome movement in mammalian cells.     

Molecular weight dependency of heparin inhibition of microtubule assembly in vitro

Low molar ratios of heparin inhibited in vitro assembly of bovine brain microtubule proteins and disassembled preformed microtubules. Addition of purified microtubule-associated proteins counteracted the assembly inhibition by heparin. Our results suggest that the polyanion heparin affects microtubule assembly by binding to the microtubule-associated proteins. This complex can not support nucleation or stabilize the microtubule structure although it still can associate with the tubulin polymer. In the presence of heparin, the critical concentration needed for microtubule assembly was increased. Furthermore, the absolute assembly difference induced by heparin, the delta A350, was only dependent on the concentration and the molecular weight of heparin, not of the total microtubule protein concentration, or the addition of microtubule-associated proteins. Commercial, standard heparin (Mr 6000-25 000) had an I50 of about 0.1/tubulin dimer. The heparin fraction(s) with a high molecular weight had a stronger effect than those with lower molecular weight. Substoichiometric amounts of taxol completely relieved the inhibition of assembly by heparin, although aberrant forms were present. These microtubules had a reduced amount of coassembled microtubule-associated proteins, and furthermore contained heparin.