Enhanced Tubulin Polymerization Research Articles

Avermectin B1a Shows Potential Anti-Proliferative and Anticancer Effects in HCT-116 Cells via Enhancing the Stability of Microtubules.

Avermectins are a group of macrocyclic lactones that are commonly used as pesticides to treat pests and parasitic worms. Some members of the avermectin family, such as ivermectin, have been found to exhibit anti-proliferative activity toward cancer cells. This study aimed to investigate the potential anti-cancer activities of avermectin B1a using the HCT-116 colon cancer cell line. The MTT assay was used to calculate the IC50 by incubating cells with increasing doses of avermectin B1a for 24, 48, and 72 h. Flow cytometry was used to evaluate apoptosis following the 24 h incubation of cells. The migration capacity of the HCT-116 cells in the absence or presence of avermectin B1a was also investigated. Finally, tubulin polymerization in the presence of avermectin B1a was evaluated. Avermectin B1a presented anti-proliferative activity with an IC50 value of 30 μM. Avermectin B1a was found to promote tubulin polymerization at 30 μM. In addition, avermectin B1a induced apoptosis in HCT-116 cells and substantially diminished their ability to migrate. Avermectin B1a exhibits significant anti-cancer activity and enhances tubulin polymerization, suggesting that it can be used as a promising microtubule-targeting agent for the development of future anticancer drugs.

Viriditoxin Stabilizes Microtubule Polymers in SK-OV-3 Cells and Exhibits Antimitotic and Antimetastatic Potential.

Microtubules play a crucial role in mitosis and are attractive targets for cancer therapy. Recently, we isolated viriditoxin, a cytotoxic and antibacterial compound, from a marine fungus Paecilomyces variotii. Viriditoxin has been reported to inhibit the polymerization of bacterial FtsZ, a tubulin-like GTPase that plays an essential role in bacterial cell division. Given the close structural homology between FtsZ and tubulin, we investigated the potential antimitotic effects of viriditoxin on human cancer cells. Viriditoxin, like paclitaxel, enhanced tubulin polymerization and stabilized microtubule polymers, thereby perturbing mitosis in the SK-OV-3 cell line. However, the morphology of the stabilized microtubules was different from that induced by paclitaxel, indicating subtle differences in the mode of action of these compounds. Microtubule dynamics are also essential in cell movement, and viriditoxin repressed migration and colony formation ability of SK-OV-3 cells. Based on these results, we propose that viriditoxin interrupts microtubule dynamics, thus leading to antimitotic and antimetastatic activities.

Involvement of hyperglycemia in the development of platelet procoagulant response: the role of aldose reductase and platelet swelling.

: Rise in mean platelet volume (MPV) has been demonstrated to be associated with increased platelet reactivity. In diabetes patients, augmented MPV was proposed to contribute to increased risk of thrombotic complications. Therefore, the aim of this study was to investigate whether under hyperglycemic conditions, aldose reductase (AR)-mediated sorbitol formation and associated rise in cell volume, which subsequently results in platelet hyperactivation. Platelets were obtained from 30 healthy volunteers and 13 patients with diabetes. We evaluated changes in platelet size, their reactivity (measured as aggregation and secretion), and sorbitol content evoked by glucose. Measurement of procoagulant activity and thromboelastography were performed to assess how hyperglycemia affects coagulation. We have found that incubation of platelets with glucose (>10 mmol/l) leads to increased MPV, potentiation of collagen-evoked platelet aggregation, secretion, and procoagulant response (measured as platelet-dependent thrombin generation and phosphatidylserine expression). Glucose-treated platelets had higher sorbitol content and demonstrated enhanced tubulin polymerization. All the above-mentioned phenomena were reduced following the blocking of AR or by vincristine (microtubule destabilizing agent). Thromboelastography measurements demonstrated that hyperglycemia is associated with reduction of clotting time (R) and increase in the alpha angle (reflects platelet activation). Addition of sorbinil (AR inhibitor) or vincristine normalized R variable and alpha angle. The hyperglycemic conditions may accelerate platelet-related thrombin generation through the activation of polyol pathway, enhanced tubulin polymerization and associated with it rise in platelet volume.

Cytosolic Cl- Affects the Anticancer Activity of Paclitaxel in the Gastric Cancer Cell Line, MKN28 Cell

Background/Aims: Our previous study revealed that cytosolic Cl^- affected neurite elongation promoted via assembly of microtubule in rat pheochromocytoma PC12D cells and Cl^-–induced blockade of intrinsic GTPase enhanced tubulin polymerization in vitro. Paclitaxel (PTX) is a microtubule-targeted chemotherapeutic drug and stabilizes microtubules resulting in mainly blockade of mitosis at the metaphase-anaphase transition and induction of apoptosis. In the present study, we tried to clarify whether the cytosolic Cl^- affected PTX ability to inhibit cell growth in the gastric cancer cell line, MKN28. Methods: To clarify the cytosolic Cl^- action on PTX-induced cell death and metaphase-anaphase transition in the gastric cancer cell line, MKN28 cell, and PTX-induced tubulin polymerization, we performed cell proliferation assay, cytosolic Cl^- concentration measurement, immunofluorescence microscopy, and in vitro tubulin polymerization assay. Results: The decline of cytosolic Cl^- weakened the cytotoxic effect of PTX on cell proliferation of MKN28 cells, which could pass through the metaphase-anaphase transition. Moreover, in vitro PTX-induced tubulin polymerization was diminished under the low Cl^- condition. Conclusions: Our results strongly suggest that the upregulation of cytosolic Cl^- concentration would enhance the antitumor effect of PTX, and that the cytosolic Cl^- would be one of the key targets for anti-cancer therapy.

Understanding the Sequence of Chemomechanical Transitions in Kinesin-5

The kinesin-5 KSP/Eg5 pauses at microtubule plus-ends and enhances tubulin polymerization by increasing the growth rate and decreasing the catastrophe rate (Chen et al. 2015). The end pausing resembles that of the kinesin-8, kip3p, but has the opposite effect on microtubule dynamics. Upon initial binding to the microtubule lattice, kinesin-5 was reported to pause for 1-second pause before stepping (Krzysiak et al. 2008). However, this “initial pause” predicted from biochemical experiments have not been observed in our single-molecule motility experiments. Compared to transport kinesins, kinesin-5 is less processive but paradoxically less load-dependent, suggesting that kinesin-5 may use different gating mechanisms during processive walking. We measured a kcat of 12 s-1 for the kinesin-5 monomer ATPase and a K0.5 of 0.9 μM, which generates a kbiATPase of 13 μM-1 s-1. The bimolecular microtubule-encounter rate kbiADP was 9 μM-1 s-1 which predicts a monomer chemical processivity of kbi ratio ∼1.4. This suggests that for each ATP turnover, kinesin-5 monomer readily detaches from the microtubule. The ADP off-rate of the bound head was 40 s-1, consistent with rate limiting hydrolysis. In half-site ADP release experiments with processive dimers, we measured maximal rates of 13 s-1 in ATP and 2.1 s-1 in ATPγS, which matched the single-molecule stepping rate in each nucleotide, suggesting that hydrolysis precedes attachment of the tethered head, consistent with recent work on kinesin-1. Hence, we propose that kinesin-5 processivity is gated by the race between detachment of the bound-head in the ADP-Pi state and attachment of the tethered-head. This study provides insights into gating mechanism with respect to processivity on the microtubule wall and in the possible strong-binding states at the microtubule plus-end.

Indomethacin derivatives as tubulin stabilizers to inhibit cancer cell proliferation

Cyclooxygenase (COX) inhibitor Indomethacin analogs exhibited more potent cancer cell growth inhibition and apoptosis inducing activities than the parental compound. The anti-proliferative mechanism investigation of the analogs revealed that they inhibited tubulin polymerization at high concentrations whereas enhanced polymerization at low concentrations. The two opposite activities might antagonize each other and impaired the anti-proliferative activity of the derivatives eventually. In this study, we further performed lead optimization based on the structure activity relationship (SAR) generated. One of the new Indomethacin derivatives compound 11 {2-(4-(benzyloxy)phenyl)-N-(1-(4-bromobenzoyl)-3-(2-((2-(dimethylamino)ethyl)amino)-2-oxoethyl)-2-methyl-1H-indol-5-yl)acetamide} inhibited the proliferation of a panel of cancer cell lines with IC50s at the sub-micromole levels. Further study revealed that the compound only enhanced tubulin polymerization and was a tubulin stabilizer.

Synthesis and identification of α-cyano bis(indolyl)chalcones as novel anticancer agents

Microwave-assisted synthesis of 23 α-cyano bis(indolyl)chalcones (6a–w) and their in vitro anticancer activity against three human cancer cell lines have been discussed. Among the synthesized chalcones, compound 6n was found to be the most potent and selective against A549 lung cancer cell line (IC50=0.8μM). In a preliminary mechanism of action studies some α-cyano bis(indolyl)chalcones were found to enhance tubulin polymerization suggesting these compounds could act as microtubule stabilizing agents.

Abstract 4495: Fisetin, a dietary flavonoid, binds to and disrupts microtubule dynamic instability in prostate cancer cells.

Abstract Taxanes are the first class of chemotherapy drugs shown to improve survival in advanced prostate cancer (PCa). Taxanes bind β-tubulin and stabilize microtubules, resulting in mitotic arrest and cell death. Therefore, disruption of microtubule function may provide an effective therapy for human PCa, which has been observed in clinical settings using paclitaxel. However, these drugs are associated with severe side effects and are sensitive to resistance mechanisms. Thus, search for novel and non-toxic tubulin targeting agents is urgently needed. We hypothesized that fisetin may offer PCa chemotherapeutic and/or chemopreventive effects by interfering with microtubule assembly, either by inhibiting or enhancing tubulin polymerization. Using an in vitro microtubule polymerization assay, we observed that fisetin enhanced tubulin polymerization whose kinetics paralleled that of paclitaxel. Because acetylation of α-tubulin is considered as a marker of stable microtubule structure, we next tested whether increased acetylated α-tubulin correlates with increased microtubule stability in PC-3 and DU-145 cells. We found that fisetin (20-80 μM) treatment increased α-tubulin acetylation in a dose dependent manner in PC-3 and DU-145 cells. To further investigate the stabilization of the microtubule network in fisetin-treated cells, we evaluated microtubule resistance to cold-induced depolymerization. Immunofluorescence labeling revealed that fisetin-treated PC-3 and DU-145 cells were resistant to cold-induced microtubule depolymerization. To identify microtubule-dependent tumor-specific pathways modulated by fisetin, we determined its effect on MAP2 expression in PC-3 and DU-145 cells. We found increased expression of MAP-2 with fisetin treatment. Stabilization of microtubules resulted in arrest of the cells in the G2/M phase of cell cycle and subsequent cell growth inhibition. Activation of microtubule-stabilizing proteins in PCa cells may inhibit their proliferation and correlate with a delay or inhibition of cell migration. Therefore, we investigated the efficacy of fisetin on the invasion and metastasis of PCa using scratch wound assay. We found that fisetin treatment (20-80 μM) for 24 hours inhibited PC-3 cells migration and invasion, which suggested fisetin as an anti-metastatic agent. As a mechanism for these effects we observed that fisetin increased the expression of nuclear migration protein Nud C in PC-3 and DU-145 cells in a dose dependent manner. Because Nud C protein regulates microtubule dynamics in the cell, the result reveal anti-microtubule effects of fisetin. The data reported here provide the first evidence of the dietary flavonoid fisetin as a microtubule stabilizing agent in PCa. Our observation suggests that fisetin could be used for the treatment of advanced PCa alone or as an adjuvant with other chemotherapy drugs. Citation Format: Eiman Mukhtar, Vaqar M. Adhami, Deeba N. Syed, Hasan Mukhtar. Fisetin, a dietary flavonoid, binds to and disrupts microtubule dynamic instability in prostate cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4495. doi:10.1158/1538-7445.AM2013-4495

Microtubule assembly-derived by dimerization of TPPP/p25. Evaluation of thermodynamic parameters for multiple equilibrium system from ITC data

BackgroundThe disordered Tubulin Polymerization Promoting Protein/p25 (TPPP/p25) modulates the dynamics and stability of the microtubule system. In this paper the role of dimerization in its microtubule-related functions is established, and an approach is proposed to evaluate thermodynamic constants for multiple equilibrium systems from ITC measurements. MethodsFor structural studies size exclusion chromatography, SDS-PAGE, chemical cross-linking, circular dichroism, fluorescence spectroscopy and isothermal titration calorimetry were used; the functional effect was analyzed by tubulin polymerization assay. Numerical simulation of the multiple equilibrium was performed with Mathematica software. ResultsThe dimerization of TPPP/p25 is promoted by elevation of the protein concentration and by GTP addition. The dimeric form displaying enhanced tubulin polymerization promoting activity is stabilized by disulfide bond or chemical cross-linking. The GTP binding to the dimeric form (Kd-GTP=200μM) is tighter with one order of magnitude than to the monomeric one leading to the enrichment of the dimers. A mathematical model elaborated for the multiple equilibrium of the TPPP/p25-GTP system was validated by fitting the GTP-dependent changes of ellipticity and fluorescence signal in the course of TPPP/p25 titrations. The evaluation of the equilibrium constants rendered it possible to determine the thermodynamic parameters of the association of different TPPP/p25 forms with GTP from ITC measurements. Conclusions/General SignificanceThe dimerization of TPPP/p25 with favorable physiological functional potency is proposed to play significant role in the fine tuning of TPPP/p25-mediated microtubule assembly; the unfolded monomers might be involved in the formation of pathological inclusions characteristic for Parkinson's disease and other synucleinopathies.

Potent Taccalonolides, AF and AJ, Inform Significant Structure–Activity Relationships and Tubulin as the Binding Site of These Microtubule Stabilizers

The taccalonolides are a class of microtubule stabilizing agents isolated from plants of the genus Tacca. In efforts to define their structure-activity relationships, we isolated five new taccalonolides, AC-AF and H2, from one fraction of an ethanol extract of Tacca plantaginea. The structures were elucidated using a combination of spectroscopic methods, including 1D and 2D NMR and HR-ESI-MS. Taccalonolide AJ, an epoxidation product of taccalonolide B, was generated by semisynthesis. Five of these taccalonolides demonstrated cellular microtubule-stabilizing activities and antiproliferative actions against cancer cells, with taccalonolide AJ exhibiting the highest potency with an IC(50) value of 4.2 nM. The range of potencies of these compounds, from 4.2 nM to >50 μM, for the first time provides the opportunity to identify specific structural moieties crucial for potent biological activities as well as those that impede optimal cellular effects. In mechanistic assays, taccalonolides AF and AJ stimulated the polymerization of purified tubulin, an activity that had not previously been observed for taccalonolides A and B, providing the first evidence that this class of microtubule stabilizers can interact directly with tubulin/microtubules. Taccalonolides AF and AJ were able to enhance tubulin polymerization to the same extent as paclitaxel but exhibited a distinct kinetic profile, suggesting a distinct binding mode or the possibility of a new binding site. The potencies of taccalonolides AF and AJ and their direct interaction with tubulin, together with the previous excellent in vivo antitumor activity of this class, reveal the potential of the taccalonolides as new anticancer agents.

A new diaryl urea compound, D181, induces cell cycle arrest in the G1 and M phases by targeting receptor tyrosine kinases and the microtubule skeleton

Receptor tyrosine kinases (RTKs) modulate a variety of cellular events, including cell proliferation, differentiation, mobility and apoptosis. In addition, RTKs have been validated as targets for cancer therapies. Microtubules are another class of proven targets for many clinical anticancer drugs. Here, we report that 1-(4-chloro-3-(trifluoromethyl) phenyl)-3-(2-cyano-4-hydroxyphenyl)urea (D181) functions as both a receptor tyrosine kinase inhibitor and a tubulin polymerization enhancer. D181 displayed potent inhibitory activities against a panel of RTKs, including Flt3, VEGFR, cKit, FGFR1 and PDGFRβ. D181 also enhanced tubulin polymerization and modified the secondary structure of tubulin proteins to disrupt their dynamic instability. Because of synergistic cooperation, D181 strongly inhibited the proliferation of various cancer cell lines, induced LoVo cell cycle arrest in the G1 and M phases and suppressed tumor growth in nude mice bearing human LoVo and HT29 xenografts. Our studies have provided a new, promising lead compound and novel clues for multi-target anticancer drug design and development.

Abstract IA9-1: Is taxol resistance related to tubulin isotypes?

Abstract Microtubules are an important component of the euraryotic cytoskeleton and are essential for normal cell division. They are also a validated target for antitumor drugs, such as taxol, that bind to the – tubulin subunit in the microtubule polymer. Endogenous resistance or the development of drug resistance during treatment with taxol is a serious clinical problem. Understanding the emergence of taxol resistance and being able to predict patient response would enhance the efficacy of the drug. A number of mechanisms, such as overexpression of P-glycoprotein that acts as an ATP-dependent drug-efflux pump, or mutations in the drug-binding site in tubulin, have been described. Of particular interest to our research, is the aberrant expression of β-tubulin isotypes in cancer. Tubulin is composed of α- and β-tubulin subunits that exist as heterodimers. In humans, there are seven β-tubulins and eight β-tubulins, each the product of a distinct gene located on different chromosomes. Each isotype can undergo extensive posttranslational modifications such as acetylation, phosphorylation, polyglutamylation, polyglycylation and reversible tyrosination, all of which add to the complexity of the tubulin/microtubule system. Most of the divergence between the isotypes is found in the last 15-20 amino acids at the carboxyl end of tubulin and in addition, most posttranslational modifications of tubulin occur in this region. For the most part, tubulin isotypes have been detected at the mRNA level or at the protein level with isotype-specific antibodies. Our laboratory has developed proteomic methods, including highresolution isoelectrofocusing, CNBr cleavage and mass spectrometry that allow us to determine the isotype content and posttranslational modifications of tubulin in cells and tissues in a single experiment. We have utilized taxol, which enhances tubulin polymerization, to isolate sufficient tubulin from cells and are developing methods to quantify tubulin isotype expression by mass spectrometry. The overexpression of βIII-tubulin has been associated with resistance to taxanes in a range of tumor types and in taxol-resistant cells generated in our laboratory. βIII-tubulin is normally expressed only in neuronal cells. However, it is aberrantly expressed in numerous malignant tissues and could be a meaningful prognostic factor for treatment outcome. As has been shown by others in different tumors, in our laboratory an immunohistochemical analysis of 47 tumor specimens from head and neck patients determined that overexpression of β-III tubulin was highly prognostic of survival. Citation Information: Clin Cancer Res 2010;16(14 Suppl):IA9-1.

Tubulin polymerization modifies cardiac sodium channel expression and gating

Treatment with the anticancer drug taxol (TXL), which polymerizes the cytoskeleton protein tubulin, may evoke cardiac arrhythmias based on reduced human cardiac sodium channel (Na(v)1.5) function. Therefore, we investigated whether enhanced tubulin polymerization by TXL affects Na(v)1.5 function and expression and whether these effects are beta1-subunit-mediated. Human embryonic kidney (HEK293) cells, transfected with SCN5A cDNA alone (Na(v)1.5) or together with SCN1B cDNA (Na(v)1.5 + beta1), and neonatal rat cardiomyocytes (NRCs) were incubated in the presence and in the absence of 100 microM TXL. Sodium current (I(Na)) characteristics were studied using patch-clamp techniques. Na(v)1.5 membrane expression was determined by immunocytochemistry and confocal microscopy. Pre-treatment with TXL reduced peak I(Na) amplitude nearly two-fold in both Na(v)1.5 and Na(v)1.5 + beta1, as well as in NRCs, compared with untreated cells. Accordingly, HEK293 cells and NRCs stained with anti-Na(v)1.5 antibody revealed a reduced membrane-labelling intensity in the TXL-treated groups. In addition, TXL accelerated I(Na) decay of Na(v)1.5 + beta1, whereas I(Na) decay of Na(v)1.5 remained unaltered. Finally, TXL reduced the fraction of channels that slow inactivated from 31% to 18%, and increased the time constant of slow inactivation by two-fold in Na(v)1.5. Conversely, slow inactivation properties of Na(v)1.5 + beta1 were unchanged by TXL. Enhanced tubulin polymerization reduces sarcolemmal Na(v)1.5 expression and I(Na) amplitude in a beta1-subunit-independent fashion and causes I(Na) fast and slow inactivation impairment in a beta1-subunit-dependent way. These changes may underlie conduction-slowing-dependent cardiac arrhythmias under conditions of enhanced tubulin polymerization, e.g. TXL treatment and heart failure.

Cevipabulin (TTI-237): Preclinical and clinical results for a novel antimicrotubule agent

Antimitotic agents are among the most effective drugs for the treatment of solid tumors and metastatic cancer. These drugs promote cell death by interfering with the crucial structural and regulatory function of microtubules in cells. Most of the agents of clinical relevance are natural products or semisynthetic derivatives thereof, and they fall into two major classes: microtubule stabilizers such as the taxanes, which enhance tubulin polymerization, and microtubule destabilizers such as the Vinca alkaloids, which lead to the depolymerization of existing microtubules. While these drugs are effective in inhibiting the progression of certain types of tumors, their utility is limited in part by incomplete tumor responses and/or significant side effects. In addition, inherent resistance is encountered in many tumor types, or acquired resistance may occur as a result of multiple cycles of therapy. Cevipabulin (TTI-237) is a novel, small synthetic molecule with an unusual biological mode of action. It appears to bind at the vinca site, but exhibits some properties similar to those of taxane-site ligands, such as enhancing tubulin polymerization. The compound works against a variety of tumors, including those resistant to paclitaxel and vincristine. Furthermore, cevipabulin is stable and water-soluble, and can be administered i.v. or p.o. in saline. It can be synthesized in bulk quantities efficiently. Based on these properties, cevipabulin was selected for clinical development.

The Tumor Suppressor CYLD Regulates Microtubule Dynamics and Plays a Role in Cell Migration

The familial cylindromatosis tumor suppressor CYLD is known to contain three cytoskeleton-associated protein glycine-rich (CAP-Gly) domains, which exist in a number of microtubule-binding proteins and are responsible for their association with microtubules. However, it remains elusive whether CYLD interacts with microtubules and, if so, whether the interaction is mediated by the CAP-Gly domains. In this study, our data demonstrate that CYLD associates with microtubules both in cells and in vitro, and the first CAP-Gly domain of CYLD is mainly responsible for the interaction. Knockdown of cellular CYLD expression dramatically delays microtubule regrowth after nocodazole washout, indicating an activity for CYLD in promoting microtubule assembly. Our data further demonstrate that CYLD enhances tubulin polymerization into microtubules by lowering the critical concentration for microtubule assembly. In addition, we have identified by wound healing assay a critical role for CYLD in mediating cell migration and found that its first CAP-Gly domain is required for this activity. Thus CYLD joins a growing list of CAP-Gly domain-containing proteins that regulate microtubule dynamics and function.

SB401, a pollen‐specific protein from Solanum berthaultii, binds to and bundles microtubules and F‐actin

We characterize a novel, pollen-specific, microtubule-associated protein, SB401, found in Solanum berthaultii. This protein binds to and bundles taxol-stabilized microtubules and enhances tubulin polymerization in a concentration-dependent manner, particularly at lower temperatures. Electron microscopy revealed that the protein decorates the entire length of microtubules. Cross-linking and electrophoresis studies showed that SB401 protein forms dimers, and suggest that dimerization could account for bundling. Double immunofluorescent staining of pollen tubes of S. berthaultii showed that SB401 protein co-localized with cortical microtubule bundles. SB401 protein also binds to and bundles actin filaments, and could connect actin filaments to microtubules. SB401 protein had a much higher affinity for microtubules than for actin filaments. In the presence of both cytoskeletal elements, the protein preferentially bound microtubules to form bundles. These results demonstrate that SB401 protein may have important roles in organizing the cytoskeleton in pollen tubes.

Evaluation of the Apoptotic and Antiproliferative Activities of Paclitaxel in Ehrlich Ascites Tumor Cells

ABSTRACTTaxol (paclitaxel, PAC) is a diterpene with antitumor activity against a variety of human neoplasms. PAC cytotoxicity is thought to derive mainly from a stabilization of microtubules as a result of enhanced tubulin polymerization that leads to an accumulation of cells in G2/M phase of cell cycle. In this study, the cytotoxic effect occurred by PAC in tumor cells in vitro were examined. Mouse mammary gland carcinoma-derived Ehrlich Ascites Tumor Cells (EATC) were employed as tumor cell line. Results of the experiments were evaluated with cell kinetic parameters such as growth rate, mitotic index, apoptotic index and DNA fragmentation. IC30 and IC50 concentrations of PAC (6 μg/ml and 12 μg/ml, respectively) were applied to the cell line for 0–24 hours. The proliferation rate of EATC was inhibited by PAC concentrations compared to control with increasing treatment time (2–24 hours). The inhibition of growth rate was higher in 12 μg/ml PAC treatment than those in 6 μg/ml treatment. In our studies, when the mitotic index parameter data was evaluated to determine which phase of the cell cycle was affected by PAC to cause the repression of cell reproduction, it was found that PAC exposes its cytotoxic effect by causing cell accumulation at mitosis. The accumulation of the cells resulted in an increase in mitotic index values, which was an expected consequence of PAC treatment. In addition to the effect of PAC on mitotic cell accumulation, the results on its effect on apoptotic index parameter support the findings about its repressive function on cell reproduction and its effect on mitotic index increase. It was observed that depending on the drug treatments, increase of mitotic index, inhibition of growth rate and apoptotic index in EATC were increased with respect to control, being of statistically significant (p<0.01-p<0.001). And the DNA fragmentation levels of EATC induced by PAC increased significantly.

Association of Ebola Virus Matrix Protein VP40 with Microtubules

Viruses exploit a variety of cellular components to complete their life cycles, and it has become increasingly clear that use of host cell microtubules is a vital part of the infection process for many viruses. A variety of viral proteins have been identified that interact with microtubules, either directly or via a microtubule-associated motor protein. Here, we report that Ebola virus associates with microtubules via the matrix protein VP40. When transfected into mammalian cells, a fraction of VP40 colocalized with microtubule bundles and VP40 coimmunoprecipitated with tubulin. The degree of colocalization and microtubule bundling in cells was markedly intensified by truncation of the C terminus to a length of 317 amino acids. Further truncation to 308 or fewer amino acids abolished the association with microtubules. Both the full-length and the 317-amino-acid truncation mutant stabilized microtubules against depolymerization with nocodazole. Direct physical interaction between purified VP40 and tubulin proteins was demonstrated in vitro. A region of moderate homology to the tubulin binding motif of the microtubule-associated protein MAP2 was identified in VP40. Deleting this region resulted in loss of microtubule stabilization against drug-induced depolymerization. The presence of VP40-associated microtubules in cells continuously treated with nocodazole suggested that VP40 promotes tubulin polymerization. Using an in vitro polymerization assay, we demonstrated that VP40 directly enhances tubulin polymerization without any cellular mediators. These results suggest that microtubules may play an important role in the Ebola virus life cycle and potentially provide a novel target for therapeutic intervention against this highly pathogenic virus.

HIV-1 Tat protein enhances Microtubule polymerization

BackgroundHIV infection and progression to AIDS is characterized by the depletion of T cells, which could be due, in part, to apoptosis mediated by the extra-cellular HIV-encoded Tat protein as a consequence of Tat binding to tubulin. Microtubules are tubulin polymers that are essential for cell structure and division. Molecules that target microtubules induce apoptosis and are potent anti-cancer drugs. We studied the effect on tubulin polymerization of three Tat variants: Tat HxB2 and Tat Eli from patients who are rapid progressors (RP) and Tat Oyi from highly exposed but persistently seronegative (HEPS) patients. We compared the effect on tubulin polymerization of these Tat variants and peptides corresponding to different parts of the Tat sequence, with paclitaxel, an anti-cancer drug that targets microtubules.ResultsWe show that Tat, and specifically, residues 38–72, directly enhance tubulin polymerization. We demonstrate that Tat could also directly trigger the mitochondrial pathway to induce T cell apoptosis, as shown in vitro by the release of cytochrome c from isolated mitochondria.ConclusionsThese results show that Tat directly acts on microtubule polymerization and provide insights into the mechanism of T cell apoptosis mediated by extra-cellular Tat.

Dimethylarsinic acid targets tubulin in mitotic cells to induce abnormal spindles

AbstractDimethylarsinic acid (DMA) is the most effective inducer of cell‐cycle disruption among the arsenic compounds and their metabolites. The present study was conducted to gain further insight into cell‐cycle disruption induced by DMA. The inhibition of cell proliferation and the mitotic arrest induced by DMA were significant and dose‐dependent in Chinese hamster V79 cells and the two seemed to be closely related. At less than 140 µM the DMA did not inhibit the proliferation of cells, but it significantly induced mitotic arrest. An indirect immunofluorescence assay using anti‐α‐tubulin antibodies revealed that DMA induced the formation of abnormal spindles in the metaphase cells even at 350 µM with 5 h of treatment. At 1.4 mM DMA no metaphase cells could form a definite spindle structure. The spindle figures were similar to those induced by colchicine (125 nM) or vinblastine (110 nM), major antimitotic agents. In DMA‐treated interphase cells, the microtubule networks were indistinguishable from those of normal cells. With the tubulin‐assembly assay estimated by turbidity, DMA at less than 200 µM suppressed tubulin assembly in a dose‐dependent manner, whereas at more than 700 µM it enhanced tubulin polymerization remarkably with or without addition of excess guanosine‐5′‐triphosphate. According to the above findings, we discussed the possibility that DMA, a primary metabolite of inorganic arsenic in mammals, is related to arsenic carcinogenicity. Copyright © 2001 John Wiley &amp; Sons, Ltd.