Microtubules and Cell Division: Potential Pharmacological Targets in Cancer Therapy.

Microtubules are a well-known target in cancer chemotherapy because of their critical role in cell division. Chromosome segregation during mitosis depends on the establishment of the mitotic spindle apparatus through microtubule dynamics. The disruption of microtubule dynamics through the stabilization or destabilization of microtubules results in the mitotic arrest of the cells. Microtubule-targeted drugs, which interfere with microtubule dynamics, inhibit the growth of cells at the mitotic phase and induce apoptotic cell death. The principle of microtubule-targeted drugs is to arrest the cells at mitosis and reduce their growth because cancer is a disease of unchecked cell proliferation. Many anti-microtubule agents produce significant inhibition of cancer cell growth and are widely used as chemotherapeutic drugs for the treatment of cancer. The drugs that interact with microtubules generally bind at one of the three sites vinblastine site, taxol site, or colchicine site. Colchicine binds to the interface of tubulin heterodimer and induces the depolymerization of microtubules. The colchicine binding site on microtubules is a much sought-after target in the history of anti-microtubule drug discovery. Many colchicine-binding site inhibitors have been discovered, but their use in the treatment of cancer is limited due to their dose-limiting toxicity and resistance in humans. Combination therapy can be a new treatment strategy to overcome these drawbacks of currently available microtubule-targeted anticancer drugs. This review discusses the significance of microtubules as a potential pharmacological target for cancer and stresses the necessity of finding new microtubule inhibitors to fight the disease.

Investigation on the Anticancer and Antibacterial Mechanism of Thiazoline/Imidazoline Derived Palladium(II) Complexes.

Biological activities of a series of palladium(II) complexes (M1-M9) bearing N∩ N, N∩ S, and N∩ O chelating ligands are reported. The palladium complexes were tested for their cytotoxic properties against human cervical cancer (HeLa) cells and antibacterial activity against Gm+ve and Gm-ve bacteria. Among the palladium complexes studied (M1-M9), the complex M5, M8, and M9 were found to be more effective in inhibiting the proliferation of HeLa cells. Hence, these complexes were further investigated for their potential role in cellular damage and apoptosis. DCFDA staining, Rhodamine 123 staining and DNA cleavage assay revealed that complex M5, M8 and M9 induced apoptotic cell death in HeLa cells through ROS generation, DNA damage and mitochondrial depolarization. Computational and titration studies also indicated strong electrostatic interaction with DNA groove. Most of the complexes exhibited good antibacterial activity against both Gm+ve and Gm-ve bacteria. The antibacterial activity of the compounds could not be correlated with their anticancer activity indicating a differential mechanism at their effective concentrations. The detailed study on the antibacterial mechanism of the most potent complex M7 revealed that it exerted its antibacterial activity by inhibiting the function of FtsZ and perturbing the localization of the Z-ring at the mid cell.

MD simulation-based screening approach identified tolvaptan as a potential inhibitor of Eg5.

We discovered tolvaptan as a new Eg5 inhibitor using molecular dynamics simulation-based virtual screening. The Eg5-monastrol, Eg5-ispinesib, and Eg5-STLC complexes with "closed" L5 conformation obtained in MD simulation were used to generate a combined pharmacophore model, and this model was used during the process of virtual screening. Further, the MD simulation for 1 µs showed that the binding of tolvaptan to Eg5 was stable due to the closure of the α2/L5/α3 pocket. Tolvaptan belongs to the class of drugs called vaptans which are non-peptide vasopressin receptor antagonists. Since our virtual search for mitotic inhibitors identified tolvaptan as a potential candidate, we were interested in unraveling its antimitotic mechanism. Tolvaptan bound to purified Eg5-437H with a dissociation constant of 27 ± 3.8 µM. Tolvaptan inhibited the growth of HeLa cells through the mitotic block, and around 70% of these mitotic cells exhibited a characteristic monopolar spindle. Tolvaptan bound to goat brain tubulin with a dissociation constant of 103 ± 13 µM. The binding location of tolvaptan on tubulin overlapped with that of colchicine, according to molecular docking analysis. The combination of tolvaptan with STLC augmented mitotic bock with monopolar cells, whereas its combination with vinblastine increased mitotic block with bipolar cells. Since tolvaptan is found to have a significant cytotoxic effect on HeLa cells, it can be developed as a prospective anticancer agent either alone or in combination with other antimitotic drugs. Tolvaptan was identified as an inhibitor of Eg5 in a MD simulation-based virtual screening using a combined pharmacophore model.

Cytotoxic mechanism of tioconazole involves cell cycle arrest at mitosis through inhibition of microtubule assembly.

Tioconazole is one of the drugs used to treat topical mycotic infections. It exhibited severe toxicity during systemic administration; however, the molecular mechanism behind the cytotoxic effect was not well established. We employed HeLa cells as a model to investigate the molecular mechanism of its toxicity and discovered that tioconazole inhibited HeLa cell growth through mitotic block (37%). At the half-maximal inhibitory concentration (≈ 15 µM) tioconazole apparently depolymerized microtubules and caused defects in chromosomal congression at the metaphase plate. Tioconazole induced apoptosis and significantly hindered the migration of HeLa cells. Tioconazole bound to goat brain tubulin (K d, 28.3 ± 0.5 µM) and inhibited the assembly of microtubules in the in vitro assays. We report for the first time that tioconazole binds near to the colchicine site, based on the evidence from in vitro tubulin competition experiment and computational analysis. The conformation of tubulin dimer was found to be "curved" upon binding with tioconazole in the MD simulation. Tioconazole in combination with vinblastine synergistically inhibited the growth of HeLa cells and augmented the percentage of mitotic block by synergistically inhibiting the assembly of microtubules. Our study indicates that the systemic adverse effects of tioconazole are partly due to its effects on microtubules and cell cycle arrest. Since tioconazole is well tolerated at the topical level, it could be developed as a topical anticancer agent in combination with other systemic anticancer drugs. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10616-021-00516-w.

Securinine induces mitotic block in cancer cells by binding to tubulin and inhibiting microtubule assembly: A possible mechanistic basis for its anticancer activity.

AIM: Analysis of the anticancer and antimitotic activity of the plant derived alkaloid securinine along with its effect on the organization of cellular microtubules as well as its binding with purified goat brain tubulin in-vitro. MATERIALS AND METHODS: The cytotoxicity of securinine on different cell lines was conducted using SRB assay. The effect of securinine on the cellular microtubules was analyzed using immunofluorescence microscopy. The binding of securinine on purified goat brain tubulin was evaluated using fluorescent spectroscopy. KEY FINDINGS: Securinine effectively prevented the proliferation of cervical, breast and lung cancer cells with an IC50 of 6, 10 and 11 µM respectively and induced minimal toxicity in HEK cell line. Securinine at concentrations higher than IC50 induced significant depolymerization in interphase and mitotic microtubules and it suppressed the reassembly of cold depolymerized spindle microtubules in HeLa cells. In the wound healing assay, securinine effectively suppressed the migration of HeLa cells to close the wound. Securinine bound to tubulin with a Kd of 9.7 µM and inhibited the assembly of tubulin into microtubules. The treatment with securinine induced a mitochondrial dependent ROS response in HeLa cells which enhanced the cytotoxic effect of securinine. The result from gene expression studies indicates that securinine induced apoptosis in MCF-7 cells through p53 dependent pathway. SIGNIFICANCE: Considering the strong anticancer and anti-metastatic property and low toxicity in non-malignant cell lines, we suggest that securinine can be used as a chemotherapeutic drug either alone or in combination with other known anticancer molecules.

The natural anthraquinone dye purpurin exerts antibacterial activity by perturbing the FtsZ assembly.

There is an increasing demand to discover novel antibacterial drugs to counter the ever-evolving genetic machinery of bacteria. The cell division protein FtsZ plays a vital role in bacterial cytokinesis and has been recognized as an effective antibacterial drug target. In this study, we have shown that the madder dye purpurin inhibited bacterial cytokinesis through perturbation of FtsZ assembly. Purpurin inhibited the growth of bacterial cells in a concentration-dependent manner and induced bacterial cell filamentation. Microscopy studies showed that it inhibited the localization of the Z ring at the midcell, and FtsZ was dispersed throughout the cells. Further, purpurin bound firmly to FtsZ with a dissociation constant of 11 µM and inhibited its assembly in vitro. It reduced the GTP hydrolysis by binding closer to the nucleotide-binding site of FtsZ. Purpurin inhibited the proliferation of mammalian cancer cells at higher concentrations without disturbing the polymerization of tubulin. The results collectively suggest that the natural anthraquinone purpurin can potently inhibit the growth of bacteria and serve as a lead molecule for the development of antibacterial agents.

Vanadocene dichloride induces apoptosis in HeLa cells through depolymerization of microtubules and inhibition of Eg5.

Vanadocene dichloride (VDC), a vanadium containing metallocene dihalide exhibits promising anticancer activity. However, its mechanism of action remains elusive as several diverse targets and pathways have been proposed for its anticancer activity. In this study, we observed that VDC inhibited the proliferation of mammalian cancer cells and induced apoptotic cell death by altering the mitochondrial membrane potential and the expression of bcl2 and bax. Probing further into its anticancer mechanism, we found that VDC caused depolymerization of interphase microtubules and blocked the cells at mitosis with considerable proportion of cells exhibiting monopolar spindles. The reassembly of cold depolymerized microtubules was strongly inhibited in the presence of 10 µM VDC. VDC perturbed the microtubule-kinetochore interactions during mitosis as indicated by the absence of cold stable spindle microtubules in the cells treated with 20 µM VDC. Using goat brain tubulin, we found that VDC inhibited the steady-state polymer mass of microtubules and bound to tubulin at a novel site with a Kd of 9.71 ± 0.19 µM and perturbed the secondary structure of tubulin dimer. In addition, VDC was also found to bind to the mitotic kinesin Eg5 and inhibit its basal as well as microtubule stimulated ATPase activity. The results suggest that disruption of microtubule assembly dynamics and inhibition of the ATPase activity of Eg5 could be a plausible mechanism for the antiproliferative and antimitotic activity of VDC.Graphic abstract.

Sertaconazole induced toxicity in HeLa cells through mitotic arrest and inhibition of microtubule assembly.

Econazole, miconazole, and sertaconazole, the structurally related azoles with imidazole moiety, were evaluated for their cytotoxicity and their ability to bind to mammalian tubulin. Our results indicated that sertaconazole and econazole bound to goat brain tubulin with a dissociation constant of 9 and 19 µM respectively, while miconazole did not bind to goat brain tubulin. Econazole, miconazole, and sertaconazole inhibited the proliferation of HeLa cells with an IC50 of 28, 98, and 38 µM respectively with sertaconazole alone inducing a mitotic block in the treated cells. Since sertaconazole bound to goat brain tubulin with higher affinity and blocked the cells at mitosis, we hypothesized that its cytotoxic mechanism might involve inhibition of tubulin and econazole which did not block the cells at mitosis may have additional targets than tubulin. Sertaconazole inhibited the polymerization of tubulin in HeLa cells and the in vitro assembled goat brain tubulin. Competitive tubulin-binding assay using colchicine and computational simulation studies showed that sertaconazole bound closer to the colchicine site and induced the tubulin dimer to adopt a "bent" conformation which is incompetent for the polymerization. Results from RT-PCR analysis of the A549 cells treated with sertaconazole indicated activation of apoptosis. Sertaconazole significantly inhibited the migration of HeLa cells and showed synergistic antiproliferative potential with vinblastine. Collectively, the results suggest that sertaconazole which is already in clinical practice could be useful as a topical chemotherapy agent for the treatment of skin cancers in combination with other systemic anticancer agents.

Benserazide Perturbs Kif15-kinesin Binding Protein Interaction with Prolonged Metaphase and Defects in Chromosomal Congression: A Study Based on in silico Modeling and Cell Culture.

The interaction of Kif15 with kinesin binding protein (KBP) is critical for its microtubule localization, bundling of kinetochore microtubules and proper alignment of chromosomes at the metaphase plate. The Kif15-KBP structure was prepared from the crystal structure of Kif15 and nonhomologous model of KBP through docking. Benserazide was retrieved when we did a screening of the ZINC Drug Database using the pharmacophore model generated from the potential binding site on Kif15 in an effort to identify molecules for repurposing as Kif15 inhibitors. Live cell imaging of HeLa cells revealed that benserazide delayed metaphase to anaphase-onset by 47±10 min compared to control cells. Benserazide treatment perturbed the kinetochore and microtubule interaction and inhibited the proliferation of HeLa cells with an IC50 of 101 µM with a mitotic block of 12 %. It did not bind to tubulin in the in vitro assays suggesting that the observed effects could be due to its perturbation of Kif15-KBP interaction.

Biochemical and toxicological investigation of karanjin, a bio-pesticide isolated from Pongamia seed oil.

Karanjin, a furanoflavonol from Pongamia pinnata L is used in agricultural practices for its pesticidal, insecticidal and acaricidal activities. It is commercially available as a bio-pesticide targeting a wide variety of pests. The present study was intended to evaluate the biochemical interactions of karanjin with bovine serum albumin (BSA) and study its toxicological effects on mammalian and bacterial cell lines. Karanjin bound to BSA at a single site with a dissociation constant of 19.7 µM. Evaluation of BSA-karanjin interactions at three different temperatures indicated the involvement of static mode of quenching. Binding experiments in the presence of warfarin and computational docking analysis indicated that karanjin bound closer to the warfarin binding site located in the Subdomain IIA of BSA. Using Förster resonance energy transfer analysis the distance between TRP 213 of BSA and karanjin was found to be 20 Å. Collective results from synchronous fluorescence spectra analysis, differential scanning calorimetry, and circular dichroism analysis indicated that binding of karanjin induced conformational changes in the secondary structure of BSA. Karanjin exhibited low toxicity against human cervical cancer cells and normal mouse fibroblast L929 cells and modestly inhibited the growth of B. subtilis and E. coli cells. The data presented in this study provides insights for understanding the binding interactions of karanjin with BSA and its possible toxicological effects on mammalian cell lines and bacteria.

Carbon dot festooned and surface passivated graphene-reinforced chitosan construct for tumor-targeted delivery of TNF-α gene.

Gene therapy is a promising alternative that ensures effective treatment and cure for cancer. Here, we report graphene-reinforced chitosan (CS) construct based non-viral vector for tumor-targeted gene therapy. The therapeutic gene, pDNA-TNF-α, was loaded on to chitosan-carboxylated graphene oxide (CS-CGO) construct via electrostatic interaction. The pDNA-TNF-α-CS-CGO thus obtained was further passivated with 4,7,10-trioxa-1,13-tridecanediamine for protecting the vector from the mononuclear phagocyte system that contributes to the prolongation of circulation half-life. The surface passivated carrier (PEG-pDNA-TNF-α-CS-CGO) then festooned with the folic acid derived carbon dots (C-dots) for targeting folate receptors that are overexpressed in most of the cancer cells. The results of TEM images and zeta potential values ensured the occurrence of desired changes in each stage of C-dot-PEG-pDNA-TNF-α-CS-CGO formulation. After 14 days of incubation, the anti-angiogenesis effect was observed for final formulation in the chorioallantoic membrane. The results of in vitro gene expression study in cancer cell line show a comparatively higher transfection efficacy of the developed system (C-dot-PEG-pDNA-TNF-α-CS-CGO) than pDNA-TNF-α. The efficiency of the developed gene delivery system was further confirmed using a developed and validated artificial tumor cell apparatus.

Zerumbone, a cyclic sesquiterpene, exerts antimitotic activity in HeLa cells through tubulin binding and exhibits synergistic activity with vinblastine and paclitaxel.

OBJECTIVES: The aim of this study was to elucidate the antimitotic mechanism of zerumbone and to investigate its effect on the HeLa cells in combination with other mitotic blockers. MATERIALS AND METHODS: HeLa cells and fluorescence microscopy were used to analyse the effect of zerumbone on cancer cell lines. Cellular internalization of zerumbone was investigated using FITC-labelled zerumbone. The interaction of zerumbone with tubulin was characterized using fluorescence spectroscopy. The Chou and Talalay equation was used to calculate the combination index. RESULTS: Zerumbone selectively inhibited the proliferation of HeLa cells with an IC50 of 14.2 ± 0.5 µmol/L through enhanced cellular uptake compared to the normal cell line L929. It induced a strong mitotic block with cells exhibiting bipolar spindles at the IC50 and monopolar spindles at 30 µmol/L. Docking analysis indicated that tubulin is the principal target of zerumbone. In vitro studies indicated that it bound to goat brain tubulin with a Kd of 4 µmol/L and disrupted the assembly of tubulin into microtubules. Zerumbone and colchicine had partially overlapping binding site on tubulin. Zerumbone synergistically enhanced the anti-proliferative activity of vinblastine and paclitaxel through augmented mitotic block. CONCLUSION: Our data suggest that disruption of microtubule assembly dynamics is one of the mechanisms of the anti-cancer activity of zerumbone and it can be used in combination therapy targeting cell division.

Indirubin, a bis-indole alkaloid binds to tubulin and exhibits antimitotic activity against HeLa cells in synergism with vinblastine.

Indirubin, a bis-indole alkaloid used in traditional Chinese medicine has shown remarkable anticancer activity against chronic myelocytic leukemia. The present work was aimed to decipher the underlying molecular mechanisms responsible for its anticancer attributes. Our findings suggest that indirubin inhibited the proliferation of HeLa cells with an IC50 of 40 µM and induced a mitotic block. At concentrations higher than its IC50, indirubin exerted a moderate depolymerizing effect on the interphase microtubular network and spindle microtubules in HeLa cells. Studies with goat brain tubulin indicated that indirubin bound to tubulin at a single site with a dissociation constant of 26 ±â€¯3 µM and inhibited the in vitro polymerization of tubulin into microtubules in the presence of glutamate as well as microtubule-associated proteins. Molecular docking analysis and molecular dynamics simulation studies indicate that indirubin stably binds to tubulin at the interface of the α-ß tubulin heterodimer. Further, indirubin stabilized the binding of colchicine on tubulin and promoted the cysteine residue modification by 5,5'-dithiobis-2-nitrobenzoic acid, indicating towards alteration of tubulin conformation upon binding. In addition, we found that indirubin synergistically enhanced the anti-mitotic and anti-proliferative activity of vinblastine, a known microtubule-targeted agent. Collectively our studies indicate that perturbation of microtubule polymerization dynamics could be one of the possible mechanisms behind the anti-cancer activities of indirubin.

Antibacterial and anticancer activity of the purified cashew nut shell liquid: implications in cancer chemotherapy and wound healing.

The cashew nut shell liquid (CNSL) from the cashew nut shell of Anacardium occidentale L. has been used to treat skin infections, cracks on soles of feet and cancerous ulcers. In this study, we have purified the technical CNSL, systematically evaluated its anticancer, antibacterial and wound healing activity. The LC-MS data revealed that the purified CNSL contains the compounds, cardanol, anacardic acid and methylcardol. It inhibited the proliferation of HeLa cells with an IC50 of 0.004%(v/v) and caused moderate mitotic block with spindle abnormality. It induced apoptosis in HeLa cells and accelerated wound closure in L929 cells. It inhibited the growth of Bacillus subtilis with an IC50 of 0.35%(v/v) and the treated cells exhibited elongated morphology indicating that suppression of cell division is one of the possible mechanisms of its action. The study suggests that the purified CNSL might have potential applications in anticancer and antibacterial drug development.

Berberine Induces Toxicity in HeLa Cells through Perturbation of Microtubule Polymerization by Binding to Tubulin at a Unique Site.

Berberine has been used traditionally for its diverse pharmacological actions. It exhibits remarkable anticancer activities and is currently under clinical trials. In this study, we report that the anticancer activity of berberine could be partly due to its inhibitory actions on tubulin and microtubule assembly. Berberine inhibited the proliferation of HeLa cells with an IC50 of 18 µM and induced significant depolymerization of interphase and mitotic microtubules. At its IC50, berberine exerted a moderate G2/M arrest and mitotic block as detected by fluorescence-activated cell sorting analysis and fluorescence microscopy, respectively. In a wound closure assay, berberine inhibited the migration of HeLa cells at concentrations lower than its IC50, indicating its excellent potential as an anticancer agent. In vitro studies with tubulin isolated from goat brain indicated that berberine binds to tubulin at a single site with a Kd of 11 µM. Berberine inhibited the assembly of tubulin into microtubules and also disrupted the preformed microtubules polymerized in the presence of glutamate and paclitaxel. Competition experiments indicated that berberine could partially displace colchicine from its binding site. Results from fluorescence resonance energy transfer, computational docking, and molecular dynamics simulations suggest that berberine forms a stable complex with tubulin and binds at a novel site 24 Å from the colchicine site on the ß-tubulin. Data obtained from synchronous fluorescence analysis of the tryptophan residues of tubulin and from the Fourier transform infrared spectroscopy studies revealed that binding of berberine alters the conformation of the tubulin heterodimer, which could be the molecular mechanism behind the depolymerizing effects on tubulin assembly.

Plumbagin-silver nanoparticle formulations enhance the cellular uptake of plumbagin and its antiproliferative activities.

Colloidal silver nanoparticles (AgNPs) have attracted much attention in recent years as diagnostics and new drug delivery system in cancer medicine. To study the effects of plumbagin (PLB), a relatively non-toxic napthaquinone isolated from the roots of Plumbago indica in human cervical cancer cell line and developed a formulation to enhance its cytotoxic activities. Silver nanoparticles were synthesised by chemical reduction method and complexed with PLB. Both the AgNPs and the complex PLB-AgNPs were characterised by dynamic light scattering, high-resolution scanning electron microscopy and transmission electron microscopy. The amount of PLB and PLB-AgNPs internalised was determined by ultra-violet-visible spectrophotometer. Cell inhibition was determined by sulphorhodamine B assay. Mitotic index was determined by Wright-Giemsa staining. Apoptosis induction was assessed by western blot using cleaved poly adenosine diphosphate-ribose polymerase antibody. The scanning electron microscope analysis indicated an average particle size of 32±8 nm in diameter. Enhanced internalisation of PLB into the HeLa cells was observed in PLB-AgNPs. PLB inhibited proliferation of cells with IC50 value of about 18±0.6 µM and blocked the cells at mitosis in a concentration-dependent manner. PLB also inhibited the post-drug exposure clonogenic survival of cells and induced apoptosis. The antiproliferative, antimitotic and apoptotic activities were also found to be increased when cells were treated with PLB-AgNPs. The authors results support the idea that AgNP could be a promising and effective drug delivery system for enhanced activity of PLB in cancer treatment.

Indicine N-oxide binds to tubulin at a distinct site and inhibits the assembly of microtubules: a mechanism for its cytotoxic activity.

Indicine N-oxide, a pyrrolizidine alkaloid present in the plant Heliotropium indicum had shown promising cytotoxic activity in various tumor models. The compound exhibited severe toxicity to hepatocytes and bone marrow cells. The present work was aimed to evaluate the molecular mechanism of the toxicity of indicine N-oxide. We found that indicine N-oxide inhibited the proliferation of various cancer cell lines in a concentration dependent manner with IC50 ranging from 46 to 100 µM. At the half maximal inhibitory concentration it blocked the cell cycle progression at mitosis without significantly altering the organization of the spindle and interphase microtubules. The toxicities of the compound at higher concentrations are attributed to its severe depolymerizing effect on both the interphase and spindle microtubules. Binding studies using purified goat brain tubulin indicated that indicine N-oxide binds to tubulin at a distinct site not shared by colchicine or taxol. It decreased the polymer mass of both purified tubulin and MAP-rich tubulin. It was found to induce cleavage of DNA using pUC18 plasmid. The interactions of indicine N-oxide on DNA were also confirmed by computational analysis; which predicted its binding site at the minor groove of DNA. These studies bring to light that the toxicities of indicine N-oxide were due to its DNA damaging effects and depolymerization of microtubules.

Griseofulvin stabilizes microtubule dynamics, activates p53 and inhibits the proliferation of MCF-7 cells synergistically with vinblastine.

BACKGROUND: Griseofulvin, an antifungal drug, has recently been shown to inhibit proliferation of various types of cancer cells and to inhibit tumor growth in athymic mice. Due to its low toxicity, griseofulvin has drawn considerable attention for its potential use in cancer chemotherapy. This work aims to understand how griseofulvin suppresses microtubule dynamics in living cells and sought to elucidate the antimitotic and antiproliferative action of the drug. METHODS: The effects of griseofulvin on the dynamics of individual microtubules in live MCF-7 cells were measured by confocal microscopy. Immunofluorescence microscopy, western blotting and flow cytometry were used to analyze the effects of griseofulvin on spindle microtubule organization, cell cycle progression and apoptosis. Further, interactions of purified tubulin with griseofulvin were studied in vitro by spectrophotometry and spectrofluorimetry. Docking analysis was performed using autodock4 and LigandFit module of Discovery Studio 2.1. RESULTS: Griseofulvin strongly suppressed the dynamic instability of individual microtubules in live MCF-7 cells by reducing the rate and extent of the growing and shortening phases. At or near half-maximal proliferation inhibitory concentration, griseofulvin dampened the dynamicity of microtubules in MCF-7 cells without significantly disrupting the microtubule network. Griseofulvin-induced mitotic arrest was associated with several mitotic abnormalities like misaligned chromosomes, multipolar spindles, misegregated chromosomes resulting in cells containing fragmented nuclei. These fragmented nuclei were found to contain increased concentration of p53. Using both computational and experimental approaches, we provided evidence suggesting that griseofulvin binds to tubulin in two different sites; one site overlaps with the paclitaxel binding site while the second site is located at the alphabeta intra-dimer interface. In combination studies, griseofulvin and vinblastine were found to exert synergistic effects against MCF-7 cell proliferation. CONCLUSIONS: The study provided evidence suggesting that griseofulvin shares its binding site in tubulin with paclitaxel and kinetically suppresses microtubule dynamics in a similar manner. The results revealed the antimitotic mechanism of action of griseofulvin and provided evidence suggesting that griseofulvin alone and/or in combination with vinblastine may have promising role in breast cancer chemotherapy.

Benomyl and colchicine synergistically inhibit cell proliferation and mitosis: evidence of distinct binding sites for these agents in tubulin.

Benomyl, a tubulin-targeted antimitotic antifungal agent, belongs to the benzimidazole group of compounds, which are known to inhibit the binding of colchicine to tubulin. Therefore, benomyl was thought to bind at or near the colchicine-binding site on tubulin. However, recent mutational studies in yeast and fluorescence studies involving competitive binding of benomyl and colchicine on goat brain tubulin suggested that benomyl may bind to tubulin at a site distinct from the colchicine-binding site. We set out to examine whether colchicine and benomyl bind to tubulin at distinct sites using a human cervical cancer (HeLa) cell line with the thinking that these agents should exert either additive or synergistic activity on cell proliferation if their binding sites on tubulin are different. We found that benomyl and colchicine synergistically inhibited the proliferation of HeLa cells and blocked their cell cycle progression at mitosis. The synergistic activity of benomyl and colchicine was also apparent from their strong depolymerizing effects on both the spindle and interphase microtubules when used in combinations, providing further evidence that these agents bind to tubulin at different sites. Using NMR spectroscopy, we finally demonstrated that benomyl and colchicine bind to tubulin at different sites and that the binding of colchicine seems to positively influence the binding of benomyl to tubulin and vice versa. Further, an analysis of the saturation transfer difference NMR data yielded an interesting insight into the colchicine-tubulin interaction. The data presented in this study provided a mechanistic understanding of the synergistic effects of benomyl and colchicine on HeLa cell proliferation.

Kinetic stabilization of microtubule dynamic instability by benomyl increases the nuclear transport of p53.

Using time-lapse confocal microscopy and enhanced green fluorescent protein-tubulin transfected MCF-7 cells, we found that a tubulin-targeted antimitotic agent, benomyl at its half-maximal proliferation inhibitory concentration (5 microM) strongly suppressed the rate and extent of growing and shortening excursions of individual microtubules in MCF-7 cells without noticeably depolymerizing the microtubule network or decreasing the polymerized mass of tubulin. Further, benomyl treatment caused an increase in the acetylation level of microtubules suggesting that it stabilizes microtubules. Under the conditions that suppressed the dynamic instability, a sharp increase in the nuclear accumulation of p53 in MCF-7 cells was observed in the presence of benomyl. Up regulation of bax and the increased nuclear accumulation of p21 upon benomyl treatment confirmed the activation of p53. Cisplatin caused an increase in the translocation of p53 into the nucleus in the presence of lower effective concentrations of benomyl while a decrease in the nuclear accumulation of p53 was observed in the presence of high concentrations of benomyl suggesting that the stabilized microtubules assist in the nuclear transportation of p53. Furthermore, increased localization of the light chain of the minus end directed motor protein dynein was detected on the microtubules in the benomyl-treated cells indicating that the suppression of microtubule dynamics may influence the binding of dynein on the microtubules and dynein-mediated cargo transport. Together the data indicate that benomyl inhibits mitosis primarily by suppressing the dynamic instability of microtubules and support the hypothesis that the kinetic stabilization of microtubules enhances the microtubule-mediated transport of p53 into the nucleus.