Microtubules, composed of αβ-tubulin heterodimers, exhibit diverse structural and functional properties in different cell types. The diversity in the microtubule structure originates from tubulin heterogeneities, namely tubulin isotypes and their post-translational modifications (PTMs). These heterogeneities confer differential stability to microtubules and provide spatial cues for the functioning of the cell. Furthermore, the altered expressions of tubulin isotypes and PTMs are prominent factors for the development of resistance against some cancer drugs. In this review, we summarize our current knowledge of the tubulin isotypes and PTMs and how, together, they control the cellular functions of the microtubules. We also describe how cancer cells use this tubulin heterogeneity to acquire resistance against clinical agents and discuss existing attempts to counter the developed resistance.

Twenty-three combretastatin A-4 (CA-4) analogues were synthesized by judiciously incorporating a functional N-heterocyclic motif present in Celecoxib (a marketed drug) while retaining essential pharmacophoric features of CA-4. Combretastatin-(trifluoromethyl)pyrazole hybrid analogues, i.e., 5-trimethoxyphenyl-3-(trifluoromethyl)pyrazoles with a variety of relevantly substituted aryls and heteroaryls at 1-position were considered as potential tubulin polymerization inhibitors. The cytotoxicity of the compounds was evaluated using MCF-7 cells. Analog 23 (C-23) was found to be the most active among the tested compounds. It showed pronounced cytotoxicity against HeLa, B16F10, and multidrug-resistant mammary tumor cells EMT6/AR1. Interestingly, C-23 displayed significantly lower toxicity toward noncancerous cells, MCF10A and L929, than their cancerous counterparts, MCF-7 and B16F10, respectively. C-23 depolymerized interphase microtubules, disrupted mitotic spindle formation, and arrested MCF-7 cells at mitosis, leading to cell death. C-23 inhibited the assembly of tubulin in vitro. C-23 bound to tubulin at the colchicine binding site and altered the secondary structures of tubulin. The data revealed the importance of (trimethoxyphenyl)(trifluoromethyl)pyrazole as a cis-restricted double bond-alternative bridging motif, and carboxymethyl-substituted phenyl as ring B for activities and interaction with tubulin. The results indicated that the combretastatin-(trifluoromethyl)pyrazole hybrid class of analogues has the potential for further development as anticancer agents.

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Objectives To examine the antiproliferative effect of a rationally designed, novel noscapine analogue, 9-((perfluorophenyl)methylene) aminonoscapine, '9-PAN') on MDA-MB-231 breast cancer cell line, and to elucidate the underlying mechanism of action. Methods The rationally designed Schiff base-containing compound, 9-PAN, was characterized using IR, NMR and mass spectra analysis. The effect of the compound on cell viability was studied using an MTT assay. Cell cycle and cell death analyses were performed using flow cytometry. Binding interactions of 9-PAN with tubulin were studied using spectrofluorometry. Reactive oxygen species (ROS) generation and mitochondrial membrane potential (MMP) were investigated using the probes, DCFDA and rhodamine-123, respectively. Immunofluorescence imaging was used to visualize cellular microtubules. Key findings 9-PAN inhibited cell proliferation (IC 50 of 20 AE 0.3 µM) and colony formation (IC 50 , 6.2 AE 0.3 µM) by arresting the cells at G 2 /M phase of the cell cycle. It bound to tubulin in a concentration-dependent manner without considerably altering the tertiary conformation of the protein or the polymer mass of the microtubules in vitro. The noscapinoid substantially damaged cellular microtubule network and induced cell death, facilitated by elevated levels of ROS. Conclusions 9-PAN exerts its antiproliferative effect by targeting tubulin and elevating ROS level in the cells.

Here, we have synthesized a copper complex of plumbagin (Cu-PLN) and investigated its antiproliferative activities in different cancer cells. The crystal structure of Cu-PLN showed that the complex was square planar with a binding stoichiometry of 1:2 (Cu/Plumbagin). Cu-PLN inhibited the proliferation of human cervical carcinoma (HeLa), human breast cancer (MCF-7), and murine melanoma (B16F10) cells with half-maximal inhibitory concentrations (IC50) of 0.85 ± 0.05, 2.3 ± 0.1, and 1.1 ± 0.1 μM, respectively. Plumbagin inhibited the proliferation of HeLa, MCF-7, and B16F10 cells with IC50 of 7 ± 0.1, 8.2 ± 0.2, and 6.2 ± 0.4 μM, respectively, showing that Cu-PLN is a stronger antiproliferative agent than plumbagin. Interestingly, Cu-PLN showed much stronger toxicity against breast carcinoma and skin melanoma cells than noncancerous breast epithelial and skin fibroblast cells, indicating its specific cytotoxicity toward cancer cells. A short exposure of Cu-PLN triggered microtubule disassembly in cultured cancer cells, and the complex also inhibited the polymerization of purified tubulin much more strongly than plumbagin. Furthermore, Cu-PLN inhibited the binding of colchicine to tubulin. In addition to microtubule depolymerization, the antiproliferative mechanism of Cu-PLN involved induction of reactive oxygen species, reduction of the mitochondrial membrane potential, and DNA damage. Moreover, the cytotoxic effects of Cu-PLN reduced significantly in cells pre-treated with N-acetyl cysteine, suggesting that reactive oxygen species generation is crucial in Cu-PLN’s mode of action. Thus, the complexation of plumbagin with copper yields a promising antitumor agent having a stronger antiproliferative activity than cisplatin, a widely used anticancer drug.

Indibulin, a microtubule-depolymerizing agent, produces minimal neurotoxicity in animals. It is also less cytotoxic toward differentiated neuronal cells than undifferentiated cells. We found that the levels of β-III tubulin, acetylated tubulin, and polyglutamylated tubulin were significantly increased in differentiated neuroblastoma cells (SH-SY5Y). Since neuronal cells express β-tubulin isotypes differently from other cell types, we explored the binding of indibulin to different β-tubulin isotypes. Our molecular docking analysis suggested that indibulin binds to β-III tubulin with lower affinity than to other β-tubulin isotypes. We therefore studied the implications of different β-tubulin isotypes on the cytotoxic effects of indibulin, colchicine, and vinblastine in differentiated SH-SY5Y cells. Upon depletion of β-III tubulin in the differentiated cells, the toxicity of indibulin and colchicine significantly increased, while sensitivity to vinblastine was unaffected. Using biochemical, bioinformatics, and fluorescence spectroscopic techniques, we have identified the binding site of indibulin on tubulin, which had not previously been established. Indibulin inhibited the binding of colchicine and C12 (a colchicine-site binder) to tubulin and also increased the dissociation constant of the interaction between tubulin and colchicine. Indibulin did not inhibit the binding of vinblastine or taxol to tubulin. Interestingly, indibulin antagonized colchicine treatment but synergized with vinblastine treatment in a combination study performed in MDA-MB-231 cells. The results indicate that indibulin is a colchicine-site binder and that the efficacy of colchicine-site binders is affected by the β-III tubulin levels in the cells.

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