Copper-Plumbagin Complex Produces Potent Anticancer Effects by Depolymerizing Microtubules and Inducing Reactive Oxygen Species and DNA Damage.

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.

ß-III Tubulin Levels Determine the Neurotoxicity Induced by Colchicine-Site Binding Agent Indibulin.

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 ß-III tubulin, acetylated tubulin, and polyglutamylated tubulin were significantly increased in differentiated neuroblastoma cells (SH-SY5Y). Since neuronal cells express ß-tubulin isotypes differently from other cell types, we explored the binding of indibulin to different ß-tubulin isotypes. Our molecular docking analysis suggested that indibulin binds to ß-III tubulin with lower affinity than to other ß-tubulin isotypes. We therefore studied the implications of different ß-tubulin isotypes on the cytotoxic effects of indibulin, colchicine, and vinblastine in differentiated SH-SY5Y cells. Upon depletion of ß-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 ß-III tubulin levels in the cells.

Crocin, a carotenoid, suppresses spindle microtubule dynamics and activates the mitotic checkpoint by binding to tubulin.

Crocin, a constituent of the saffron spice, exhibits promising antitumor activity in animal models and also inhibits the proliferation of several types of cancer cells in culture. Recently, we have shown that crocin binds to purified tubulin at the vinblastine site, depolymerizes microtubules and induces a mitotic block in cultured cells. Here, we extend our previous suggestion and explore the cellular effects of crocin to further understand its mechanism of action. In a kinetic study, we observed that the crocin-induced depolymerization of microtubules preceded both DNA damage and reactive oxygen species generation indicating that depolymerizing microtubules is the primary action of crocin. Crocin also inhibited the growth of cold-depolymerized microtubules in HeLa cells indicating that it can inhibit microtubule dynamics. Using fluorescence recovery after photobleaching, crocin was found to suppress the spindle microtubule dynamics in live HeLa cells. Further, crocin treatment resulted in activation of spindle assembly checkpoint proteins, BubR1 and Mad2. Similar to other microtubule-targeting agents, crocin also perturbed the localization of end-binding protein EB1 from the growing microtubule ends and enhanced the acetylation of remaining microtubules. Further, crocin was found to bind to purified tubulin with a dissociation constant of 12 ±â€¯1.5 µM. The results suggested that crocin exerted its antiproliferative effect primarily by inhibiting the assembly and dynamics of microtubules. Importantly, the combination of crocin with known anticancer agents like combretastatin A-4, cisplatin, doxorubicin or sorafenib, exerted a strong synergistic cytotoxic effect in HeLa cells indicating that crocin may enhance the effectiveness of other anticancer agents.

Drug-Clinical Agent Molecular Hybrid: Synthesis of Diaryl(trifluoromethyl)pyrazoles as Tubulin Targeting Anticancer Agents.

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.