Azaindole derivatives are inhibitors of microtubule dynamics, with anti-cancer and anti-angiogenic activities

Abstract

Background and purpose: Drugs targeting microtubules are commonly used for cancer treatment. However, the potency of microtubule inhibitors used clinically is limited by the emergence of resistance. We thus designed a strategy to find new cell-permeable microtubule-targeting agents.

Experimental approach: Using a cell-based assay designed to probe for microtubule polymerization status, we screened a chemical library and identified two azaindole derivatives, CM01 and CM02, as cell-permeable microtubule-depolymerizing agents. The mechanism of the anti-tumour effects of these two compounds was further investigated both in vivo and in vitro.

Key results: CM01 and CM02 induced G2/M cell cycle arrest and exerted potent cytostatic effects on several cancer cell lines including multidrug-resistant (MDR) cell lines. In vitro experiments revealed that the azaindole derivatives inhibited tubulin polymerization and competed with colchicines for this effect, strongly indicating that tubulin is the cellular target of these azaindole derivatives. In vivo experiments, using a chicken chorioallantoic xenograft tumour assay, established that these compounds exert a potent anti-tumour effect. Furthermore, an assay probing the growth of vessels out of endothelial cell spheroids showed that CM01 and CM02 exert anti-angiogenic activities.

Conclusions and implications: CM01 and CM02 are reversible microtubule-depolymerizing agents that exert potent cytostatic effects on human cancer cells of diverse origins, including MDR cells. They were also shown to inhibit angiogenesis and tumour growth in chorioallantoic breast cancer xenografts. Hence, these azaindole derivatives are attractive candidates for further preclinical investigations.

Figure 1

Effect of CM01 and CM02 on microtubule network organization. (A) Immunofluorescence analysis of cell microtubules. HeLa cells were incubated for 2 h with 0.25% DMSO (panel a, vehicle control), 5 μM colchicine (panel b), 25 μM CM01 (panel c) or 25 μM CM02 (panel d). Cells were then permeabilized, fixed and stained for tubulin (green) as described in Methods. Nuclei were stained with Hoechst (blue) Bar = 10 μm. (B) α-Tubulin partition between soluble and insoluble fractions. HeLa cells were treated for 2 h with 0.25% DMSO (vehicle control), 2 μM colchicine and increasing concentrations of CM01 as indicated. Insoluble (I) and soluble (S) fractions were prepared as described in Methods. Equivalent volumes of extracts (20 μL) were separated on 8% SDS-PAGE, and subjected to immunoblot analysis using monoclonal antibody specific for total α-tubulin. (C) same as (B), but with CM02.

Figure 2

Effect of CM01 and CM02 on microtubule polymerization in vitro. (A) Microtubule protein (MTP) (upper graph) and pure tubulin (lower graph) polymerization assay. Tubulin was allowed to polymerize at 37°C, at the indicated conditions. Fluorescence of DAPI bound to microtubules was measured to monitor microtubule polymerization. Experiments were performed in triplicate, in the presence of increasing concentrations of CM01 or CM02, as indicated. Results are presented as mean ± SEM. (B) Effect of CM01 and CM02 on the binding of [³H]-colchicine and [³H]-vinblastine to MTPs. [³H]-colchicine 50 nM or [³H]-vinblastine 30 nM were used to competed with 100 μM CM01, CM02, colchicine and vinblastine as described in Methods. Each value represents the mean ± SEM from triplicate determinations.

Figure 3

Cell cycle distribution after treatment of HeLa cells with CM01 or CM02. HeLa cells were incubated for 16 h with DMSO (control), colchicine (2 μM), CM01 or CM02 (1 μM and 25 μM, as indicated). Cell cycle parameters were analysed by flow cytometry, as described in Methods. The upper panel shows the graphs obtained for 1 and 25 μM, as indicated, of CM01 and CM02 (red), compared with that obtained for DMSO (blue). Values (lower table) are expressed as percentage of the total cell population.

Figure 4

Azaindole derivatives inhibit capillary-like tube formation. HUVEC cells were seeded on Matrigel and compounds at the indicated concentrations were added after cell attachment (one hour later). Tubule formation was observed by phase-contrast microscopy at 24 h. Bar = 200 μm.

Figure 5

Quantitative analysis of endothelial sprouting in response to azaindole derivatives. FGF2 (100 ng·mL⁻¹) was added at day 0 to collagen-embedded HMEC-GFP spheroids in the presence of CM01 or CM02 at different concentrations (0.1 μM to 25 μM). After 3 days of culture, the spheroids were observed under an epifluorescence microscope. (A) Overlay of phase contrast and fluorescence observations at the indicated concentrations of compounds. (B) Measure of the mean total sprout length of endothelial spheroids, performed by quantitative microscopy image analysis. In each condition, data represent the mean values ± SEM of multiple spheroids (n > 10) from one representative experiment out of two.

Figure 6

Anti-tumour effect of azaindole derivatives. MDA-MB-231 cells were xenografted on chick embryo chorioallantoic membrane (CAM). After treatment with either vehicle (DMSO), colchicine, CM01 or CM02, tumours were excised and weighed. The lower CAM was also dissected and fixed and the number of GFP-fluorescent nodules was counted, as described in the Methods section. (A) Representative pictures of tumours at the end of the different treatments. (B) Effect of the different treatments on tumour weight (means ± SEM of six samples). (C) Effect of the different treatments on the nodule number detected in the lower CAM (means ± SEM of six samples). *P < 0.05; **P < 0.01, significantly different from control values using Mann–Whitney test. Bar = 0.8 mm.