Novel microtubule polymerization inhibitor with potent antiproliferative and antitumor activity

Abstract

Microtubule-stabilizing and microtubule-destabilizing agents are commonly used as anticancer agents. Although highly effective, success with these agents has been limited due to their relative insolubility, cumbersome synthesis/purification, toxic side effects, and development of multidrug resistance. Hence, the identification of improved agents that circumvent one or more of these problems is warranted. We recently described the rational design of a series of triazole-based compounds as antimitotic agents. Members of this N-substituted 1,2,4-triazole family of compounds exhibit potent tubulin polymerization inhibition and broad spectrum cellular cytotoxicity. Here, we extensively characterize the in vitro and in vivo effects of our lead compound from the series 1-methyl-5-(3-(3,4,5-trimethoxyphenyl)-4H-1,2,4-triazole-4-yl)-1H-indole, designated T115. We show that T115 competes with colchicine for its binding pocket in tubulin, produces robust inhibition of tubulin polymerization, and disrupts the microtubule network system inside the cells. In addition, T115 arrests human cancer cells in the G(2)-M phase of cell cycling, a hallmark of microtubule destabilizing drugs. T115 also inhibits cell viability of several cancer cell lines, including multidrug-resistant cell lines, in the low nanomolar range. No cytotoxicity was observed by T115 against normal human skin fibroblasts cell lines, and acute toxicity studies in normal nontumor-bearing mice indicated that T115 is well-tolerated in vivo (maximum total tolerated dose, 400 mg/kg). In a mouse xenograft model using human colorectal (HT-29) and prostate (PC3) cancer cells, T115 significantly inhibited tumor growth when administered i.p. Taken together, our results suggest that T115 is a potential drug candidate for cancer chemotherapy.

Figure 1

Effect of T115 (1-methyl-5-(3-(3,4,5-trimethoxyphenyl)-4H-1,2,4-triazole-4-yl)-1Hindole; A) on tubulin binding of [³H]colchicine (B), [³H]vinblastine (C) and [³H]paclitaxel (D). Tubulin (>99% pure) was incubated with tritiated tubulin binders in the presence of either unlabeled drugs or T115 at indicated concentrations for 1 hr at 37°C. Data points represent means ± S.E. obtained from three independent experiments.

Figure 2

Effect of T115 on microtubule polymerization in vitro. Tubulin (>99% pure, 0.3mg/assay) was exposed to T115, CA-4 or colchicine at concentrations ranging from 0.1µM- 10µM (vehicle control: 0.1% DMSO). Absorbance at 340 nm was recorded at 37°C every min. for 60 min.

Figure 3

Effect of T115 on cell cycle progression. HeLa cells were treated with 0.1% DMSO (vehicle control) or T115 at indicated concentrations for 24 hr, trypsinized, fixed and stained with propidium iodide. A. Cell distribution (X-axis, arbitrary unit) versus DNA content (Y-axis, arbitrary unit). B. The percentage of cells in each mitotic phase pre- and post-treatment.

Figure 4

Effect of T115 on microtubule network organization. HeLa (A) or MCF-7 (B) cells were grown in DMEM containing either 0.1% DMSO, CA-4 or T115 at indicated concentrations for 24 hr, stained for α-tubulin using FITC-conjugated secondary antibody (green), and visualized by fluorescent microscopy with a 40X oil immersion lens. Cell nuclei were stained with DAPI (blue). Images are representative of two independent experiments.

Figure 5

In vivo activity of T115 in mice bearing HT-29 colorectal (A) or PC3 prostate (B) xenografts. NCR nu⁻/nu⁻ mice were inoculated with 1 × 10⁶ HT-29 or PC3 cells and after tumor formation treated with either T115 (60 and 90 mg/kg) or vehicle i.p. Treatments were given on days 0, 2, 4, 6 and 8 as described in Materials and Methods. The tumor volumes were recorded every other day using digital caliper. Each point represents mean tumor volume for the six animals in each group.