Anticancer activity of a novel small molecule tubulin inhibitor STK899704

Abstract

We have identified the small molecule STK899704 as a structurally novel tubulin inhibitor. STK899704 suppressed the proliferation of cancer cell lines from various origins with IC50 values ranging from 0.2 to 1.0 μM. STK899704 prevented the polymerization of purified tubulin in vitro and also depolymerized microtubule in cultured cells leading to mitotic arrest, associated with increased Cdc25C phosphorylation and the accumulation of both cyclin B1 and polo-like kinase 1 (Plk1), and apoptosis. Unlike many anticancer drugs such as Taxol and doxorubicin, STK899704 effectively displayed antiproliferative activity against multidrug-resistant cancer cell lines. The proposed binding mode of STK899704 is at the interface between αβ-tubulin heterodimer overlapping with the colchicine-binding site. Our in vivo carcinogenesis model further showed that STK 899704 is potent in both the prevention and regression of tumors, remarkably reducing the number and volume of skin tumor by STK899704 treatment. Moreover, it was significant to note that the efficacy of STK899704 was surprisingly comparable to 5-fluorouracil, a widely used anticancer therapeutic. Thus, our results demonstrate the potential of STK899704 to be developed as an anticancer chemotherapeutic and an alternative candidate for existing therapies.

Fig 1. STK899704 suppressed the growth of a variety of human cancer cell lines.

(A) Chemical structure of STK899704. (B) Antiproliferative effect of STK899704 on HeLa cells. Cells were seeded at 2 x 10³ cells in 96-well plate and treated with various concentrations of STK899704 for 4 days. Cell growth was determined by MTT assay. (C) Inhibitory effects of STK899704 on the growth of various cancer cell lines. Data were fitted with dose-response curve by using Graphpad Prism software.

Fig 2. Antimitotic effect of STK899704.

(A) Flow cytometric analysis for cell cycle distribution. HeLa cells were treated with the indicated concentrations of STK899704 for 24 h. Treated cells were then stained with propidium iodide (PI) and processed for cell cycle analysis. Data are representative of three independent experiments. (B) Mitotic index. HeLa cells were treated with the indicated concentrations of STK899704 for 17 h. Cells were then stained with Hoechst 33342 and mitotic cells were counted. At least 100 cells were counted from the different regions. (C) Cell cycle related protein expression. HeLa cells were treated with DMSO control, 200 ng/ml nocodazole (Noc), or indicated concentrations of STK899704 for 17 h. Treated cells were lysed and subjected to immunoblot analysis with antibodies against cyclinB1, Plk1, Cdc25C, histone H3, and phospho-histone H3 (S10). β-actin was used as a loading control. (D) Reversible effect of STK899704. HeLa cells were treated with nocodazole (200 ng/ml) or STK899704 (1 or 5 μM) for 17 h. Cells were washed twice and released into fresh DMEM without nocodazole or STK899704. Cells were then stained with PI at the indicated time and processed for cell cycle analysis. Each Bar indicates mean ± SD from three independent experiments.

Fig 3. STK899704 inhibited tubulin polymerization and mitotic spindle organization.

(A) Tubulin polymerization assay. The effect of STK899704 (5 μM) on polymerization of purified tubulin in vitro was examined in a GTP-containing buffer. DMSO was used as a negative control. Tubulin-targeting agents Taxol (5 μM) and vinblastine (5 μM) were also used as controls for tubulin-stabilizing and tubulin-destabilizing agents, respectively. Assembly of tubulin into microtubules was determined by the degree of turbidity at 340 nm. (B) Immunofluorescence staining of microtubules in HeLa cells. Cells were treated with DMSO, Taxol (100 nM), nocodazole (200 ng/ml), or indicated concentrations of STK899704 for 17 h. Cells were then fixed and stained with Alexa Fluor 488-conjugated anti-tubulin antibody and Hoechst 33342 to visualize α-tubulin and DNA, respectively. Scale bar, 10 μm. (C) Fraction of mitotic cells. At least 100 cells from (B) were counted from the different regions. Percentages of normal metaphase, misaligned, multipolar, and tubulin aggregate phenotypes were shown. (D) Proposed binding model of STK899704 on tubulin. The αβ-tubulin heterodimer from PDB entry 1SA0 is shown as ribbon (gray, α-tubulin; cyan, β-tubulin). STK899704 is presented in red stick while colchicine is shown in green. (E) Effect of STK899704 on tubulin binding. Tubulin binding was tested with a SPA-based competition assay. Error bars represent mean ± SDs from three independent experiments.

Fig 4. STK899704 triggered programmed cell death.

(A) Effect of STK899704 on DNA fragmentation. HeLa cells were treated with 200 ng/ml nocodazole (Noc), 100 nM colchicine (Col), or indicated concentrations of STK899704. Treated cells were then stained with PI and processed for cell cycle analysis at 24 and 48 h. (B) Antagonistic effect of Z-VAD-FMK on STK899704-induced cell death. HeLa cells were treated with DMSO or STK899704 (STK, 1 or 5 μM) in the presence or absence of Z-VAD-FMK (50 μM). Cells were then stained with PI and processed for cell cycle analysis at 24 and 48 h. (C) Effect of STK899704 on the levels of activated caspases. HeLa cells were treated as in (A) and then subjected to immunoblot analysis with antibodies against caspase-3, caspase-7, caspase-8, caspase-9, and PARP. GAPDH was used as a loading control. Each bar indicates mean ± SD of subG₁ population from three independent experiments.

Fig 5. The antitumor activity of STK899704 in vivo carcinogenesis model.

After tumor initiation by DMBA treatment, mice were further subjected to TPA challenge for tumor promotion twice a week. Either STK899704 or 5-fluorouracil (5-FU) was applied topically 30 minutes after TPA treatment for 15 weeks. (A) The tumor development and growth were assessed during STK899704 or 5-FU treatment and compared to TPA-treated control group at 15th week. Average number (B) and volume (C) of skin tumors per group were weekly recorded and calculated during treatment. (D) Average weight of tumors was measured in each group of mice at the end of the experiment. Error bars show mean ± SDs for each group of mice.