WTC-01, a novel synthetic oxime-flavone compound, destabilizes microtubules in human nasopharyngeal carcinoma cells in vitro and in vivo

Abstract

Background and purpose: Dynamic polymerization of microtubules is essential for cancer cell growth and metastasis, and microtubule-disrupting agents have become the most successful anti-cancer agents in clinical use. Besides their antioxidant properties, flavonoids also exhibit strong microtubule-disrupting activity and inhibit tumour growth. We have designed, synthesized and tested a series of oxime/amide-containing flavone derivatives. Here we report the evaluation of one compound, WTC-01 for its anti-proliferative effects in human cancer cells.

Experimental approach: We used a range of cancer cell lines including two human nasopharyngeal carcinoma (NPC) cell lines, measuring proliferation, cell cycle and apoptosis, along with caspase levels and mitochondrial membrane potentials. Assays of tubulin polymerisation in vitro and computer modelling of the colchicine binding site in tubulin were also used. In mice, pharmacokinetics and growth of NPC-derived tumours were studied.

Key results: WTC-01 was most potent against proliferation of NPC cells (IC50 = 0.45 μM), inducing accumulation of cells in G2 /M and increasing apoptosis, time- and concentration-dependently. The colchicine competition-binding experiments and computer modelling results suggested that WTC-01 causes microtubule disruption via binding to the colchicine-binding site of tubulin resulting in mitochondrial membrane damage and cell apoptosis via activation of caspase-9/-3 without noticeable activation of the caspase-8. Notably, our in vivo studies demonstrated that at doses of 25 and 50 mg·kg(-1) , WTC-01 exhibited good pharmacokinetic properties and completely inhibited the growth of NPC-TW01 cells in a xenograft nude mouse model.

Conclusions and implications: WTC-01, a new synthetic oxime-containing flavone, exhibited potent anti-tumour activity against NPC cells and merits further investigation.

Figure 1

The chemical structure of WTC-01 (Z)-6-[2-hydroxyimino-2-(4-methoxyphenyl)ethoxy]-2-phenyl-4H-1-benzopyran-4-one (WTC-01).

Figure 2

WTC-01 induced a concentration- and time-dependent G2/M cell arrest in human NPC lines.NPC-TW01 (A, C) and HONE-1 (B, D) cells were treated with IC₅₀ (0.45 μM) of WTC-01 (A, B) for the indicated times or different concentrations (0.225, 0.45 and 0.9 μM) of WTC-01(C, D) for 24 h. The cell cycle population ratios were determined by flow cytometry.

Figure 3

WTC-01 triggers caspase-9-dependent apoptosis in human NPC cells.(A) Time-dependent activation of pro- and anti-apoptotic proteins following WTC-01 treatment. NPC-TW01 and HONE-1 cells were treated with 0.45 μM of WTC-01 for the indicated times. The protein levels of apoptosis-related proteins were evaluated using Western blot analysis with β-actin as an internal control. (B) Kinetics of caspase-8 and -9 activation in NPC-TW01 and HONE-1 cells. Cells were treated with 0.45 μM of WTC-01 for different periods and caspase activity was analysed. Data represent means ± SD for three determinations. (C) Caspase inhibition affects the cytotoxicity of WTC-01 in NPC-TW01 cells. NPC-TW01 cells were pretreated with 100 μM of caspase-8 inhibitor (Z-IEHD-FMK-TFA) or caspase-9 inhibitor (Z-IETD-FMK), respectively, for 1 h followed by WTC-01(4.5 μM, at 10-fold the IC₅₀) for another 72 h. Cell viability was determined by MTT assay.

Figure 4

WTC-01 treatment disrupts the mitochondrial membrane potential in human NPC cells.NPC-TW01 (A) and HONE-1 (B) cells were treated with 0.45 μM of WTC-01 for the indicated times and mitochondrial membrane potential was measured with the dye, DiOC₆. Data are presented as log fluorescence intensity. Inset table is the geometric mean value.

Figure 5

WTC-01 treatment interferes with microtubule assembly in NPC cells.In vitro (A) and ex vivo (B) analysis of WTC-01 inhibition of tubulin polymerization. (A) In vitro tubulin polymerization was performed in the absence or present of 20 nM colchicine or WTC-01 and level of polymerized tubulin was measured. (B) Cancer cells were treated either with paclitaxel (40 nM), colchicine (800 nM) or various concentrations of WTC-01 for 5.5 h and the amount of polymerized tubulin was measured by Western blots. (C) The colchicine competition-binding SPAs (upper panel) were conducted and the inhibition constants (K_i; lower panel) were calculated using the Cheng-Prusoff equation. WTC-01 strongly competes with [³H] colchicine for binding to the same pocket in tubulin (D). Overlay of the docking pose of WTC-01 (yellow) with the bound orientation of colchicine (blue) in the crystal structure of tubulin. Computer simulation results suggest WTC-01 is able to dock into the colchicine-binding site of tubulin. Some tubulin amino acid residues interacting with WTC-01 are shown as stick structures. The dashed lines indicate hydrogen-bonding interactions.

Figure 6

WTC-01 treatment reduces the migratory ability of human NPC cells. Confluent cell cultures of NPC-TW01 (A) and HONE-1 (B) were wounded by scratching with a plastic micropipette tip, suspended cells were then removed by PBS washes and then treated with different concentrations (0.25,0.5 and 1 x IC₅₀) of WTC-01. Cells were photographed at the indicated times after wounding by phase contrast microscopy (×100). The percentage of area wound healing of cells was quantified, and values shown are means ± SD from triplicate experiments. *P < 0.05, **P < 0.01, significantly different as indicated; one-way ANOVA.

Figure 7

In vivo anti-tumour activity of WTC-01 in a human NPC-TW01 xenograft model. (A) The pharmacokinetics of WTC-01, after i.p. injection. Mice (n = 4) were injected with 25 or 50 mg·kg⁻¹ of WTC-01 and blood samples were drawn at the indicated times. Serum was isolated and the amount of WTC-01 in the serum sample was measured. (B) Nude mice (n = 5) were subcutaneously injected with NPC-TW01 cells. After 7 days, the mice received i.p. injection of 25 or 50 mg·kg⁻¹ WTC-01 once every 3 days. Vincristine was used as a positive control at a dose of 10 mg·kg⁻¹ once a week. Tumour size was measured every three day Data shown are means ± SD. *P < 0.05 compared with control group; Student's t-test.