Novel microtubule-targeted agent 6-chloro-4-(methoxyphenyl) coumarin induces G2-M arrest and apoptosis in HeLa cells

Aim: To identify a novel coumarin analogue with the highest anticancer activity and to further investigate its anticancer mechanisms. Methods: The viability of cancer cells was investigated using the MTT assay. The cell cycle progression was evaluated using both flow cytometric and Western blotting analysis. Microtubule depolymerization was observed with immunocytochemistry in vivo and a tubulin depolymerization assay in vitro. Apoptosis was demonstrated using Annexin V/Propidium Iodide (PI) double-staining and sub-G(1) analysis. Results: Among 36 analogues of coumarin, 6-chloro-4-(methoxyphenyl) coumarin showed the best anticancer activity (IC50 value about 200 nmol/L) in HCT116 cells. The compound had a broad spectrum of anticancer activity against 9 cancer cell lines derived from colon cancer, breast cancer, liver cancer, cervical cancer, leukemia, epidermoid cancer with IC50 value of 75 nmol/L-1.57 mu mol/L but with low cytotocitity against WI-38 human lung fibroblasts (IC50 value of 12.128 mu mol/L). The compound (0.04-10 mu mol/L) induced G(2)-M phase arrest in HeLa cells in a dose-dependent manner, which was reversible after the compound was removed. The compound (10-300 mu mol/L) induced the depolymerization of purified porcine tubulin in vitro. Finally, the compound (0.04-2.5 mu mol/L) induced apoptosis of HeLa cells in dose-and time-dependent manners. Conclusion: 6-Chloro-4-(methoxyphenyl) coumarin is a novel microtubule-targeting agent that induces G(2)-M arrest and apoptosis in HeLa cells.