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

Abstract

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 (IC 50 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 IC 50 value of 75 nmol/L-1.57 μmol/L but with low cytotocitity against WI-38 human lung fibroblasts (IC 50 value of 12.128 μmol/L). The compound (0.04-10 μmol/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 μmol/L) induced the depolymerization of purified porcine tubulin in vitro. Finally, the compound (0.04-2.5 μmol/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.

Physalin B not only inhibits the ubiquitin-proteasome pathway but also induces incomplete autophagic response in human colon cancer cells in vitro

“…The inhibitory effect of physalin B on the growth of HCT116 cells was evaluated using the MTT viability assay as described previously [23] . Briefly, the cells were seeded onto plastic 96-well cell culture plates and cultured at 37 °C.…”

Establishing a High-content Analysis Method for Tubulin Polymerization to Evaluate Both the Stabilizing and Destabilizing Activities of Compounds

“…In fluorescent microscopic studies, either the α- or β-tubulin can be labeled directly with a tubulin antibody-conjugated fluorescent probe, or indirectly via a secondary antibody. Such studies, while straightforward, are limited in throughput, and they lack adequate methods for quantification (e.g., [ 8 , 9 ]). Quantitative cell-based assays for assessing microtubule activities of compounds are often achieved by measuring the indirect effects on cell cycle which result from a disruption of microtubule networks.…”

Enhanced uptake, high selective and microtubule disrupting activity of carbohydrate fused pyrano-pyranones derived from natural coumarins attributes to its anti-malarial potential

“…Coumarins and their synthetic derivatives have shown to be effective antimicrobial and anticancer agents [2, 3]. Furthermore, different derivatives of coumarin have been shown to inhibit proliferation via targeting microtubule dynamics [4, 5]. Tubulin is identified as one of the potential targets of coumarin derivatives.…”