Decrease of cyclin D1 in the human lung adenocarcinoma cell line A-427 by 7-hydroxycoumarin

Coumarin in vivo has antitumor activity in various types of cancer. In vitr o, coumarin and 7-hydroxycoumarin, its major biotransformation product in h umans, inhibit the proliferation of several human tumor cell lines. The mol ecular mechanisms of these effects are unknown. To gain information about t hese mechanisms, we studied the effects of coumarin and 7-hydroxycoumarin i n the human lung adenocarcinoma cell line A-427 on the inhibition of: (i) c ell proliferation; (ii) cell cycle progression; and (iii) expression of cyc lins DI, E and A. The inhibitory concentrations 50 (IC50) of both compounds were estimated by cytostatic assays of tetrazolium MTT) reduction. The eff ects on cell cycle progression were assayed with propidium iodide and BrdU using DNA histograms and multiparametric flow cytometry. The percentages of cells expressing cyclins DI, E, and A were estimated by means of bivariate flow cytometry using propidium iodide, and FITC-conjugated monoclonal anti bodies for each cyclin. The IC50 (+/- S.E.M. n = 3) of 7-hydroxycoumarin an d coumarin at 72 It exposure, were 100 +/- 4.8 and 257 +/- 8.8 mug/ml, resp ectively. 7-hydroxycoumarin at the concentration of 160 mug/ml (I mM), inhi bited the G(1)/S transition of the cell cycle, an action consistent with th e cytostatic effect. No significant decreases of cyclins E and A were obser ved. in contrast, cyclin D1 significantly decreased, which appears to indic ate an action of 7-hydroxycoumarin in early events of phase G(1). However, messenger RNA of cyclin D1, assayed by RT-PCR, did not change. This suggest s a posttranscriptional effect. The effects of coumarin were not significan t. Cyclin DI is overexpressed in many types of cancer, and its inhibition h as been proposed as a pharmacological and therapeutic target for novel anti tumor agents. Knowledge of the decrease of cyclin DI by 7-hydroxycoumarin m ay lead to its use in cancer therapy, as well as to the development of more active compounds. (C) 2001 Elsevier Science Ireland Ltd. All rights reserv ed.