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 vitro, coumarin and 7-hydroxycoumarin, its major biotransformation product in humans, inhibit the proliferation of several human tumor cell lines. The molecular mechanisms of these effects are unknown. To gain information about these mechanisms, we studied the effects of coumarin and 7-hydroxycoumarin in the human lung adenocarcinoma cell line A-427 on the inhibition of: (i) cell proliferation; (ii) cell cycle progression; and (iii) expression of cyclins D1, E and A. The inhibitory concentrations 50 (IC(50)) of both compounds were estimated by cytostatic assays of tetrazolium (MTT) reduction. The effects on cell cycle progression were assayed with propidium iodide and BrdU using DNA histograms and multiparametric flow cytometry. The percentages of cells expressing cyclins D1, E, and A were estimated by means of bivariate flow cytometry using propidium iodide, and FITC-conjugated monoclonal antibodies for each cyclin. The IC(50) (+/-S.E.M. n=3) of 7-hydroxycoumarin and coumarin at 72 h exposure, were 100+/-4.8 and 257+/-8.8 microg/ml, respectively. 7-Hydroxycoumarin at the concentration of 160 microg/ml (1 mM), inhibited the G(1)/S transition of the cell cycle, an action consistent with the cytostatic effect. No significant decreases of cyclins E and A were observed. In contrast, cyclin D1 significantly decreased, which appears to indicate 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 suggests a posttranscriptional effect. The effects of coumarin were not significant. Cyclin D1 is overexpressed in many types of cancer, and its inhibition has been proposed as a pharmacological and therapeutic target for novel antitumor agents. Knowledge of the decrease of cyclin D1 by 7-hydroxycoumarin may lead to its use in cancer therapy, as well as to the development of more active compounds.

Expression of urokinase-type plasminogen activator in an experimental model of hepatocarcinoma.

Hepatocellular carcinoma (HCC) is the most common primary malignant tumor of the liver. Molecular genetic analyses have clarified that accumulation of genome changes provides important steps in carcinogenesis. Urokinase type plasminogen activator (uPA) forms part of an important enzymatic system that degraded the extracellular matrix in process of invasion and metastasis. In order to study the kinetics of uPA cellular expression during this process, we used specific polyclonal antibodies against uPA in an immunohistochemistry assay in liver sections from a HCC in rats. The neoplastic transformation induced with this model was preceded by the appearance of numerous hyperplastic nodules during early stages, after time lesions progressed to well-differentiated HCC. The morphological changes of premalignant and malignant lesions were associated with a progressive increment of uPA expression, which reached its peak at 5 and 6 months after the administration of the carcinogenic drugs. Of the enzymatic markers analyzed, the gamma glutamyl transpeptidase showed correlationship with the histological findings. Our results suggest that the increase in the uPA expression should not only be considered as the hallmark of metastasis, but may also be related to early events in the neoplastic transformation and with the proliferation of vessels and biliary ducts.