RESUMO

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.

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The tumour necrosis factor alpha (TNF-alpha) is produced by mononuclear phagocytes as a defence mechanism against malignant cells. However, these cells can evade destruction by TNF-alpha. The present study evaluates in three lung cancer cell lines (small cell carcinoma NCI-H69, adenocarcinoma A-427, squamous carcinoma SK-MES-1) and one erythroleukaemia (K-562) cell line the following evasion mechanisms: (1) inhibition of TNF-alpha production, in indirect and direct co-cultures with monocytes; (2) the expression of type I and type II receptors for TNF-alpha (TNFRI and TNFRII) by tumour cell lines, using indirect immunofluorescence and flow cytometry; (3) the sensitivity of tumour cell lines to the toxic action of recombinant human TNF-alpha (rhTNF-alpha). With the exception of cell line NCI-H69, the other tumour cell lines liberated soluble factors that inhibited TNF-alpha production in monocytes. This effect occurred even after membrane contact with the A-427 and SK-MES-1 cell lines. Erythroleukaemia K-562 cells expressed both types of receptors for TNF-alpha, whereas the NCI-H69 cells expressed only TNFRI, and the A-427 and SK-MES-1 cells expressed no receptors. Lines NCI-H69, A-427 and K-562 were insensitive to the cytotoxic action of rhTNF-alpha. In conclusion, different lung cancer cell lines may evade destruction by TNF-alpha by various mechanisms that range from blocking TNF-alpha production by monocytes to blocking the cytotoxic action of this molecule. For selecting the most effective immunotherapy, knowledge of the evasion mechanisms would be useful.

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Integrins are receptors that mediate cell adhesion and the formation of signaling complex. Changes in the expression of integrins are required during the following steps in the generation of metastases: a) angiogenesis; b) detachment from the primary tumor; c) tumor cell-platelet interaction; d) adhesion to vascular endothelium and e) proliferation. There is a correlation between invasive capability and changes in the expression of some proteins that are clustered in focal adhesion sites, as FAK, CD82, CD9 or CD63. Both, integrin blocking (using antibodies or RGD containing peptides), as well as induced changes in the expression of integrin-associated molecules, are able to inhibit formation of metastases. Discovery and characterization of molecules that regulate the adhesive capability of tumor cells, will lead to development of antimetastasic therapies. In the search of tumor dissemination inhibitors, integrins and some integrin-associated molecules are important pharmacological targets.

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Recientemente se han demostrado correlaciones significativas entre la expresión de algunas moléculas de adhesión y la capacidad de células de producir metástasis. Por ejemplo, se ha observado una correlación entre la expresión de la integrina a6/ß1 en células cancerosas pulmonares y la producción de metástasis. También se ha observado correlación entre la expresión de algunas moléculas de adhesión en células de melanoma maligno y su capacidad de producir metástasis pulmonares. En este trabajo estudiamos la acción in vitro de la cumarina en el melanoma murino B16-F10, productor de metástasis pulmonares, sobre la expresión de dos moléculas de adhesión, ICAM-1 y LFA-1. No se observó disminución en la expresión de la molécula de adhesión LFA-1, y la expresión de ICAM-1 disminuyó de manera uniforme con todas las concentraciones de cumarina estudiadas. Estos resultados no explican los efectos antimetastásicos producidos por la cumarina en modelos animales de metástasis pulmonares experimentales y espontáneas, ni los efectos antimetastásicos en humanos. Es necesario, por tanto, estudiar el efecto de la cumarina en la expresión de otras integrinas. Este tipo de estudios permite el desarrollo de nuevas estrategias en la búsqueda de mejores agentes antineoplásicos que disminuyan en mayor grado el número y tamaño de metástasis, y retarden importantemente su producción; contribuye, adenomás, al conocimiento de la fisiopatogenia de estos tumores malignos

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