In vitro effects of fenretinide on cell-matrix interactions.

BACKGROUND: Understanding the molecular basis of the metastatic spread of cancer and the underlying mechanisms is crucial for the development and appropriate clinical use of novel therapeutic agents directed at prevention of metastasis. Retinoids have been reported to inhibit cell proliferation, modulate cell differentiation, enhance apoptosis and to prevent the conversion of in situ cancer to locally invasive malignancy by suppressing the invasive process as well as by inhibiting angiogenesis. Fenretinide (4-HPR), a synthetic derivative of retinoic acid, is less toxic than natural retinoids and is active in the prevention and treatment of a variety of tumours in animal models. Its efficacy in cancer chemoprevention and therapy has been investigated in clinical trials. MATERIALS AND METHODS: In order to evaluate the effects of 4-HPR on the late stages of tumour progression, chemically transformed BALB/c 3T3 cells, showing a fully malignant phenotype, were exposed to 4-HPR (0.25-10 microM; 72 hours pre-treatment) and then analysed for in vitro invasive ability. The possible mechanisms of action responsible for the anti-invasive activity of 4-HPR were investigated, analysing cellular adhesion, motility, and proteolytic capability. RESULTS: Data showed that 4-HPR significantly inhibited the invasive phenotype of chemically transformed cells; the reduction in Matrigel invasion was dose-dependent and seemed not to be related to cytotoxic effects or reduction in cell proliferation rates induced by 4-HPR assayed doses. The 4-HPR-induced decrease in chemotactic motility of transformed cells correlated well with the invasion inhibition. 4-HPR, at active concentrations, differently affected cell adhesion to the extracellular matrix, depending on the coating substrate used (laminin, collagen IV, fibronectin and vitronectin). 4-HPR treatment significantly enhanced cell adhesion to laminin, while reducing cell-vitronectin attachment. It did not modify the attachment of the cells to fibronectin and collagen IV. Zymographic analysis failed to demonstrate 4-HPR involvement in the modulation of the activity and expression of gelatine degrading enzymes. CONCLUSION: These data suggest that 4-HPR inhibits tumour cell invasion through a basement-like matrix, by suppressing chemotactic motility and by altering cell-matrix interactions.

Effects of the protease inhibitor antipain on cell malignant transformation.

BACKGROUND: Several natural products have been found to exhibit a chemopreventive activity both in in vivo and in vitro experimental systems. Among them, protease inhibitors seem to play a key role in the regulation of growth and phenotypic expression of transformed cells as well as in the regulation of the late events of carcinogenesis. We evaluated the effect of antipain (AP), a natural protease inhibitor, on chemically induced BALB/c 3T3 cell transformation, on invasion and chemotactic motility of transformed cells and on their gelatinase expression. METHODS: BALB/c 3T3 cells were plated and exposed to 2.5 micrograms/ml 3-MCA or 50 micrograms/ml, 1,2-DBE. The effect of a non-cytotoxic dosage of AP (10 microM) was studied by: a) pretreating cells with AP for 48 hours before the carcinogen exposure; b) adding AP simultaneously to the carcinogen treatment; c) chronic addition of AP at each medium change throughout the experimental duration. The effectiveness of the treatment was analysed as the ability to reduce or inhibit the occurrence of transformed foci. Modulation of the invasive phenotype by anti-transforming dosages of AP was evaluated by in vitro Matrigel invasion assay. Gelatin zymography was performed in order to assess AP regulation of proteolytic enzymes, such as metalloproteases, involved in invasion and metastasis. RESULTS: AP treatment can reduce the transformation rate both in 3-MCA- and 1,2-DBE-initiated cells. Its effectiveness depends on the administration schedule, and chronic addition seems to be the most effective treatment. The concentration of AP, which is effective in the antitransformation assay, is not able to significantly affect the migration and invasion of chemically transformed cells or their gelatinase activity. CONCLUSIONS: AP can suppress chemically induced BALB/c 3T3 cell transformation through mechanisms which do not involve modulation of the invasive phenotype.