Antimetastatic, antineoplastic, and toxic effects of 4-hydroxycoumarin in a preclinical mouse melanoma model.

PURPOSE: We have previously reported that in vitro treatment of B16-F10 melanoma cells with 4-hydroxycoumarin (4-HC) decreases their metastatic potential. However, the antimetastatic efficacy of 4-HC in vivo is unknown; therefore, we investigated the antimetastatic and antineoplastic effects of 4-HC in a mouse melanoma model. Based on the findings, the immunomodulatory and toxic effects of 4-HC were also studied. METHODS: Experimental metastasis assay was performed in C57BL/6 mice that received 4-HC before intravenous injection of B16-F10 cells. Antitumor and antimetastatic efficacy of 4-HC was assessed in mice implanted subcutaneously with melanoma cells. Possible immunostimulant and toxic effects of 4-HC were studied in healthy mice. RESULTS: 4-HC reduced the number of experimental lung metastases. Moreover, 4-HC diminished primary tumor growth and increased survival time in mice bearing melanoma tumors. Treatments also decrease spontaneous lung metastases in the same animals. Different to other coumarins, the antitumor effect of 4-HC seems to be unrelated to immunostimulation, since plasma concentrations of cytokines remained unchanged. In contrast, toxic histological changes in nephrons and bronchiolar epithelium and a pronounced anticoagulant effect were found in 4-HC treated animals. CONCLUSIONS: These results show that 4-HC not only exhibit antimetastatic effect in vivo, but also effectively reduces tumor growth and improves survival, even when it produce toxic effects. Although the molecular mechanism of 4-HC actions needs to be further defined, our data suggest that 4-HC may lead to the development of agents that could be used as adjuvants in the therapy of melanoma.

Aged garlic extract induces proliferation and ameliorates gentamicin-induced toxicity in LLC-PK1 cells.

Gentamicin (GM)-induced nephrotoxicity limits the use of this antibiotic. It has been shown that aged garlic extract (AGE) and S-allylcysteine (SAC), the most abundant organosulfur compound in AGE, ameliorate GM-induced nephrotoxicity in rats. The present communication evaluated the effect of AGE and SAC on proliferation and on GM-induced toxicity and genotoxicity of porcine kidney epithelial cell line (LLC-PK1 cells). The cells were preincubated with different concentrations of AGE or SAC for 12 h before incubation with 8 mm GM for an additional 72 h. At the end of this time, cell viability, genotoxicity and proliferation were evaluated. AGE stimulated cell proliferation and protected LLC-PK1 cells from GM-mediated toxicity and genotoxicity. SAC partially prevented only GM-induced genotoxicity. These results suggest that the stimulation of cell proliferation could possibly be one of the mechanisms involved in the in vitro protective effect of AGE in GM-induced toxicity of LLC-PK1 cells.

Influence of serum-free medium on the expression of glutamate transporters and the susceptibility to glutamate toxicity in cultured cortical neurons.

The presence of glia and glial glutamate transporters seems to modify glutamate-mediated toxicity in neuronal cultures. In this work we cultured cortical cells in serum-containing medium and in a serum-free medium (Neurobasal medium + B27 supplement) and studied the expression of the glutamate transporters GLAST, GLT, and EAAC by immunocytochemistry and RT-PCR. The proportion of glial cells was below 10% in the Neurobasal medium and 46% in the serum-containing medium. Semiquantitative evaluation of the mRNA for the glutamate transporters showed similar amounts in cells grown in serum-free and serum-containing media. We detected immunoreactivity for the three transporters in both media, but EAAC was coexpressed with the neuronal marker MAP2, whereas GLAST and GLT predominated in nonneuronal cells. When the cultures were treated with glutamate for 15 min, the cultures in serum-containing medium showed a clear concentration-dependent neuronal death, whereas cells primed in this medium and switched to Neurobasal medium, as well as cells grown only in the latter, were less sensitive to glutamate concentrations up to 1 mM. A similar difference in the sensitivity to excitotoxicity was observed when the glutamate uptake inhibitor L-trans-2,4-pyrrolidine-dicarboxylate was applied during 6 hr, although the accumulation of extracellular glutamate was similar in the two media. We conclude that glutamate transporters with the culture conditions studied are sensitive to glutamate uptake inhibition and that Neurobasal/B27 medium protects cells against excitotoxicity.