No abstract

We have previously shown that a low Magnesium (Mg)-containing diet reversibly inhibits the growth of primary tumors that develop after the injection of Lewis lung carcinoma (LLC) cells in mice. Here we investigate some of the mechanisms responsible for the Mg-dependent regulation of tumor development by studying cell cycle regulation, tumor angiogenesis, and gene expression under Mg deficiency. The inhibition of LLC tumor growth in Mg-deficient mice is due to a direct effect of low Mg on LLC cell proliferation and to an impairment of the angiogenic switch. We also observed an increase of nitric oxide synthesis and oxidative DNA damage. Complementary DNA arrays reveal that Mg deficiency modulates tumor expression of genes involved in the control of cell cycle, stress response, proteolysis, and adhesion. Our results suggest that Mg has multiple and complex roles in tumor development.

Mammary epithelial cells (HC11) chronically adapted to grow in a low-magnesium (0.05 mM vs. 0.5 mM) or in a high-magnesium (40 mM) medium were used to investigate on the mechanisms of cell magnesium transport under conditions of non-physiological magnesium availability. Magnesium influx was higher in low-magnesium cells compared to control or high-magnesium cells, whereas magnesium efflux was higher in high-magnesium cells compared to control and low-magnesium cells. Magnesium efflux was partially inhibited by imipramine, inhibitor of the Na(+)/Mg(2+) exchange. Using a monoclonal antibody detecting a approximately 70 kDa protein associated with Na(+)/Mg(2+) exchange activity, we found that the expression levels of this protein were proportional to magnesium efflux capacity, that is, high-magnesium cells > control cells > low-magnesium cells. As for magnesium influx, this was abolished by Co(III)hexaammine, inhibitor of magnesium channels. Surprisingly, we found that cells grown in low magnesium upregulated mRNA for the magnesium channel TRPM6, but not for other channels like TRPM7 or MagT1. TRPM6 mRNA was also rapidly upregulated or downregulated in HC11 cells deprived of magnesium or in low-magnesium cells re-added with magnesium, respectively. TRPM6 protein levels, as assessed by Western blot and immunofluorescence, underwent similar changes under comparable conditions. We propose that mammary epithelial cells adapt to decreased magnesium availability by upregulating magnesium influx via TRPM6, and counteract increased magnesium availability by increasing magnesium efflux primarily via Na(+)/Mg(2+) exchange. These results show, for the first time, that TRPM6 contributes to regulating magnesium influx in mammary epithelial cells, similar to what is known for intestine and kidney.

Low Mg availability reversibly inhibited the growth of mammary epithelial HC11 cells by increasing the number of cells in the G0/G1 phase of the cell cycle. Because low Mg has been reported to promote oxidative reactions, we considered that low Mg-dependent growth arrest was mediated by oxidative stress. Surprisingly, both dichlorofluorescein-detectable reactive oxygen species and hydrogen peroxide-induced oxidative DNA damage were found to be lower in cells cultured in low Mg than in cells grown under control or high-Mg conditions. Gene expression profiling of low- and high-Mg cells showed the modulation of several genes, some regulating cell proliferation. In addition, low Mg cells also displayed overexpression of glutathione S-transferase (GST), leading to increased enzymatic activity. Of note, GST has been shown to modulate cell growth; therefore, we suggest that in low-Mg cells, GST upregulation might have a dual role in protecting against oxidative stress and in modulating cell proliferation.