The organic and inorganic components of the supplement are extracted from the marine red algae Lithothamnion calcareum, whose

mineral extract has shown growth-inhibitory effects on human colon carcinoma cells [19] as well as inhibition of liver tumor formation in C57BL6 mice [20]. Referring to CF formulation, previous studies have demonstrated its ability to furnish effective in vitro antioxidant protection [21]. At the same time, the capability of CF to modulate O2 availability and buy Afatinib mitochondrial respiratory metabolism has been evidenced in endothelial cells [22]. All these observations led us to investigate see more the potential role of CF as hypoproliferative agent in vitro. For this purpose, we analyzed the effect of CF on cell growth, viability, glycolytic profile, and apoptosis on three human leukemia cell lines, Jurkat, U937, and K562. Eighteen percent of malignancies are of hematological Niraparib origin [17]; moreover, leukemic cells are highly glycolytic [23], though these cells reside within the bloodstream at higher oxygen tensions than cells in most normal tissue. In the present study we reported evidence that CF showed antiproliferative effect on the above mentioned leukemia cell lines due to apoptosis induction and tumor metabolism modifications. Methods Cellfood™ The supplement (liquid) was kindly provided by Eurodream srl (La Spezia, Italy) and stored at room temperature. CF was diluted in phosphate buffered

saline (PBS) and sterilized using a 0.45 μm syringe-filter before use. Cell culture Three human leukemia cell lines were used in this study, Jurkat (acute lymphoblastic leukemia), U937 (acute myeloid leukemia), and K562 (chronic myeloid leukemia in blast crisis). Cells were grown in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum, 1% L-glutamine and 1% penicillin/streptomycin 100 U/ml, and incubated in a CO2 incubator (37°C, 5% CO2 and humidified atmosphere). Cell culture reagents were Low-density-lipoprotein receptor kinase from VWR International (Milan, Italy). Lymphocytes were isolated from blood samples

provided by healthy volunteers by centrifugation in the presence of Lymphoprep™ (Axis-Shield PoC AS, Oslo, Norway), and were cultured as described above with the addition of 10 μg/ml of phytohemagglutinin (Sigma-Aldrich, Milan, Italy). A single dose of CF (final concentration 5 μl/ml) was administered to leukemia cells or lymphocytes; cells were collected after 24, 48, and 72 h of CF administration. Untreated cells served as controls. Trypan blue cell counting was performed at each experimental time point to evaluate the viable cell number. Cell viability assay Cell proliferation and viability were analyzed at 450 nm by the WST-1 reagent (4-[3-(4-lodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate) (Roche Diagnostics GmbH, Mannheim, Germany). The assay was based on the cleavage of the tetrazolium salt WST-1 by mitochondrial dehydrogenases in viable cells.