Effects of iron on the cytotoxicity of nitric oxide and the associated cytolysis by natural killer cells of MCF-7 human breast cancer cells

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Natural killer (NK) cells and nitric oxide (NO) are both important components of the natural or innate immune response. Natural killer (NK) cell cytolysis is associated with synthesis of NO, and this NO can affect cellular iron metabolism. Iron has been reported to promote carcinogenesis and neoplastic cell growth. In the present study, we investigated the role of iron in NO-mediated cytotoxicity and NK cell cytolysis. MCF-7 breast cancer cells were treated with S-nitroso-N-acetyl-penicillamine (SNAP), a NO donor, or SNAP plus ferric chloride. MCF-7 cells and NK-92MI human NK cells were co-incubated for 4 hours in media in the absence or presence of ferric chloride. Cell proliferation was determined by ³[H]-thymidine incorporation. NK-92MI lysis of MCF-7 cells was assessed with a ⁵¹Cr-release assay. NO concentration was measured with the Griess Reagent System. Cell transferrin receptor, ferritin and intracellular iron were also quantatively measured. We found that SNAP significantly suppressed the growth of MCF-7 breast cancer cells in a dose-dependent manner (IC50 of 50um, 3 days of incubation). NO up-regulated the expression of the transferrin receptor and depleted intracellular iron in MCF-7 cells. The addition of ferric chloride significantly decreased the SNAP-induced cytotoxicity to the MCF-7 breast cancer cells. The ⁵¹Cr-release assay showed that NK-92MI cells lysed MCF-7 cells (10.9+4.5% and 5.0+1.8% specific lysis of MCF-7 cells at NK-92MI to MCF-7 ratios of 10:1 and 2.5:1, respectively). Exogenous 400um of FeCl₃ significantly decreased the cytotoxic activity with 7.5+4.4% (p<0.05) and 2.0+2.1% (p<0.05) specific lysis at NK-92MI to MCF-7 ratios of 10:1 and 2.5:1, respectively. Nitrite, a stable and nonvolatile breakdown products of NO, was also found in the NK-92MI culture supernatants. The concentration of nitrite was dramatically increased when the MCF-7 target cells were co-incubated with the NK-92MI cells, and the formation of NO by NK-92MI was not inhibited by the addition of iron. We conclude that one mechanism of NO-mediated cytotoxicity is by depletion of intracellular iron, and NK-92MI cytolysis is associated with the production of NO. Therefore, iron not only protects tumor cells from exogenous NO cytotoxicity, but also prevent NK cells cytolysis of tumor cells by inhibiting the cytotoxicity of the NO produced by the NK cells. This is the first time that iron has been shown to decrease NK cell cytolysis of tumor cells.