Bioeffects And Molecular Mechanisms Of A New Nitroxide, L-nnp In Mcf-7 And Mda-mb-231 Cells

Nitroxyl nitroxide radicals are a series of stable free radical compounds, characterized as water-soluble, membrane-permeable with low molecular weight, and mimic metal-independent superoxide dismutase. They show protection from oxidative injury and ionizing radiation by inhibiting the formation of oxygen and hydroxyl radical, and alleviating the oxidative stress of nitric oxide, nitroxyl and peroxynitrite. However, nitroxyl nitroxide radicals were found to be pro-oxidative and inhibied the growth of different tumor cell lines. Tempol (4-hydroxy-2, 2, 6, 6-tetramethylpiperidine 1-oxyl), as a typify of nitroxyl nitroxide radicals, was reported to exert a pro-oxidative effect in tumor cells by increasing the intracellular H2O2 concentration mediated by the cytochrome P450 system and was found to be significantly more effective in inhibiting the growth of neoplastic cells, especially in tumor cells with multidrug-resistant phenotype or with loss of hormone receptors.In the present research, we investigate the cytotoxicity and its mechanism of a new synthetic nitroxyl nitroxide radicals, tert-butyl-2 (4, 5-dihydrogen-4, 4,5,5-tetramethyl-3-O-1H-imidazole-3-cationic-1-oxyl-2-pyrrolidine-1-carboxyli c ester), L-NNP in MCF-7 and MDA-MB-231 cells.Firstly, we focus on the cytotoxicity of L-NNP and its candidate targets. The MTT and clone formation assay were used to investigate the cell survival. The cell death and cell circle were measured by flow cytometric assay. Janus green B staining and Hoechst 33258 staining were used to observe the cell morphologic alteration; the transmission electron microscope assay was used to observe cell ultrastructure. The LDH release and PI intake assay were used to assess the damage of cell membrane. The agarose gel electrophoresis, single cell elecrophoresis andγ-H2AX immunofluroscence were used to investigate L-NNP induced DNA double strands break. The flow cytometric assays of JC-1 and fluorescence microscope were used to measure intracellular reactive oxygen species and the mitochondrial membrane potential. The biochemical assays were used to measure intracellular lipid oxidation, anti-oxidant ability and mitochondrial respiratory chain function.L-NNP treatment resulted in a significant growth inhibition in two breast cancer cell lines, and L-NNP exerted more cytotoxicity in MDA-MB-231 cell line than MCF-7 cell line. The same concentration of L-NNP (50μg/ml ) resulted in high cell death percentage in these two breast cancer cell lines more than Docetaxel did, and which followed a concentration- and time-dependant way. L-NNP induced biphase cell cycle change, including a short, mild G1 phase blocking and, S phase blocking delayed and significantly. Morphologic research of MCF-7 and MDA-MB-231 cells treated by L-NNP for 48 h revealed typical oncotic morphologic alterations, including significant cell swelling and cell structure damage. Membrane damage, DNA double strand break, mitochondrial structure and function loss, these phenomena came out in the L-NNP treated group with a concentration-and time-dependant mode. The cell biochemisry assays showed that intracellular ATP level and SDH activity decrease, MDA production increase, and antioxidative ablity decend. Moreover, L-NNP induced reactive oxygen species (ROS) increasing significantly in a concentration- and time-dependant way in two breast cancer cell lines. It seems that L-NNP exposure could induce oxidative stress and damage in MCF-7 and MDA-MB-231 cells. Therefore, we assume that L-NNP might via a significant increase reactive oxygen species, which resulted in oxidative damage, and caused cell oncosis.In order to prove our hypothesis, the ROS scavenger, NAC was used to probe the mechanism of L-NNP originated oncosis in MCF-7 cells. Pretreatment of MCF-7 cells for 1h with NAC 10 mM prior to 6 h of L-NNP exposure, then analyze the reactive oxygen specices level with flow cytometric assay. Co-treatment of MCF-7 cells with NAC 10 mM and L-NNP (30 and 50μg/ml) for 48 h, then analysis the changes of cell survival and cell morphologic by MTT, Janus green B and Hoechst 33258 staining, respecitvely. In additional, the effect of L-NNP on Caspase-3 activity was examined. Western blot was used to detect the expression of p53, Bax, Porimin, NF-κB and Bcl-2 proteins. We found that the L-NNP induced reactive oxygen species production was inhibited significantly in MCF-7 cells by NAC pretreatment. The co-incubation of NAC and L-NNP enhanced cell survival rate and amended the changes of morphology significantly compared to L-NNP group, it suggested that ROS is very important factor when L-NNP induced oncosis in MCF-7 cells. And, we found that Caspase-3 activity, p53 and Bax protein expression were inhibited, but NF-κBp65 and Bcl-2 protien expression increased by L-NNP treatment, NAC inhibited up-regulation of NF-κBp65 and Bcl-2 protein induced by L-NNP. More interestingly, the specific inhibitor of NF-κB (Bay-11-7082) also inhibited up-regulation of NF-κBp65 and Bcl-2 protein induced by L-NNP. It suggests that intracellular reactive oxygen species is an initiator of cytotoxicity of L-NNP, then it activates Caspase-independent NF-κB/Bcl-2 pathway and results in cell oncosis in breast cancer cells.Next, in order to elucidate the molecular mechanism of L-NNP induced oncosis in cancer cells in early stage, human oligo gene chip, real-time PCR, western blot and ELISA assay were performed. Compared to the control, 26 genes were up-regulated and 74 genes were down-regulated after 30μg/ml L-NNP 6 h treatment. We confirmed that ACCN2, S100A6 and IGF-1 mRNA expression increased induced by L-NNP with RT-PCR. ACCN2, S100A6 and IGF-1 protein expression also increased from western blot results. It indicated that ACCN2, S100A6 and IGF-1 involved in early responds in the process of oncosis induced by L-NNP. We confirmed that IL-2, IL-4, IL-10 protein level increased significantly after L-NNP treatment in a concentration- and time-dependant way with ELISA assay. It suggests that L-NNP caused intracellular ROS increasing results in NF-κB/Bcl-2 pathway activation, and then leads cytokines level secretion, which involving in or regulating oncosis cascade reaction in breast cancer cells.In order to explore the anticancer effect of L-NNP in vivo, human liver cancer cell line HepG2 cell bearing nude mouse mode was used to examine the anticancer effect of L-NNP. The results exhibited that 4 mg/ml L-NNP extended life span of tumor bearing nude mouse, and 2 mg/ml L-NNP partially inhibted tumor growth compared with 2 mg/ml 5-FU.In conclusion, L-NNP displays anticancer effects in vitro and in vivo. L-NNP activates Caspase independent NF-κB/Bcl-2 pathway via ROS mediated oxidative stress reaction, and induces cell membrane, mitochondrial and DNA damage; eventually results in cell oncosis in cancer cells. It suggests that L-NNP will become a cancidate of anti-cancer drug in future.