2-phenyl-4-4-5-5-tetramethylimidazoline-1-oxyl-3-oxide and pimagedine

Anoikis, a detachment-induced apoptosis, is a principal mechanism of inhibition of tumor cell metastasis. Tumor cells can acquire anoikis resistance which is frequently observed in metastatic lung cancer. This phenomenon becomes an important obstacle of efficient cancer therapy. Recently, signaling mediators such as caveolin-1 (Cav-1) and nitric oxide (NO) have garnered attention in metastasis research; however, their role and the underlying mechanisms of metastasis regulation are largely unknown. Using human lung carcinoma H460 cells, we show that NO impairs the apoptotic function of the cells after detachment. The NO donors sodium nitroprusside and diethylenetriamine NONOate inhibit detachment-induced apoptosis, whereas the NO inhibitors aminoguanidine and 2-(4-carboxyphenyl) tetramethylimidazoline-1-oxyl-3-oxide promote this effect. Resistance to anoikis in H460 cells is mediated by Cav-1, which is significantly down-regulated after cell detachment through a non-transcriptional mechanism involving ubiquitin-proteasomal degradation. NO inhibits this down-regulation by interfering with Cav-1 ubiquitination through a process that involves protein S-nitrosylation, which prevents its proteasomal degradation and induction of anoikis by cell detachment. These findings indicate a novel pathway for NO regulation of Cav-1, which could be a key mechanism of anoikis resistance in tumor cells.

Topics: Animals; Anoikis; Caveolin 1; Cell Line, Tumor; Cyclic N-Oxides; Free Radical Scavengers; Guanidines; Humans; Imidazoles; Lung Neoplasms; Neoplasm Metastasis; Nitric Oxide; Nitric Oxide Donors; Nitric Oxide Synthase; Nitroprusside; Nitroso Compounds; Proteasome Endopeptidase Complex; Ubiquitin

The aim of this work was to characterize the radioadaptive response at the molecular level.. We used wild-type (wt) p53 and mutated (m) p53-containing cells derived from the human lung cancer H1299 cell line, which is p53-null. Cellular radiation sensitivities were determined with a colony-forming assay. The accumulations of p53, the human homolog of endogenous murine double minute 2 (Hdm2), and inducible nitric oxide synthase were analyzed with Western blotting. Quantification of chromosomal aberrations was estimated by scoring dicentrics per cell.. In wtp53 cells, it was demonstrated that the lack of p53 accumulation was coupled with the activation of Hdm2 after low-dose irradiation (0.02 Gy). Although NO radicals were only minimally induced in wtp53 cells irradiated with a challenging irradiation (6 Gy) alone, NO radicals were seen to increase about two- to fourfold after challenging irradiation subsequent to a priming irradiation (0.02 Gy). Under similar irradiation conditions with a priming and challenging irradiation in wtp53 cells, induction of radioresistance and a depression of chromosomal aberrations were observed only in the absence of 5, 5'-(2, 5-Furanidiyl)bis-2-thiophenemethanol (RITA) or Nutlin-3 (p53-Hdm2 interaction inhibitors), aminoguanidine (an inducible nitric oxide synthase inhibitor), and c-PTIO (an NO radical scavenger). On the other hand, in p53 dysfunctional cells, a radioadaptive response was not observed in the presence or absence of those inhibitors. Moreover radioresistance developed when wtp53 cells were treated with isosorbide dinitrate (an NO-generating agent) alone.. These findings suggest that NO radicals are initiators of the radioadaptive response, acting through the activation of Hdm2 and the depression of p53 accumulations.

Topics: Cell Line, Tumor; Cell Survival; Chromosome Aberrations; Cyclic N-Oxides; Free Radical Scavengers; Genes, p53; Guanidines; Humans; Imidazoles; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Piperazines; Proto-Oncogene Proteins c-mdm2; Radiation Tolerance; Time Factors; Tumor Suppressor Protein p53

That irradiated cells affect their unirradiated 'bystander' neighbors is evidenced by reports of increased clonogenic mortality, genomic instability, and expression of DNA-repair genes in the bystander cell populations. The mechanisms underlying the bystander effect are obscure, but genomic instability suggests DNA double-strand breaks (DSBs) may be involved. Formation of DSBs induces the phosphorylation of the tumor suppressor protein, histone H2AX and this phosphorylated form, named gamma-H2AX, forms foci at DSB sites. Here we report that irradiation of target cells induces gamma-H2AX focus formation in bystander cell populations. The effect is manifested by increases in the fraction of cells in a population that contains multiple gamma-H2AX foci. After 18 h coculture with cells irradiated with 20 alpha-particles, the fraction of bystander cells with multiple foci increased 3.7-fold. Similar changes occurred in bystander populations mixed and grown with cells irradiated with gamma-rays, and in cultures containing media conditioned on gamma-irradiated cells. DNA DSB repair proteins accumulated at gamma-H2AX foci, indicating that they are sites of DNA DSB repair. Lindane, which blocks gap-junctions, prevented the bystander effect in mixing but not in media transfer protocols, while c-PTIO and aminoguanidine, which lower nitric oxide levels, prevented the bystander effect in both protocols. Thus, multiple mechanisms may be involved in transmitting bystander effects. These studies show that H2AX phosphorylation is an early step in the bystander effect and that the DNA DSBs underlying gamma-H2AX focus formation may be responsible for its downstream manifestations.

Topics: Bystander Effect; Coculture Techniques; Cyclic N-Oxides; DNA; DNA Damage; Enzyme Inhibitors; Fibroblasts; Guanidines; Hexachlorocyclohexane; Histones; Humans; Imidazoles; Insecticides; Nitric Oxide; Nitric Oxide Synthase; Phosphorylation; Radiation, Ionizing