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Nitric oxide, NO, which is generated by various components of the immune system, has been presumed to be cytotoxic. However, NO has been proposed to be protective against cellular damage resulting during ischemia reperfusion. Along with NO there is often concomitant formation of superoxide/hydrogen peroxide, and hence a synergistic relationship between the cytotoxic effects of nitric oxide and these active oxygen species is frequently assumed. To study more peroxide or to hypoxanthine/xanthine oxidase resulted in the degradation of the dopamine uptake and release mechanism. As was observed in the case of the V79 cells, the presence of NO essentially abrogated this peroxide-mediated cytotoxic effect on mesencephalic cells.Nitric oxide, NO, is one of the proposed cytotoxic species produced by the immune surveillance system (1, 2). Many reports also suggest that during the ischemia reperfusion event, NO mediates tissue injury (3)(4)(5)(6)(7)(8)(9)(10)(11)(12). However, it has been suggested that NO can function as a protective agent on the basis of two lines of evidence: (i) NO synthase (NOS) inhibitors increase tissue damage during in vivo ischemia reperfusion within the cerebral cortex, and (ii) in more direct evidence, NO has been shown to prevent damage during ischemia reperfusion events in both brain and heart (13-18). Additional studies provide evidence that NO, though present, plays a minimal role in the pathological effects associated with ischemia reperfusion injury (19,20) or tumor necrosing factor-mediated cytotoxicity (21). Unfortunately, the exact role(s) NO plays in cytotoxicity in vivo is not clear because there are a number of different physiological functions NO or the NOS inhibitors could affect simultaneously. It has been proposed that NO or reactive nitrogen oxide species can directly cause cell death. However, primary neuronal cell cultures exposed to concentrations of NO as high as 1 mM show no adverse effects (22, 23). Many of the biological events in which NO has been proposed as a toxin occur concurrently with the production of reactive oxygen species-e.g., immune response and ischemia reperfusion injury.To clarify the relationship between the cytotoxic properties of the reactive oxygen species and NO, it is necessary to simplify the system. We now show that NO released from a series of compounds known as the "NONOates" (24) Abstract Service Registry Number 136587-13-8) were synthesized and assayed for NO production via chemiluminescence technique as described (24).Enzyme Analysis/Controls. The activity of XO was monitored in the absence and presence of 1 mM DEA/NO by two different assays. (i) Superoxide-induced reduction of ferricytochrome c to ferrocytochrome c was monitored spectrophotometrically at 550 nm (25). The reaction was carried out in a 1-ml volume in aerated 50 mM phosphate buffer (pH 7.8) containing 50 ,AM diethylenetriaminepentaacetic acid. HX was maintained at 2.5 mM and ferricytochrome c at 20 ,uM. The reactions were initiated with the addition of XO (final con...

or superoxide, can generate reactive nitrogen oxide spe-AS was found to be cytotoxic to Chinese hamster V79 cies (RNOS) 2 , which can oxidize nitrate, or nitrosate, lung fibroblast cells over a concentration range of 2-4 mM. The presence of equimolar ferricyanide (Fe(III)-other biomolecules (2, 3). RNOS are proposed to medi-(CN 6 ) 30 ), which converts NO 0 to NO, afforded dramatic ate various toxic and cytotoxic mechanisms. In conprotection against AS-mediated cytotoxicity. Treat-trast, NO can protect cells from damage caused by reacment of V79 cells with L-buthionine sulfoximine to re-tive oxygen species (ROS) (4-6), as a result of different duce intracellular glutathione markedly enhanced AS chemical reactions than those involving RNOS (7-10).cytotoxicity, which suggests that GSH is critical for NO participates in chemical reactions associated cellular protection against the toxicity of NO 0 . Fur-with both oxidative and nitrosative stress which can ther experiments showed that low molecular weight have deleterious consequences in vivo. Studies suggest transition metal complexes associated with the forma-that there exists a balance between NO and two of tion of reactive oxygen species are not involved in AS-the major reactive nitrogen oxide species, dinitrogen mediated cytotoxicity since metal chelators had no ef-trioxide (N 2 O 3 ) and peroxynitrite (ONOO 0 ) (11-13) fect. However, under aerobic conditions, AS was more that can determine the toxicological outcome under toxic than under hypoxic conditions, suggesting that oxygen dramatically enhanced AS-mediated cytotoxicity. At a molecular level, AS exposure resulted in DNA 2 Abbreviations used: RNOS, reactive nitrogen oxide species; AS, double strand breaks in whole cells, and this effect was Angelis's salt; DETAPAC, diethylenetriaminepentaacetic acid; DF, desferrioxime; TPH, tempol-H; NADPH, b-nicotinamide adenine dinucleotide phosphate; PBS, phosphate-buffered saline; GSH, intra-1 To whom correspondence and reprint requests should be ad-cellular glutathione; ER, enhancement ratio; ESR, electron spin resonance. dressed. Fax: (301) 480-2238. 66

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To characterize the ionizing radiation (IR) enhancing effects and underlying mechanisms of the CDK4/6 inhibitor abemaciclib in non-small cell lung cancer (NSCLC) cells and IR enhancement by abemaciclib in a variety of NSCLC cell lines was assessed by clonogenic assay, flow cytometry, and target inhibition verified by immunoblotting. IR-induced DNA damage repair was evaluated by γH2AX analysis. Global metabolic alterations by abemaciclib and IR combination were evaluated by LC/MS mass spectrometry and YSI bioanalyzer. Effects of abemaciclib and IR combination were studied by xenograft tumor regrowth delay, xenograft lysate immunoblotting, and tissue section immunohistochemistry. Abemaciclib enhanced the radiosensitivity of NSCLC cells independent of RAS or EGFR status. Enhancement of radiosensitivity was lost in cell lines deficient for functional p53 and RB protein. After IR, abemaciclib treatment inhibited DNA damage repair as measured by γH2AX. Mechanistically, abemaciclib inhibited RB phosphorylation, leading to cell-cycle arrest. It also inhibited mTOR signaling and reduced intracellular amino acid pools, causing nutrient stress. , abemaciclib, when administered in an adjuvant setting for the second week after fractionated IR, further inhibited vasculogenesis and tumor regrowth, with sustained inhibition of RB/E2F activity, mTOR pathway, and HIF-1 expression. In summary, our study signifies inhibiting the CDK4/6 pathway by abemaciclib in combination with IR as a promising therapeutic strategy to treat NSCLC. Abemaciclib in combination with IR enhances NSCLC radiosensitivity in preclinical models, potentially providing a novel biomarker-driven combination therapeutic strategy for patients with NSCLC. .