Abstract 4793: NOSH-aspirin inhibits breast cancer cell growth: an effect modulated through reactive oxygen species and independent of the ER status.

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Abstract Introduction: We recently reported the synthesis and characterization of NOSH-aspirin a highly potent hybrid; that releases both nitric oxide (NO) and hydrogen sulfide (H2S) two important signalling molecules of physiological relevance. The present study was undertaken to unravel some of the underlying mechanisms of its action. Methods: Cell lines: MCF-7 (ER+), MDA-MB-231 (ER-, PR-, does not over express Her2), HMEpC, normal human mammary epithelial; Cell growth: MTT; Cell cycle phase distribution: Apoptosis and cell cycle: Flow cytometry; Proliferation: PCNA; ROS: measured hydrogen peroxide and super oxide by flow cytometry using DCFDA and DHE dyes. Xenografts: NuNu mice (N=5/group) implanted with MCF-7 and MDA-MB 231, after 10 days randomly divided animals and gavaged daily with NOSH-ASA (100 mg/kg body weight) or vehicle. Tumor volume and animal weight were recorded every 3 days. After 4 weeks of treatment, mice were sacrificed, tumors excised, weighed, and fixed in 10% buffered formalin. Results: NOSH-aspirin's IC50 in nM at 24h for cell growth inhibition were MCF-7 (285±20), MDA-MB-231 (98±9), HMEpC (20,000±3,000); aspirin's IC50 in all cell lines was greater than 5,000,000 nM at 24h. Cell proliferation at 2xIC50 was decreased 31±4 % in ER+ cells and 27.8 2% in ER- cells. This was accompanied by a G0-G1 phase cell cycle arrest (59.5% at IC50 for ER+ cells and 66.3% at IC50 for ER- cells). NOSH-aspirin at the doses studied, did not cause appreciable apoptosis at 24hr, at 2xIC50 the values being 32.7±3% in ER(+) and 24±2% in ER(-) cells. These results are in line with our data showing that in PC9 lung cancer cells stably expressing cytochrome c-GFP (used for mitochondrial cytochrome c release) NOSH-aspirin caused partial release of cytochrome c up to 3 hours when followed in real -time by fluorescence microscopy and image analysis. NOSH-ASA increased intracellular H2O2 and superoxide anions. Pretreatment of cells with N-acetylcysteine, blocked these. Xenografts: NOSH-aspirin had no effect on the weight of the mice and there were no overt signs of toxicity. Tumor volume was reduced as a function of treatment time. The growth rate of ER(-) xenografts was significantly greater than that of ER(+) ones and NOSH-aspirin had a greater effect on the ER(-) vs ER(+) xenografts. Tumor mass for each group were, ER(+): 0.46±0.056g untreated and 0.21±0.035g treated, (55% reduction, P=0.022), ER(-): 1.2±0.33g untreated and 0.11±0.058g treated (91% reduction, P=0.006). NOSH-aspirin inhibited growth of these cancer cell xenografts as a result of reduced proliferation (decreased PCNA expression), and induction apoptosis (increased number of TUNEL positive cells), and induction of ROS. Conclusions: NOSH-aspirin preferentially affects cancer cells and has potent anti-cancer properties. It targets parameters important in determining cellular mass and merits further evaluation. Citation Format: Diandra E. Nesbitt, Mitali Chattopadhyay, Federica Vannini, Thuy-Tien C. Le, Ravinder Kodela, Niharika Nath, Khosrow Kashfi. NOSH-aspirin inhibits breast cancer cell growth: an effect modulated through reactive oxygen species and independent of the ER status. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4793. doi:10.1158/1538-7445.AM2013-4793 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.