Abstract 1848: Ultra-high concentrations of gaseous nitric oxide show rapid cytotoxic capabilities against colon, breast, pancreatic and other cancer cells in vitro
Authors: Confino Hila , Goldshtein Matan , Puyesky Shani , Yarkoni Shay , Avniel Amir , Lisi Steve , Wolf Ido 
Published: June 2022

Abstract
Background: Nitric oxide (NO) acts as a signaling molecule in various pathological conditions, including cancer. High concentrations of NO are known to have an anti-tumor effect. It is anticipated that NO induces cancer cell cytotoxicity through DNA damage induction, leading to apoptotic cell death. To examine the cytotoxic effect of gaseous NO (gNO) on various cancer types, we exposed 6 cancer cell lines to gNO at ultra-high concentrations, of 10,000-100,000 ppm.
Methods: Four mouse cancer cell lines, colon (CT26), breast (4T1), melanoma (B16F10) and lung (LLC1), and 2 human cancer cell lines, pancreatic (Panc02.03) and ovarian (OVCAR-3), were exposed directly to gNO at 10,000 - 100,000 ppm for 10 - 600 seconds (n=3). Air and nitrogen controls were used to assess whether the effect is mediated by gas flow or lack of oxygen (n=3). Cell viability was measured 24 hours after exposure by an XTT-based cell proliferation method. Additionally, cells were examined after exposure using Annexin V and Propidium Iodide cell apoptosis assays to determine cell death mechanisms.
Results: gNO resulted in significant death of cancer cells in a dose and time dependent manner as revealed by XTT assay: 1. At a concentration of 100,000 ppm gNO, cell viability of CT26, 4T1, B16F10, LLC1 and Panc02.03 was reduced by ~100% after 10 seconds of exposure compared to non-treated (NT) cells (p&lt;0.001) and OVCAR-3 was reduced by 84%. 2. Exposure of cells to 25,000-50,000 ppm gNO resulted in complete eradication of CT26, 4T1, B16F10, LLC1, Panc02.03 and OVCAR-3 cell viability after 1 minute of exposure compared to NT cells (p&lt;0.001). 3. Exposure to 10,000 ppm gNO resulted in elimination of CT26, 4T1, B16F10 LLC1 and Panc02.03 after 2.5 minutes compared to NT cells (p&lt;0.001) with OVCAR-3 viability reduced by 94%. 4. Viability of cells exposed to both nitrogen and air was comparable to NT cells. Additionally, a longer exposure time to 50,000 ppm gNO resulted in an increase in late apoptosis phase for LLC1 (from ~16% for 10 seconds to ~85% for 1 minute).
Conclusion: gNO treatment resulted in a dose dependent cytotoxic response on various cancer cell lines, with higher concentrations leading to near instant cell death and lower concentrations requiring a longer exposure period to elicit cell death. Together with the known ability of NO to activate and recruit the immune system, these results suggest that gNO may be a potent therapeutic agent for tumor treatment across a range of tumor types.
Citation Format: Hila Confino, Matan Goldshtein, Shani Puyesky, Shay Yarkoni, Amir Avniel, Steve Lisi, Ido Wolf. Ultra-high concentrations of gaseous nitric oxide show rapid cytotoxic capabilities against colon, breast, pancreatic and other cancer cells in vitro [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1848.