Radiation-induced nitric oxide mitigates tumor hypoxia and radioresistance in a murine SCCVII tumor model

Abstract

Running title: IR-induced NO regulates tumor hypoxia and radioresistance.Abbreviations 3-NT, 3-nitrotyrosine; eNOS, endothelial nitric oxide synthase; ESR, electron spin resonance; H33342, Hoechst33342; iNOS, inducible nitric oxide synthase; LiNc- BuO, lithium 5,9,14,18,23,27,32,36-octa-n-butoxy-2,3-naphthalocyanine; L-NAME, N G -nitro-L-arginine methyl ester; nNOS, neuronal nitric oxide synthase ;NO, nitric oxide; NOS, nitric oxide 3 synthase; pO 2 , partial pressure of oxygen; positron emission tomography, PET. 4 ABSTRACTTumor hypoxia, which occurs mainly as a result of inadequate tissue perfusion in solid tumors, is a well-known challenge for successful radiotherapy. Recent evidence suggests that ionizing radiation (IR) upregulates nitric oxide (NO) production and that IR-induced NO has the potential to increase intratumoral circulation. However, the kinetics of NO production and the responsible isoforms for NO synthase in tumors exposed to IR remain unclear. In this study, we aimed to elucidate the mechanism by which IR stimulates NO production in tumors and the effect of IR-induced NO on tumor radiosensitivity. Hoechst33342 perfusion assay and electron spin resonance oxymetry showed that IR increased tissue perfusion and pO 2 in tumor tissue.Immunohistochemical analysis using two different hypoxic probes showed that IR decreased hypoxic regions in tumors; treatment with a nitric oxide synthase (NOS) inhibitor, L-NAME, abrogated the effects of IR. Moreover, IR increased endothelial NOS (eNOS) activity without affecting its mRNA or protein expression levels in SCCVII-transplanted tumors. Tumor growth delay assay showed that L-NAME decreased the anti-tumor effect of fractionated radiation (10 Gy × 2). These results suggested that IR increased eNOS activity and subsequent tissue perfusion in tumors. Increases in intratumoral circulation simultaneously decreased tumor hypoxia. As a result, IR-induced NO increased tumor radiosensitivity. Our study provides a new insight into the NO-dependent mechanism for efficient fractionated radiotherapy.

Activation of eNOS in endothelial cells exposed to ionizing radiation involves components of the DNA damage response pathway

“…The dose of 20 mM of L-NAME which we used in this study seems to be relatively high. However, same concentration was adopted in other studies [17,18] and was also necessary for inhibition of radiation-induced NOS activation in ex vivo samples from SCCVII tumors in our previous report [3]. In this experiment, L-NAME at 20 mM showed significant inhibition against the increase of NOS activity induced by X-irradiation, but substantial NO activity still remained; the enzyme responsible for this rest activity was unclear at present.…”

“…Among these isoforms, eNOS is the major source of NO production in endothelial cells [2]. We have previously demonstrated that ionizing radiation increases eNOS activity, and that endogenous NO production increases tissue perfusion in a murine squamous cell carcinoma SCCVII tumor model, leading to radiation-induced tumor reoxygenation [3].…”

Novel synthetic chalcones induces apoptosis in human glioblastoma cells

“…Additionally, C31 promoted some peaks of NO as well, and this could build up to a synergistic multiple-trigger of apoptosis, correlating with the reactive nitrogen species (RNS) levels and the cell cycle influence (Figueira et al 2013;Nagane et al 2013).…”

NOS Inhibition Modulates Immune Polarization and Improves Radiation-Induced Tumor Growth Delay

“…Studies have shown that steady state NO modulation within the tumor microenvironment leads to improved radiation therapeutic efficacy (20, 47, 48). Nitric oxide mediates numerous effects at the cellular and physiological levels.…”