Abstract 1474: Hepatocytes either radioprotect or sensitize colorectal cancer cells through nitric oxide-induced oxygen sparing in hypoxic microenvironment
Authors: Jiang Heng , Verovski Valeri , Law Ka Lun , Vermeersch Marieke , Storme Guy , Van den Berge Dirk , Sermeus Alexandra , Verellen Dirk , Ridder Mark De 
Published: August 2012

Abstract
Purpose: Impaired tumor oxygenation remains a major obstacle to curative radiotherapy in advanced primary tumors and metastases. In this study, tailored to the liver metastases of colorectal cancer (CRC), we compared the ability of tumor cells and normal host hepatocytes to metabolically consume oxygen and induce hypoxia. Next, we examined the possibility to re-oxygenate and thereby radiosensitize human CRC cells through the physiological mediator nitric oxide (NO) that switches the mitochondrial oxygen metabolism in hepatocytes from consumption to sparing. Methods and Materials: To model tumor-relevant hypoxia, hepatocytes and tumor cells were incubated in a tissue-mimetic co-culture system with limited oxygenation, which was monitored by fluorescence and linked to hypoxic radioresponse. To activate NO production through inducible nitric oxide synthase (iNOS), hepatocytes (or tumor cells) were exposed to a cytokine mixture of IFN-γ, TNF-α, IL-1α and LPS, and iNOS expression was examined by RT-PCR, western blotting, and NO/nitrite production. Results: Primary mouse hepatocytes consumed 10 to 40-times more oxygen than mouse CT26 and human DLD-1, HT29, HCT116 and SW480 CRC cells, suggesting that hepatocytes are the major effectors of hypoxic conditioning. At cell densities of 0.6-1.2 mln/ml, hepatocytes induced radiobiologically-relevant hypoxia within 20 min, and thereby radioprotected tumor cells 2.0 to 2.5-times. Following exposure to cytokines, hepatocytes revealed significant activation of iNOS-mediated production of NO that targeted mitochondrial aconitase and complexes II, and induced a profound respiratory self-arrest at up to 90 min. As a result, the spared oxygen caused uniform re-oxygenation and radiosensitization in a panel of CRC cell lines (by 1.9 to 2.7-times). Next, both oxygen sparing and radiosensitization could be counteracted by aminoguanidine, a metabolic iNOS inhibitor. Contrasting, all CRC cell lines showed little if any iNOS expression and therefore could not contribute to the oxygen-sparing and radiosensitizing effects of NO-producing hepatocytes. Finally, the NO donor S-nitroso-N-acetylpenicillamine and the mitochondrial inhibitors rotenone, antimycine A and sodium azide were able to block the mitochondrial respiratory chain and to reverse both metabolic hypoxia and hypoxia-induced radioresistance. Conclusion: Hepatocytes may profoundly modulate the hypoxic microenvironment and either protect or sensitize CRC cells to radiation, dependent on the mitochondrial respiratory switch. Our study for the first time provides evidence that host liver cells possess intrinsic radiosensitizing potential through NO-induced oxygen sparing.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1474. doi:1538-7445.AM2012-1474