| Summary: | There are two stable isotopes of hydrogen, protium (<sup>1</sup>H) and deuterium (<sup>2</sup>H; D). Cellular stress response dysregulation in cancer represents both a major pathological driving force and a promising therapeutic target, but the molecular consequences and potential therapeutic impact of deuterium (<sup>2</sup>H)-stress on cancer cells remain largely unexplored. We have examined the anti-proliferative and apoptogenic effects of deuterium oxide (D<sub>2</sub>O; ‘heavy water’) together with stress response gene expression profiling in panels of malignant melanoma (A375<sup>V600E</sup>, A375<sup>NRAS</sup>, G361, LOX-IMVI), and pancreatic ductal adenocarcinoma (PANC-1, Capan-2, or MIA PaCa-2) cells with inclusion of human diploid Hs27 skin fibroblasts. Moreover, we have examined the efficacy of D<sub>2</sub>O-based pharmacological intervention in murine models of human melanoma tumor growth and metastasis. D<sub>2</sub>O-induction of apoptosis was substantiated by AV-PI flow cytometry, immunodetection of PARP-1, and pro-caspase 3 cleavage, and rescue by pan-caspase inhibition. Differential array analysis revealed early modulation of stress response gene expression in both A375 melanoma and PANC-1 adenocarcinoma cells elicited by D<sub>2</sub>O (90%; ≤6 h) (upregulated:<i> CDKN1A</i>, <i>DDIT3</i>, <i>EGR1</i>, <i>GADD45A</i>, <i>HMOX1</i>, <i>NFKBIA</i>, or <i>SOD2</i> (up to 9-fold; <i>p</i> < 0.01)) confirmed by independent RT-qPCR analysis. Immunoblot analysis revealed rapid onset of D<sub>2</sub>O-induced stress response phospho-protein activation (p-ERK, p-JNK, p-eIF2α, or p-H2AX) or attenuation (p-AKT). Feasibility of D<sub>2</sub>O-based chemotherapeutic intervention (drinking water (30% w/w)) was demonstrated in a severe combined immunodeficiency (SCID) mouse melanoma metastasis model using luciferase-expressing A375-Luc2 cells. Lung tumor burden (visualized by bioluminescence imaging) was attenuated by D<sub>2</sub>O, and inhibition of invasiveness was also confirmed in an in vitro Matrigel transwell invasion assay. D<sub>2</sub>O supplementation also suppressed tumor growth in a murine xenograft model of human melanoma, and median survival was significantly increased without causing adverse effects. These data demonstrate for the first time that systemic D<sub>2</sub>O administration impairs growth and metastasis of malignant melanoma through the pharmacological induction of deuterium (<sup>2</sup>H)-stress.
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