Deuterium Oxide (D(2)O) Induces Early Stress Response Gene Expression and Impairs Growth and Metastasis of Experimental Malignant Melanoma

SIMPLE SUMMARY: Systemic administration of deuterium oxide (‘heavy water’) has shown promise in suppressing tumor growth and metastasis in mammalian cancer models, but no detailed molecular studies have revealed specific molecular pathways mediating cancer-cell-directed activities. Here, for the first time, transcriptomic analysis complemented by in vivo efficacy experiments have addressed this unresolved topic. ABSTRACT: There are two stable isotopes of hydrogen, protium ((1)H) and deuterium ((2)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 ((2)H)-stress on cancer cells remain largely unexplored. We have examined the anti-proliferative and apoptogenic effects of deuterium oxide (D(2)O; ‘heavy water’) together with stress response gene expression profiling in panels of malignant melanoma (A375(V600E), A375(NRAS), 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(2)O-based pharmacological intervention in murine models of human melanoma tumor growth and metastasis. D(2)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(2)O (90%; ≤6 h) (upregulated: CDKN1A, DDIT3, EGR1, GADD45A, HMOX1, NFKBIA, or SOD2 (up to 9-fold; p < 0.01)) confirmed by independent RT-qPCR analysis. Immunoblot analysis revealed rapid onset of D(2)O-induced stress response phospho-protein activation (p-ERK, p-JNK, p-eIF2α, or p-H2AX) or attenuation (p-AKT). Feasibility of D(2)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(2)O, and inhibition of invasiveness was also confirmed in an in vitro Matrigel transwell invasion assay. D(2)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(2)O administration impairs growth and metastasis of malignant melanoma through the pharmacological induction of deuterium ((2)H)-stress.