Deuterium Oxide (D₂O) Induces Early Stress Response Gene Expression and Impairs Growth and Metastasis of Experimental Malignant Melanoma

• Main Authors: Jana Jandova, Anh B. Hua, Jocelyn Fimbres, Georg T. Wondrak
• Format: Article
• Language: English
• Published: MDPI AG 2021-02-01
• Series: Cancers
• Subjects: malignant melanoma; deuterium oxide; heavy water; SCID mouse metastasis model; A375 melanoma xenograft model; cellular stress response
• Online Access: https://www.mdpi.com/2072-6694/13/4/605

There are two stable isotopes of hydrogen, protium (¹H) and deuterium (²H; 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 (²H)-stress on cancer cells remain largely unexplored. We have examined the anti-proliferative and apoptogenic effects of deuterium oxide (D₂O; ‘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 D₂O-based pharmacological intervention in murine models of human melanoma tumor growth and metastasis. D₂O-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 D₂O (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 D₂O-induced stress response phospho-protein activation (p-ERK, p-JNK, p-eIF2α, or p-H2AX) or attenuation (p-AKT). Feasibility of D₂O-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 D₂O, and inhibition of invasiveness was also confirmed in an in vitro Matrigel transwell invasion assay. D₂O 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 D₂O administration impairs growth and metastasis of malignant melanoma through the pharmacological induction of deuterium (²H)-stress.