Abstract
Simple SummarySystemic 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.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 (A375V600E, A375NRAS, 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: 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 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.
Highlights
There are two stable isotopes of hydrogen, protium (1 H) and deuterium (2 H (D)).Since its initial discovery as a natural heavy isotope variant of dihydrogen oxide (1 H2 O), extensive research has focused on the toxicological, biochemical, and pharmacological effects of deuterated water (2 H2 O (D2 O, referred to as ‘heavy water’)) [1]
In a panel of cultured human malignant melanoma cells (A375V600E, A375NRAS, LOX-IMVI, or G361), it was observed that culture in D2 O (90%; 24 h) induces apoptosis (Figure 1A)
A more detailed dose-response analysis (D2 O (≤90%, 24 h)) indicated that at D2 O concentrations lower than 45%, viability was maintained over the duration of the observation period (Figure 1A, bar graph; means without a common letter differ (p < 0.05); see statistical analysis (Section 4.15) for details)
Summary
There are two stable isotopes of hydrogen, protium (1 H) and deuterium (2 H (D)).Since its initial discovery as a natural heavy isotope variant of dihydrogen oxide (1 H2 O), extensive research has focused on the toxicological, biochemical, and pharmacological effects of deuterated water (2 H2 O (D2 O, referred to as ‘heavy water’)) [1]. The pharmacokinetics of tissue deuteration through systemic administration of D2 O have been studied in much detail in mammalian systems, including mice, and feasibility and toxicological consequences of long-term systemic administration of D2 O through drinking water supplementation (20% and above) have been investigated [2,3,4,5,6,7,8,9,10]. Administration of D2 O reaching up to 23% has been documented in investigational human studies for numerous purposes including determination of body water composition and use of D2 O as a potential modulator for neutron capture therapy (in the context of nuclear medicine) [8,10,11,12]. Specific molecular targets potentially mediating pharmacological effects of D2 O such as modulation of water channels (aquaporin 11 (AQP11)), inhibition of
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