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

Abstract

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.

Mefloquine induces ER stress and apoptosis in BRAFi‐resistant A375‐BRAFV600E/NRASQ61K malignant melanoma cells targeting intracranial tumors in a bioluminescent murine model

“…For generation of CRL‐1619IG‐2‐LUC2, the parental BRAF V600E /NRAS Q61 human malignant melanoma cells were CRISPR/Cas9 gene edited, generating an isogenic variant containing both BRAF V600E and mutated NRAS Q61K , followed by transduction using a lentiviral vector encoding firefly luciferase (LUC2) under control of the EF‐1alpha promoter; differential sensitivity to vemurafenib treatment as a function of NRAS mutational status was confirmed as published before 3 . All cells were maintained as published recently 33,34 …”

“…After 24 h treatment with clinical antimalarials [15 µM; amodiaquine, chloroquine, lumefantrine, piperaquine, and MQ], viability of melanoma cells was assessed by annexinV‐FITC/propidium iodide (PI) dual staining with flow cytometric analysis using an apoptosis detection kit according to manufacturer's specifications (APOAF; Sigma Aldrich) 33,35 …”

“…After 24 h treatment with clinical antimalarials [15 µM; amodiaquine, chloroquine, lumefantrine, piperaquine, and MQ], viability of melanoma cells was assessed by annexinV-FITC/propidium iodide (PI) dual staining with flow cytometric analysis using an apoptosis detection kit according to manufacturer's specifications (APOAF; Sigma Aldrich). 33,35 2.4 | Caspase-3 activation assay MQ-induced (15 µM; 24 h) caspase-3 activation was examined by flow cytometric analysis using a cleaved/activated caspase-3 (asp 175) antibody (Alexa Fluor 488 conjugate; Cell Signaling) following our published procedure. 33,35…”

“…33,35 2.4 | Caspase-3 activation assay MQ-induced (15 µM; 24 h) caspase-3 activation was examined by flow cytometric analysis using a cleaved/activated caspase-3 (asp 175) antibody (Alexa Fluor 488 conjugate; Cell Signaling) following our published procedure. 33,35…”

“…Cellular protein extraction, 4%−15% gradient sodium dodecyl sulfatepolyacrylamide gel electrophoresis gel electrophoresis (Bio-Rad Laboratories), transfer to polyvinylidene difluoride membrane, and immunoblot development were performed as published recently. 3,33 The following rabbit antihuman antibodies were used (obtained from Cell Signaling): cleaved PARP-1 (5625), p-eIF2α (3398), total eIF2α (5324), SQSTM1 (8025), LAMP1 (9091), LC3-I/II (12741), cytochrome c (11940), ATF-4 (11815), and XBP-1s (40435). Equal protein loading was examined by β-actin detection using a mouse monoclonal antibody (Sigma Aldrich); secondary antibodies: HRP-conjugated goat anti-rabbit or goat anti-mouse (Jackson ImmunoResearch Laboratories).…”