Mefloquine induces ER stress and apoptosis in BRAFi‐resistant A375‐BRAF^V600E/NRAS^Q61K malignant melanoma cells targeting intracranial tumors in a bioluminescent murine model

Abstract

Molecularly targeted therapeutics have revolutionized the treatment of BRAFV600E‐driven malignant melanoma, but the rapid development of resistance to BRAF kinase inhibitors (BRAFi) presents a significant obstacle. The use of clinical antimalarials for the investigational treatment of malignant melanoma has shown only moderate promise, attributed mostly to inhibition of lysosomal‐autophagic adaptations of cancer cells, but identification of specific antimalarials displaying single‐agent antimelanoma activity has remained elusive. Here, we have screened a focused library of clinically used artemisinin‐combination therapeutic (ACT) antimalarials for the apoptotic elimination of cultured malignant melanoma cell lines, also examining feasibility of overcoming BRAFi‐resistance comparing isogenic melanoma cells that differ only by NRAS mutational status (BRAFi‐sensitive A375‐BRAFV600E/NRASQ61 vs. BRAFi‐resistant A375‐BRAFV600E/NRASQ61K). Among ACT antimalarials tested, mefloquine (MQ) was the only apoptogenic agent causing melanoma cell death at low micromolar concentrations. Comparative gene expression‐array analysis (A375‐BRAFV600E/NRASQ61 vs. A375‐BRAFV600E/NRASQ61K) revealed that MQ is a dual inducer of endoplasmic reticulum (ER) and redox stress responses that precede MQ‐induced loss of viability. ER‐trackerTM DPX fluorescence imaging and electron microscopy indicated ER swelling, accompanied by rapid induction of ER stress signaling (phospho‐eIF2α, XBP‐1s, ATF4). Fluo‐4 AM‐fluorescence indicated the occurrence of cytosolic calcium overload observable within seconds of MQ exposure. In a bioluminescent murine model employing intracranial injection of A375‐Luc2 (BRAFV600E/NRASQ61K) cells, an oral MQ regimen efficiently antagonized brain tumor growth. Taken together, these data suggest that the clinical antimalarial MQ may be a valid candidate for drug repurposing aiming at chemotherapeutic elimination of malignant melanoma cells, even if metastasized to the brain and BRAFi‐resistant.

Drug repurposing of ivermectin abrogates neutrophil extracellular traps and prevents melanoma metastasis

“…Our in vitro screening system showed a broad-spectrum anti-parasitic compound IVM among 231 candidate small molecular compounds in the database of Drug Bank significantly reduced CTSB-induced NETs formation, with no effect on tumor cell viability. The prior investigation on repositioning drugs, such as mefloquine and albendazole have demonstrated to promote cancer treatment by targeting cell cycle arrest in melanoma cells ( 37 , 38 ). IVM is widely used in both animals and human as an FDA-approved parasiticide ( 39 ).…”

Systemic deuteration of SCID mice using the water‐isotopologue deuterium oxide (D2O) inhibits tumor growth in an orthotopic bioluminescent model of human pancreatic ductal adenocarcinoma

“…Three days after transduction, positive cells were selected in complete media containing 1 µg/mL puromycin. Luciferase expression was confirmed by measuring luciferin‐dependent luminescence (Lago CDC bioluminescent imaging system, Spectral Instruments Imaging) as published 24 …”

“…A Custom RT 2 Profiler™ array (Qiagen) assessing expression of 76 user‐specified stress response genes was employed following our published procedures 13,24–27 . Cellular total RNA was isolated using Qiagen RNeasy Mini Kit (Qiagen Sciences), and reverse transcription from 500 ng total RNA was performed using the RT 2 First Strand kit (Qiagen).…”

“…After total RNA isolation and reverse transcription RT‐qPCR was performed according to the manufacturer's protocol as published before 13,24–27 . The following primer probes were used (Thermo Fisher Scientific): human SOD2 (Hs_00167309_m1, FAM), DDIT3 (Hs_00358796_g1, FAM), PTGS2 (Hs_00153133_m1; FAM), HMOX1 (Hs_00157965_m1; FAM), HSPA1A (Hs_00359163_s1, FAM), HSPA8 (Hs_03044880_gH, FAM), RUNX1 (Hs_00231079_m1, FAM), MYC (Hs_00153408_m1, FAM) and RPS18 (housekeeping gene; Hs_01375212_g1; VIC).…”

The Drinking Water and Swimming Pool Disinfectant Trichloroisocyanuric Acid Causes Chlorination Stress Enhancing Solar UV‐Induced Inflammatory Gene Expression in AP‐1 Transgenic SKH‐1 Luciferase Reporter Mouse Skin†

“…All methods (including human skin cell culture, cell viability analysis by flow cytometry, caspase 3 activation assay, detection of intracellular oxidative stress, human epidermal reconstructs, epidermal AP-1 luciferase mouse model, comparative RT 2 Profiler TM gene expression array analysis, single RT-qPCR expression analysis and tissue immunohistochemistry) were performed according to our previously published procedures as specified in detail in the Appendix S1 (11,(39)(40)(41)(42)(43)(44)(45)(46).…”