Targeting Glycolysis with 2-Deoxy-D-Glucose and Lysosomal Integrity with L-Leucyl-L-Leucine Methyl Ester as Antimelanoma Strategy

<b>Background/Objectives</b>: Melanoma cells enhance glycolysis and expand lysosomes to support energy metabolism, proliferation, and metastasis. However, lysosomal membrane permeabilization (LMP) causes cathepsin leakage into cytosol triggering cytotoxicity. This study investigated the antimelanoma effect of 2-deoxy-D-glucose (2DG), an inhibitor of glycolytic enzyme hexokinase-2, in combination with cathepsin C-dependent LMP inducer L-leucyl-L-leucine methyl ester (LLOMe) and cathepsin C-independent LMP-inducers mefloquine and siramesine. <b>Methods</b>: The viability of A375 and B16 melanoma cells and primary fibroblasts was measured by crystal violet. Apoptosis, necrosis, and LMP were assessed by flow cytometry; caspase activation, mitochondrial depolarization, superoxide production, and energy metabolism were analyzed by fluorimetry, and expression of cathepsins and hexokinase-2 was evaluated by immunoblot. Appropriate inhibitors, antioxidant, and energy boosters were used to confirm cell death type and mechanism. <b>Results</b>: LLOMe triggered LMP, mitochondrial depolarization, and mitochondrial superoxide production, while suppressing oxidative phosphorylation. 2DG suppressed glycolysis and, together with LLOMe, synergized in ATP depletion, caspase activation, and mixed apoptosis and necrosis in A375 cells. Inhibitors of lysosomal acidification, cysteine cathepsins, and caspases, as well as antioxidant and energy boosters, reduced 2DG+LLOMe-induced toxicity. Cathepsins B, C, and D were lower, while hexokinase-2 was higher in A375 cells than fibroblasts. Accordingly, 2DG exhibited lower while LLOMe exhibited higher toxicity against fibroblasts than A375 and B16 cells. However, mefloquine and siramesine induced stronger LMP in A375 cells than in fibroblasts and showed melanoma-selective toxicity when combined with 2DG. <b>Conclusions</b>: 2DG-mediated glycolysis inhibition in combination with lysosomal destabilization induced by mefloquine and siramesine, but not with non-selectively toxic LLOMe, may be promising antimelanoma strategy.