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

Background: /Objectives: Melanoma cells enhance glycolysis and expand lysosomes to support energy metabolism, proliferation, and metastasis. However, lysosomal membrane permeabilization (LMP)-mediated release of lysosomal proteases cathepsins into cytoplasm triggers cytotoxicity. This study investigated antimelanoma effect of glycolytic inhibitor 2-deoxy-D-glucose (2DG) in combination with cathepsin C-dependent LMP inducer L-leucyl-L-leucine methyl ester (LLOMe). Methods: Viability of human melanoma A375 cells and fibroblasts treated with 2DG and/or LLOMe was measured by crystal violet. Apoptosis/necrosis/LMP were assessed by flow cytometry. Caspase activation/mitochondrial depolarization/mitochondrial superoxide production/energy metabolism were evaluated by fluorimetry. Appropriate inhibitors/antioxidant/energy boosters were used to confirm involvement of LMP/cathepsins/caspases/oxidative stress/energy stress in LLOMe+2DG-induced cytotoxicity. Expression of glycolytic enzymes and cathepsins in melanoma patients was analyzed in publicly available GEO datasets. Results: LLOMe with/without 2DG triggered LMP, mitochondrial depolarization, mitochondrial superoxide production, caspase activation, and mixed apoptosis/necrosis in A375 cells. LLOMe inhibited oxidative phosphorylation, 2DG suppressed glycolysis, which together synergized in ATP depletion. Inhibitors of lysosomal acidification/cathepsins/caspases, antioxidant, and energy boosters reduced 2DG+LLOMe-induced toxicity. Fibroblasts were highly sensitive to LLOMe but not to LMP-inducing drugs mefloquine and siramesine, whose toxicity is cathepsin B-mediated. Several glycolytic enzymes and cathepsins were increased, whereas cathepsin C expression was decreased in melanoma patients. Conclusions: LLOMe-induced LMP causes cathepsin-dependent mitochondrial dysfunction with oxidative phosphorylation suppression, which together with 2DG-inhibited glycolysis, synergizes in induction of energy stress and mixed apoptotic-necrotic cell death. Given its non-selective toxicity, possibly due to cathepsin C loss in melanoma, LLOMe should be replaced with cathepsin C-independent LMP inducers for use with 2DG in antimelanoma therapy.