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Serbian Association for Cancer Research SDIRSACR

precursors for rapid proliferation. Melanomas also exhibit increased lysosomal fragility and elevated levels of lysosomal
enzymes cathepsins. Cathepsins promote metastasis and immune evasion, but when released into the cytosol upon
lysosomal membrane permeabilization, they trigger cell death. This study aimed to evaluate the antimelanoma
potential of 2-deoxy-D-glucose (2DG), an inhibitor of the glycolytic enzyme hexokinase, in combination with L-leucyl-L-
leucine methyl ester (LLOMe), a lysosome-destabilizing agent.
Materials and Methods: Gene expression data from melanoma patients and normal skin (GEO dataset GSE3189)
were analyzed using the Mann-Whitney U test. Human A375 melanoma cells were treated with 2DG, LLOMe, or their
combination. Cell viability was assessed by MTT assay. Flow cytometry was used to assess apoptosis by detecting
phosphatidylserine externalization (Annexin V/PI staining) and DNA fragmentation (sub-G1 DNA content analysis of PI-
stained cells). Caspase activation was measured fluorometrically (ApoStat staining). The type of 2DG+LLOMe-induced
cell death was determined by assessing viability of cells pretreated with inhibitors of caspases, necroptosis, ferroptosis,
and autophagy.
Results: GEO analysis revealed elevated expression of glycolytic enzymes (hexokinase, phosphoglucose isomerase,
platelet-type phosphofructokinase, aldolase, lactate dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase,
triosephosphate isomerase, pyruvate kinase) and lysosomal proteases (cathepsins B, D, Z) in melanoma relative
to normal skin. Both 2DG and LLOMe reduced the viability of A375 cells, with a markedly enhanced effect when
combined, indicating a synergistic interaction as demonstrated by α-index >1. This cytotoxicity involved caspase
activation, phosphatidylserine exposure, and DNA fragmentation. Caspase inhibitors, but not inhibitors of necroptosis,
ferroptosis, or autophagy, reduced 2DG+LLOMe-induced cell death.
Conclusions: By demonstrating elevated expression of glycolytic enzymes and cathepsins during melanoma progression,
along with the synergistic pro-apoptotic effect of the glycolytic inhibitor 2DG and lysosomal destabilizing agent LLOMe
in melanoma cells, our study supports further investigation of dual targeting of glycolysis and lysosomal stability as a
therapeutic strategy in melanoma.

Acknowledgments and funding: This work was funded by the Ministry of Science, Technological Development and
Innovation of the Republic of Serbia (Contract No. 451-03-137/2025-03/ 200110 and 451-03-136/2025-03/200007)
and non-profit international charity “Climbers Against Cancer” (Donation number#3).

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Synergistic antimelanoma effect of shikonin-mediated glycolysis inhibition and chloroquine-induced
lysosomal destabilization in A375 melanoma cells

Miloš Mandić , Lena Aranđelović , Maja Misirkić Marjanović , Ljubica Vučićević , Mihajlo Bošnjak , Milica Kosić ,
2
1
2
1
1
1
Vladimir Trajković , Ljubica Harhaji-Trajković 2
1
1Institute of Microbiology and Immunology, Faculty of Medicine, University of Belgrade, Belgrade, Serbia
2Institute for Biological Research “Sinisa Stankovic” – National Institute of Republic of Serbia, University of Belgrade, Belgrade,
Serbia
Keywords: chloroquine, glycolysis, lysosomes, melanoma, shikonin

Background: Malignant melanoma is the most aggressive form of skin cancer, for which a fully effective therapy is
still lacking. Melanoma cells upregulate glycolysis to sustain rapid growth and proliferation, while increased lysosomal
volume and elevated activity of lysosomal enzymes cathepsins contribute to their invasiveness and chemoresistance.
However, lysosomal membrane permeabilization (LMP), followed by cathepsin release into the cytoplasm, can trigger
cell death. Shikonin (SH) is a natural inhibitor of the glycolytic enzyme pyruvate kinase M2 (PKM2). Chloroquine (CQ),
an antimalarial drug, can induce LMP and promote the release of cathepsins B and D into cytoplasm. The aim of this
study was to evaluate the antimelanoma effects of combined glycolysis inhibition by SH and lysosomal destabilization
by CQ in A375 melanoma cells.
Material and Methods: The expression levels of PKM2, cathepsin B (CTSB), and cathepsin D (CTSD) genes were
analyzed in patient-derived normal skin, benign nevi, and malignant melanoma samples from the publicly available
GEO dataset GSE3189 using the Mann-Whitney U test in GraphPad Prism. Cell viability was assessed by mitochondrial
dehydrogenase reduction assay and crystal violet staining. The type of cytotoxic interaction between the two treatments
was determined using the interaction coefficient (α), calculated as α = SFsh × SFCQ/SFsh+cq, where SF represents
the survival fraction of the indicated treatment. Apoptosis and necrosis were evaluated by flow cytometry following

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