Fig 1.
Shikonin but not lapachol promotes ferroxitosis in hypoxia.
(A) Chemical structures of naphthoquinone analogs, shikonin [5,8-dihydxroxy-2-(1-hydroxy-4methyl-3-pentenyl)-1,4-naphthoquinone], menadione [2-methyl-1,4-naphthoquinone] and lapachol [2-Hydroxy-3-(-3- methyl-2-butenyl-)-1,4-naphthoquinone]. (B) MEL103 (NRASQ61L), MEL697 (NRASQ61L BRAFV600E) and MEL526 (BRAFV600E) were treated with 20μM menadione, 5–20 μM shikonin, or 5–20 μM lapachol for 12 hours in a hypoxic chamber. Viability was determined in a resazurin-based assay. (n = 3/condition) **** P<0.0001. (C) Table depicting the ability of menadione, shikonin and lapachol to induce ferroxitosis under normoxic and hypoxic conditions (“+”—induces ferroxitosis, “_”- does not induce ferroxitosis).
Fig 2.
Lapachol inhibits glycolysis in melanoma cells.
(A) Extracellular acidification rate (ECAR) under basal (glycolysis) and maximal (glycolytic capacity) after the addition of oligomycin A (10 μM) were plotted for MEL526 and MEL 103 cell lines after the treatment of cell lines with lapachol at concentrations 0 μM, 5 μM, 10 μM, 20 μM and 40 μM overnight. Each value represents mean ECAR value ± se from five replicates (B) Oxygen consumption rate (OCR) under basal and maximal (after the addition of FCCP 30 μM) were plotted for MEL 526 and MEL103 cell lines after the treatment of cell lines with lapachol at concentrations 0 μM, 5 μM, 10 μM, 20 μM and 40 μM overnight.
Fig 3.
In silico studies of lapachol-PKM2 interactions.
(A) The structure of human muscle Pyruvate kinase M2 [1ZJH—Receptor] docked to shikonin (ligand) using Autodock 4.2 software and ligand receptor interaction diagram generated using Discovery studio 2.5. The predicted free energy of binding for shikonin -7.98 kcal/mol and predicted inhibition constant was 1.42 μM. Inset shows the site pocket with surface charge distribution on donor and acceptor atoms. (B) The structure of human Pyruvate kinase M2 docked to lapachol and ligand receptor interaction diagram generated using Discovery studio 2.5. Inset shows the site pocket with surface charge distribution on donor and acceptor atoms. (C) The 17 amino acid residues of human PKM2 protein predicted to interact with the functional groups in shikonin and the type of interaction Van der waals (pale green), H-bond (bright green), Pi-Sigma (purple) and Pi-Alkyl (pink) are highlighted. (D) The 20 amino acid residues of human PKM2 protein predicted to interact with the functional groups in lapachol and the type interaction are highlighted.
Fig 4.
Lapachol inhibits PKM2 enzyme activity.
(A) Recombinant human PKM1 enzyme was isolated and in vitro PKM1 enzyme activity was measured using a continuous assay coupled to lactate dehydrogenase. Different concentrations of lapachol (vehicle, 5 μM and 10 μM) did not show an inhibition in the activity of recombinant PKM1 invitro. (B) lapachol inhibited the activity of recombinant PKM2 in a dose dependent manner (n = 3/condition) **** P<0.0001 (C) Lapachol inhibited the pyruvate kinase activity invivo in the melanoma cell line MEL103 in a dose dependent manner (n = 3/condition)****P<0.0001.
Fig 5.
Lapachol inhibits melanoma cell proliferation.
(A) Cell proliferation was determined for MEL 526 and MEL103 melanoma cell lines and plot show the rate of proliferation in the presence of increasing concentrations of lapachol (5 μM, 10 μM, 20 μM, 40 μM) (n = 3/condition)****P<0.0001 (B) Cellular ATP levels were measured in MEL 526 and MEL103 melanoma cell lines in the presence of increasing concentrations of lapachol (5 μM, 10 μM, 20 μM, 40 μM) (n = 3/condition)****P<0.0001.
Fig 6.
Lapachol sensitizes melanoma cells to apoptosis.
(A) Annexin 7AAD-PE analysis of apoptosis carried our in MEL103 cell line. Treatment with DMSO (Vehicle) or 2, 4-di nitro phenol (DNP 10 μM) alone showed low percentage of annexin positive cells 7.9% and 8.2% respectively. Lapachol (20 μM) treatment increased annexin positive cells to 13% but the combined treatment of lapachol (20 μM) and DNP (10 μM) further increased the percentage of annexin positive cells to 20%. (B) Loss of mitochondrial membrane potential was analyzed by TMRM dye in MEL 103 melanoma cell line after treatment with DMSO (Vehicle), lapachol (20 μM), DNP (10 μM) and lapachol (20 μM) +DNP (10 μM). Mitochondrial membrane integrity in vehicle, lapachol (20 μM) and DNP (10 μM) treated cells and loss of mitochondrial membrane potential is revealed in cells treated with a combination of lapachol (20 μM) and DNP (10 μM) as reflected by the loss of TMRM fluorescence intensity.