Fig 1.
A cell proliferation based combination screening of α-Mangostin with kinase specific inhibitors.
A and B, show the screening scheme of primary screening and hit identification on SK-MEL-2 cell line. C, The dosage dependent cell proliferation assay of drugs and drug combination on SK-MEL-2 cells. D, The dosage dependent cell proliferation assay of drugs and drug combination on SK-MEL-30 cells. E, Synergistic effect of drug combination. CI (Combination Index) value at half lethal dosage IC50 indicated a strong synergistic effect of α-Mangostin and Sorafenib combination.
Fig 2.
α-Mangostin activates Sorafenib induced apoptosis.
A, Clonogenic survival assay was performed to examine the synergistic effect of the combination of Sorafenib and α-Mangostin on the survival of SK-MEL-2 cells. Cell colonies ware stained in blue. B, Colony numbers were counted. The combination of Sorafenib and α-Mangostin significantly suppressed colony formation (p = 0.001). C, TUNEL staining detects cell apoptosis. Slices were cultured in medium containing Sorafenib (5 μM), α-Mangostin (2 μM) or their combination for 3 hours and stained for TUNEL (red) or 4′, 6-diamidino-2-phenylindole (DAPI; blue). Representative images of three independent experiments are shown. D. Apoptotic cells were counted. Student’s T-test showed that a significant increase of apoptotic cells appeared in the drug combinatorial treatment (p = 0.0003).
Fig 3.
Impact of Sorafenib and α-Mangostin combination on AKT, ERK, and ER stress and autophagy markers.
A, MCL-1 expression was suppressed by Sorafenib (5 μM) and α-Mangostin (2 μM) treatment for 8 hours in SK-MEL-2 cells. B, Down regulation of phosphorylation of AKT and ERK is much stronger at three hours treatment with the combination of Sorafenib (5 μM) and α-Mangostin (2 μM) as compared to single agent treatments. C, The increased expression of phosphorylation of eIF2α were induced by the combination of Sorafenib and α-Mangostin for 8 hours, indicating ER stress. D, The relative mRNA expression level of CHOP. SK-MEL-2 cells were treated with Sorafenib (2 μM), α-Mangostin (2 μM) or their combination for 3 hours. The relative mRNA expression level of CHOP was detected by real-time polymerase chain reaction (PCR). Data shown are the means (±SEM) from at least three independent experiments. The statistical significance increase of CHOP expression was determined by Student’s T test (P = 0.02). E. Atg5 and LC3II levels were detected after the treatment of Sorefenib (5 μM) or α-Mangostin (2 μM).
Fig 4.
α-Mangostin modulates MITF expression via RXR binding.
A. Chemical structure of α-Mangostin resembles CF31, which targets RXR. B, Molecular docking shows that α-Mangostin is accommodated by the RXR ligand binding pocket. In the top panel RXR is cyan and α-Mangostin is shown as spheres. A close-up view of interactions between RXR and α-Mangostin is shown in the bottom. C. Structure of MITF promoter region. The binding sites of VDR/RXR, RAR/RXR D5 and NURR1/RXR are located at position of -912, -722, and -238 respectively. D. ATRA, a RAR activator, significantly induces expression of MIFT in a dose dependent manner but α-Mangostin blocks expression.