Figure 5.

BET inhibitors impede melanoma cell proliferation, migration, and invasion through SPP1. (A) Quantification by RT-PCR of SPP1 expression in A375 and SK-MEL-28 cells after treatment with BET inhibitor (NHWD-870), BRAF inhibitor (Vemurafenib) or MEK inhibitor (Trametinib) for 24 hours. (B-C) SPP1 expression in A375 and SK-MEL-28 cells after treatment with increasing doses of NHWD-870 for 24 hours quantified by RT-PCR (B) and western blotting (C). (D) Cell proliferation of A375 treated with vehicle (DMSO), NHWD-870 (5nM), JQ-1 (500nM) or BMS-986158 (10nM) were quantified by CCK-8 assay. (E-G) Scratch-wound healing assay (E-F) and Transwell assays (G) of A375 cells treated with vehicle, NHWD-870 (5nM), JQ-1 (500nM) or BMS-986158 (10nM). (H) Pictures of resected subcutaneous xenografted tumor in nude mice. A375 cells (10⁶) were injected subcutaneously. When the tumor reached 100mm³, NHWD-870 (1mg/kg) or vehicle (0.5% methyl cellulose + 0.1% Tween 80) were given orally once a day for five successive days and then two days off. Tumors were resected photographed at day 21 (N = 6 in each group). (I) Tumor volume were recorded twice per week by Vernier caliper measurement and calculated as ([length×width²]/2). (J) Identification by RT-PCR of SPP1 expression in tumor tissue with or without NHWD-870 treatment. All data were presented as mean ± SD of three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001.