Figure 3. The AKT inhibitor MK-2206 enhances the cytotoxic effects of carboplatin and paclitaxel after prolonged drug exposure.

(A) Acute treatment of MK-2206 enhances the anti-proliferative efficacy of carboplatin and paclitaxel treatment. Melanoma cells were either treated with vehicle, MK-2206 (5 µM), Carboplatin (1 µM), Paclitaxel (3 nM) or the combination of all three agents for 72 h, and cell numbers were quantified using Alamar blue assay. (B) Acute treatment of MK-2206 does not enhance Carboplatin and Paclitaxel-induced apoptosis. Melanoma cells were either treated with vehicle, MK-2206 (5 µM), Carboplatin (1 µM), Paclitaxel (3 nM) or the combination of all three agents for 72 h, and apoptosis levels were assessed by annexin-V staining and flow cytometry. Data show the mean of three experiments. (C) Long-term treatment of MK-2206 with Carboplatin and Paclitaxel significantly enhances the suppression of colony formation. A panel of BRAF/NRAS-WT melanoma cell lines were treated with vehicle, MK-2206 (5 µM), Carboplatin (1 µM), Paclitaxel (3 nM) or the combination of all three agents for 4 weeks. After this time, colonies were fixed and stained with crystal violet. Photographs are representative of three independent experiments. (D) Combined treatment with MK-2006, Carboplatin and Paclitaxel enhanced cytotoxicity in a panel of 3D collagen-implanted spheroids. Cells were treated with MK-2206, carboplatin and paclitaxel (3, 6 or 9 days) before being treated with calcein-AM and Ethidium bromide. Green, viable cells; red, dead cells. Lack of green staining also indicates a loss of cell viability. Magnification × 10. Statistical significance is indicated where ns>0.05, * P<0.05, ** P<0.01, *** P<0.001 and ****P<0.0001