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. Author manuscript; available in PMC: 2015 May 1.
Published in final edited form as: Pigment Cell Melanoma Res. 2014 Feb 21;27(3):465–478. doi: 10.1111/pcmr.12227

Figure 5. MK-2206/chemotherapy-induced autophagy is initially protective, followed by long-term cytotoxicity.

Figure 5

(A) Long-term treatment with the combination of MK-2206, carboplatin and paclitaxel leads to hyperactivation of autophagy and later the induction of apoptosis. Western blot showing time-dependent increases in autophagy (accumulation of LC3 II) and apoptosis (caspase 9 cleavage) following treatment with the combination of MK-2206, paclitaxel and carboplatin. (B) Inhibition of caspases partly reverses MK-2206/paclitaxel/carboplatin-induced cytotoxicity. 3D collagen-implanted spheroids of WM3918, M257 and M368 melanoma cells were treated with Carboplatin and Paclitaxel, MK-2206, MK-2206/Carboplatin/Paclitaxel, or the combination of MK-2206/Carboplatin/Paclitaxel with the caspase inhibitor z-Vad-FMK (50µM) over the course of 144 hours. Spheroids were subsequently imaged with immuno-fluorescent microscopy following treatment with calcein-AM and Ethidium bromide. Green, viable cells: red, dead cells. (C) (Upper) A panel of BRAF-WT melanoma cell lines (WM209, M257, M285, M368 and WM3918) were treated with chloroquine (12.5–50µM), MK-2206/Carboplatin/Paclitaxel or the combination of all agents for 72h. Cell numbers were quantified using Alamar blue assay and standardized against control. (Lower) Melanoma cells were treated with chloroquine alone (50µM), MK-2206/Carboplatin/Paclitaxel, or the combination of all four agents for 72h. Apoptosis was subsequently assessed by annexin-V staining and flow cytometry. (D) 3D collagen-implanted spheroids were treated with vehicle, MK-2206/Carboplatin/Paclitaxel, in the absence or presence of chloroquine (50µM) for 72h, before being treated with calcein-AM and Ethidium bromide. Green, viable cells; red, dead cells.