Given that PD0325901 may induce apoptosis in melanoma cell lines, we investigated whether a similar mechanism could account for the reduced number of viable cells in PD0325901-treated melanosphere samples . Indeed, PD0325901-treated mutant-BRAF melanospheres contained a high fraction of apoptotic
annexin V-positive cells compared to control samples. In contrast, PD0325901 treated wild type-BRAF melanospheres did not show such a dramatic increase (Figure 3D). Importantly, we found AZD1208 solubility dmso that both wild type and mutated-BRAF melanoma differentiated cells, were exquisitely sensitive to the drug, as indicated by the high fraction of sub-diploid cells detected in treated samples stained with Propidium Iodide (Figure 3E). This additional apoptosis assay confirmed that, at the level of melanospheres, only mutated-BRAF cells rapidly underwent PD0325901-induced apoptosis, while apoptotic hypodiploid DNA-cells were almost absent in the treated wild type-BRAF cells (Figure 3E). These results indicate that PD0325901 exerted strong cytotoxic activity Tanespimycin ic50 against mutant-BRAF melanospheres, and a strong cytostatic activity against wild type-BRAF melanospheres, where cytotoxicity played a minor role. In contrast, differentiated melanoma cells were efficiently killed by PD0325901, regardless
BRAF status (Figure 3E). Figure 3 Antitumor activity of PD0325901 on 17-DMAG (Alvespimycin) HCl melanospheres and their progeny. A) Cell viability (Cell
Titer Glo assay, Promega) of melanospheres with mutated- or wild type-BRAF treated with the indicated drug doses. Mean ± SD of 3 independent experiments is shown. *** p < 0,001. Cell cycle distribution (B) and immunoblot analysis of pathway activation (C) of melanospheres after a 2 day drug exposure. D) Percentage of AnnexinV positive cells in control or PD0325901-treated representative melanospheres samples with mutated- or wild type-BRAF. Mean ± SD of 3 independent experiments is shown. ** p < 0,01. E) Propidium Iodide staining and flow cytometric analysis of representative samples of melanospheres (stem) or differentiated (diff) melanoma cells with mutated- or wild type-BRAF untreated or exposed to PD0325901. The percentage of apoptotic cells with subdiployd DNA is indicated for each condition and cell type. Standard deviations of the percentages are indicated for each condition. ** ≤ 0,01, *** ≤ 0,001 compared to untreated controls. Treatment with MEK inhibitor PD0325901 results in strong antitumor activity in melanosphere-derived xenografts We investigated the activity of PD0325901 against melanosphere-generated subcutaneous xenografts. Doses of 25 or 12.