PMC

WM3629 and WM3670 cells were treated with either vehicle (control), Sorafenib (3 μM, 24 hrs) or U0126 (30 μM, 24 hrs) before being fixed, permeabilized and stained for AIF (green) and DAPI (blue). Magnification X 60.

1205Lu (V600E) and WM3629 (D594G) cells were grown as tumor xenografts in SCID mice. After tumor establishment, mice were dosed three times per week with either vehicle or sorafenib tosylate (60 mg/kg) by oral gavage for 14 days. Growth curves were normalized to the start volumes. A: Sorafenib treatment led to some regression of the established D594G mutated (WM3629) melanoma xenografts. B: Sorafenib treatment led to slowing of the growth of established BRAF V600E-mutated (1205Lu) melanoma xenografts. *Statistically different from vehicle treated animals (P<0.05).

A) Selective sorafenib-induced apoptosis in non-V600E mutated melanoma cell lines. Cells were treated with sorafenib (3 μM, 24 hrs), before being harvested, fixed and stained with propidium iodide. Figure shows sample cell cycle profiles. Extent of apoptosis induced is indicated by the sub-G1 peak. B) Sorafenib treatment enhances the loss of mitochondrial membrane potential in melanoma cells with D594G/G469E mutations. Cells were treated with Sorafenib (3 μM 8 hrs), before being stained with TMRM and analyzed by flow cytometry. C) Sorafenib selectively induces caspase-3 cleavage in D594G mutated melanoma cells. Melanoma cells were treated with Sorafenib (3 μM, 0-24 hrs), followed by protein extraction and probing for cleavage of caspase-3. Equal protein loading is shown by stripping of the blot and probing for actin expression.

A) Sorafenib potently inhibits pERK activity in the D594G BRAF mutated cells. 1205Lu (V600E) and WM3629 (D594G) mutated melanoma cells were treated with either sorafenib (0.1 −3 μM, 1 hr) or U0126 (0.3 −10 μM, 1 hr) before being extracted, resolved and probed for either pERK (pERK) or total ERK (tERK). B) Sorafenib reduces Bcl-2 expression in the D549G and G469E mutated melanoma lines in a MEK-independent manner. Cells were treated with either sorafenib (3 μM, 0-24 hrs) or U0126 (30 μM, 0-24 hrs) after which time protein was extracted, resolved and probed for expression of Bcl-2. Blots were stripped and probed for actin to demonstrate equal protein loading. C) Sorafenib selectively downregulates BAD activity in the D594G and G469E mutated melanoma cell line in a MEK-independent manner. Cells were treated with drug as in B), resulting protein extracts were probed for expression of Ser75 pBAD (pBAD) and total BAD (tBAD). Blots were stripped and probed for actin to demonstrate equal protein loading.

A) Protein expression of phospho-ERK (pERK), total ERK (tERK), phospho-MEK (pMEK), total MEK (tMEK), phospho-PTEN (pPTEN), total PTEN (PTEN), phospho-AKT (pAKT), and total AKT (AKT) in melanoma cell lines with the BRAF V600E mutation (1205Lu, 451Lu), the K601E mutation (WM3130), the D594G mutation (WM3629) and the G469E mutation (WM3670). B) The G469E and D594G mutated melanoma cells are resistant to MEK inhibition. Cells were treated with increasing concentrations of a MEK inhibitor (U0126) (3nM – 10 μM) for 72 hrs and cell proliferation was measured by the MTT assay. C). D594G mutated melanoma cells are highly sensitive to the CRAF inhibitor sorafenib. Cells were treated with increasing concentrations of sorafenib for 72 hrs, cell proliferation was measured as in B). Data shows the mean of 3 independent experiments +/- S.E. mean.

(A) Knockdown of CRAF reduces the pERK activity in D594G and G469E BRAF mutated melanoma cells. Cells with the BRAF V600E (1205Lu) or D594G (WM3629) mutation were infected with either a control lentiviral shRNA (c) or one of two clones directed against CRAF (#1 or #3). Following drug selection, cells were harvested, proteins were extracted and were probed for expression of pERK (pERK), total ERK (ERK), Bcl-2 (Bcl-2) and phoshpo-BAD (pBAD). Even protein loading was confirmed following the stripping of the blots and probing for expression of actin. B) After lentiviral infection and drug selection, cells were harvested, stained with propidium iodide and analyzed by flow cytometry. The number of sub-G1 cells is indicative of apoptosis. C) TUNEL staining of melanoma cells infected with either shRNA control (left panel) or clone #3 of the CRAF shRNA. D) CRAF knockdown reduces the tumorigenicity of WM3629 cells. WM3629 cells were infected with shRNA control, clone #1, clone #3 of the CRAF shRNA. 2 million cells were then injected into the lower back of SCID mice. Tumor volumes were measured every week over a 6-week period. **Statistically significantly different from tumor volume in control WM3629 animals (P<0.001).