Table 2.Cellular glse determinations for Plx4720 yersus a panel of cell lines B-Raf sequence ration nacasr 600 80 008 0L082 0.04 <0.002 ≥40 <0.02 23 <0.002 typ s ERK ph Raf-MEK-ERI thway.Not that certain premalignant cells such ative of B-Raf (20.21) nd it remains unclear h h c-Raf-1 03 19)Ine Raf-1.there autolimitine th link bet n the negative feedback and onc induced sene through the Raf-MEK-ERK pathwav We speculate that this cence (22).Perhaps it is the negative feedback that prevents the C 05L tro.(A)Pa with varg L PCNA 5100 are abo twith 161wi-ype)cells 3044www.pnas.og/gi/do/10.1073/pnas.0711741105 Tsai et afylation is differentially affected by a 1-h treatment with PLX4720: in cells bearing the BRAFV600E oncogene ERK, phosphorylation is potently inhibited, whereas ERK phosphorylation is unaffected in cells that lack the BRAF oncogene. This observation is consistent with previous reports of a negative feedback pathway from ERK through c-Raf-1 (13, 19). Increased ERK activity results in in￾creased phosphorylation of c-Raf-1, thereby autolimiting the flux through the Raf-MEK-ERK pathway. We speculate that this negative feedback loop is lost in BRAFV600E-bearing tumor cells, resulting in the pronounced dependence of these cells on the Raf-MEK-ERK pathway. Note that certain premalignant cells such as those found in benign nevi also demonstrate frequent expression of B-RafV600E (20, 21), and it remains unclear how these cells avoid transformation to a malignant state. Recent evidence supports the link between the negative feedback and oncogene-induced senes￾cence (22). Perhaps it is the negative feedback that prevents the 1205Lu C8161 None 100 nM 1 µM 10 µM E H & E DAPI S100 PCNA DAPI TUNEL Vehicle PLX4720 F Vehicle PLX4720 G H & E DAPI S100 PCNA DAPI TUNEL B 0 5 50 500 0.0 0.5 1.0 SbCln2 C8161 WM852 [PLX4720] nM Relative Growth 5 50 500 0.0 0.5 1.0 WM35 WM793 1205Lu [PLX4720] nM Relative Growth 1205Lu C8161 WM793 WM852 WM35 SbCln2 None 100 nM 1 µM Non 10 µM e 100 nM 1 µM 10 µM pERK β-actin pERK β-actin pERK β-actin A V600E WT None 100 nM 1 µM 10 µM 1205Lu C8161 C D T0 24' 48' 72' 0 25 50 75 100 T0 24' 48' 72' 0 25 50 75 100 Live Apoptotic Apop/Necrotic % of Population 1205Lu C8161 Fig. 3. Selectivity and antimelanoma activity of PLX4720 in vitro. (A) Panel of melanoma cell lines (V600E, left; B-Raf wild-type, right) were treated with various dosages of PLX4720, and protein extracts were subject to immunoblotting. Activity within the MAPK pathway is represented by levels of phosphorylated ERK; -actin serves as a loading control. (B) B-Raf V600E (Left) and B-Raf wild-type (Right) cells were treated PLX4720 at the indicated dosages for 72 h. Cell number was assayed by MTT analysis. (C) 1205Lu and C8161 cells were treated with 1
M PLX4720 for the times indicated and stained with Annexin V/FITC and propidium iodide (PI) for analysis of apoptosis. (D) Graphs represent raw data from the Annexin/PI assay. (E) Spheroids from 1205Lu and C8161 cells were treated with indicated dosages of PLX4720 and stained with calcein AM and ethidium bromide to assess overall viability. Green (calcein-AM) indicates live cells; red (EtBr) depicts apoptotic cells. (F) Synthetic skin was created by using 1205Lu (V600E) cells and subjected to vehicle control (Upper) or 1
M PLX4720 (Lower) for 72 h. H&E staining is depicted (Left), and immunofluorescent stains for DAPI, PCNA, and S100 are also shown. (G) Same as F, except with C8161 (B-Raf wild-type) cells. Table 2. Cellular GI50 determinations for PLX4720 versus a panel of cell lines Cell line B-Raf sequence PLX4720 proliferation GI50,
M PLX4720 P-ERK GI50,
M PD0325901 P-ERK GI50,
M COLO205 V600E 0.31 0.014 0.002 A375 V600E 0.50 0.046 0.002 WM2664 V600D 1.5 COLO829 V600E 1.7 0.044 0.002 HT716 Wild type 10 SW620 Wild type 13
40 0.002 H460 Wild type 24 Calu-6 Wild type 26 HCT116 Wild type 27 SK-MEL2 Wild type 27 23 0.002 SK-MEL3 Wild type 27 Lovo Wild type 29 H1299 Wild type 41 3044
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