Article Figures & Data
Figures
- Fig. 1.
Inhibition of MEK by U0126 induces apoptosis of melanoma cells. A, constitutive activation of ERK1/2 in cultured melanoma cells, fresh melanoma isolates, and melanocytes. Whole cell lysates from cells were subjected to Western blot analysis using an Ab against pERK1/2. The blots were subsequently reprobed with an antibody against ERK1/2. The data shown are representative of three individual experiments. B, U0126 inhibits activation/phosphorylation of ERK1/2 in melanoma cells. Whole cell lysates from Mel-RM and MM200 cells with or without treatment with U0126 (20 μmol/L) in DMEM for indicated time periods were subjected to Western blot analysis using an Ab against pERK1/2. The blots were subsequently reprobed with an antibody against ERK1/2. The data shown are representative of three individual experiments. C, U0126 induces apoptosis of melanoma cells. Mel-RM and MM200 cells treated with U0126 (20 μmol/L) for indicated intervals were subjected to either DNA laddering assays (left) or measurement of sub-G1 content by the propidium iodide method using flow cytometry (right). The data shown are either representative of three individual experiments (left) or the mean ± SE of three individual experiments (right). D, induction of apoptosis by U0126 in a panel of melanoma cell lines, fresh melanoma isolates, and melanocytes. Cells treated with U0126 (20 μmol/L) for 48 h were subjected to measurement of sub-G1 content by the propidium iodide method using flow cytometry. Columns, mean of three individual experiments; bars, SE.
- Fig. 2.
Knockdown of MEK1 by siRNA induces apoptosis of melanoma cells. A, siRNA knockdown of MEK1 inhibits activation/phosphorylation of ERK. Mel-RM and Sk-Mel-28 cells were transfected with the control or MEK1 siRNA. Twenty-four hours later, whole cell lysates were subjected to Western blot analysis of MEK1, pERK1/2, and ERK1/2 expression. The data shown are representative of three individual experiments. B, MEK1 siRNA induces apoptosis of melanoma cells. Mel-RM and Sk-Mel-28 cells were transfected with the control or MEK1 siRNA. Twenty-four hours later, the cells were switched into normal culture medicum for indicated time periods followed by measurement of sub-G1 content by the propidium iodide method using flow cytometry. Columns, mean of three individual experiments; bars, SE.
- Fig. 3.
U0126 induces mitochondrial apoptotic events. A, U0126 induces activation of Bax and Bak. Mel-RM and MM200 cells treated with U0126 (20 μmol/L) for the indicated intervals were subjected to measurement of activation of Bax and Bak by flow cytometry using antibodies that specifically recognize activated Bax and Bak, respectively. The filled and open histograms were generated from control untreated (C) and U0126-treated cells (U), respectively. The numbers represent MFIs. The data shown are representative of three individual experiments. B, U0126 induces reduction in ΔΨm. Mel-RM and MM200 cells treated with U0126 (20 μmol/L) for the indicated intervals were subjected to measurement of ΔΨm by JC-1 staining in flow cytometry. The number in each left bottom quadrant represents the percentage of cells with reduction in ΔΨm. The data shown are representative of three individual experiments. C, U0126 induces mitochondrial release of cytochrome c, Smac/DIABLO, and AIF. Mitochondrial and cytosolic fractions from Mel-RM and MM200 cells treated with U0126 (20 μmol/L) for the indicated intervals were subjected to Western blot analysis. Western blot analysis of COX IV or β-actin levels was included to show relative purity of the mitochondrial or cytosolic fractions. The data shown are representative of three individual experiments. D, overexpression of Bcl-2 inhibits U0126-induced apoptosis. Left, Bcl-2 was overexpressed in Mel-RM and Me4405 cells transfected with cDNA encoding Bcl-2, but not in the cells transfected with the vector alone. Whole cell lysates were subjected to Western blot analysis. The data shown are representative of two individual experiments. Right, Mel-RM and Me4405 cells transfected with c-DNA for Bcl-2 or vector alone were treated with U0126 (20 μmol/L) 48 h before measurement of apoptosis by the propidium iodide method using flow cytometry. Columns, mean of three individual experiments; bars, SE.
- Fig. 4.
AIF, but not the caspase cascade, plays a crucial role in U0126-induced apoptosis of melanoma. A, U0126 induces caspase activation. Whole cell lysates from Mel-RM and MM200 cells treated with U0126 (20 μmol/L) for the indicated time periods were subjected to Western blot analysis. The data shown are representative of three individual experiments. B, Mel-RM and MM200 cells with or without treatment with U0126 (20 μmol/L) for 36 h were subjected to flow cytometry analysis of caspase-3 activation using an antibody that specifically recognizes the activated form of caspase-3. The data shown are representative of three individual experiments. C, the pan-caspase inhibitor z-VAD-fmk does not block U0126-induced apoptosis of melanoma cells. Mel-RM and Sk-Mel-28 cells with or without pretreatment with z-VAD-fmk (20 μmol/L) for 1 h were treated with U0126 (20 μmol/L) for another 48 h. Apoptosis was measured by the propidium iodide method using flow cytometry. Columns, mean of three individual experiments; bars, SE. D, knockdown of AIF by siRNA inhibits U0126-induced apoptosis. Left, expression of AIF was measured by Western blot analysis of whole cell lysates from Mel-RM and Sk-Mel-28 cells 24 h after transfection with the control or AIF siRNA. The data shown are representative of three individual experiments. Right, Mel-RM and Sk-Mel-28 cells were transfected with the control or AIF siRNA. Twenty-four hours later, the cells were exposed to U0126 (20 μmol/L) for a further 48 h. Apoptosis was then measured by the propidium iodide method using flow cytometry. Columns, mean of three individual experiments; bars, SE.
- Fig. 5.
Inhibition of MEK regulates the expression of Bim, PUMA, and Mcl-1. A, U0126 alters the expression levels of Bim, PUMA, and Mcl-1in melanoma cell lines. Whole cell lysates from Mel-RM and MM200 cells treated with U0126 (20 μmol/L) for indicated time periods were subjected to Western blot analysis. The data shown are representative of three individual experiments. B, effects of MEK1 siRNA on expression of Bim, PUMA, and Mcl-1. Mel-RM and MM200 cells were transfected with the control or MEK1 siRNA. Twenty-four hours later, whole cell lysates were subjected to Western blot analysis of MEK1, pERK1/2, Bim, PUMA, Mcl-1, and ERK1/2 expression. The data shown are representative of three individual experiments. C, U0126 alters the expression levels of Bim, PUMA, and Mcl-1 in fresh melanoma isolates. Whole cell lysates from Mel-KL and Mel-WB cells treated with U0126 (20 μmol/L) for indicated time periods were subjected to Western blot analysis. The data shown are representative of two individual experiments.
- Fig. 6.
Bim, PUMA, and Mcl-1 play important roles in regulating the apoptosis of melanoma cells induced by the inhibition of MEK. A, knockdown of Bim, PUMA, and Bad by siRNA decreases the levels of expression of Bim, PUMA, and Bad, respectively, in the absence or presence of U0126. Mel-RM and Sk-Mel-28 cells were transfected with the control, Bim, PUMA, or Bad siRNA. Twenty-four hours later, the cells were treated with U0126 (20 μmol/L) for a further 24 h. Whole cell lysates were then subjected to Western blot analysis. B, inhibition of Bim or PUMA by siRNA blocks U0126-induced apoptosis. Mel-RM and Sk-Mel-28 cells were transfected with the control, Bim, PUMA, or Bad siRNA. Twenty-four hours later, the cells were exposed to U0126 (20 μmol/L) for a further 48 h. Apoptosis was then measured by the propidium iodide method using flow cytometry. Columns, mean of three individual experiments; bars, SE. C, knockdown of Mcl-1 by siRNA decreases the levels of Mcl-1 expression. Mel-RM and MM200 cells and the fresh melanoma isolate Mel-WB cells were transfected with the control or Mcl-1 siRNA. Twenty-four hours later, the cells were treated with U0126 (20 μmol/L) for a further 24 h. Whole cell lysates were then subjected to Western blot analysis. D, inhibition of Mcl-1 by siRNA induces apoptosis of melanoma (left) and sensitizes melanoma cells to U0126-induced apoptosis (right). Left, Mel-RM and MM200 cells and the fresh melanoma isolate Mel-WB cells were transfected with the control or Mcl-1 siRNA. Twenty-four hours later, the cells were switched into normal culture medium for a further 48 h followed by measurement of sub-G1 content by the propidium iodide method using flow cytometry. The data shown are the mean ± SE of three individual experiments. Right, 24 h after transfection, the cells were treated with U0126 (20 μmol/L) for 48 h followed by measurement of sub-G1 content by the propidium iodide method using flow cytometry. The data (y-axis) are expressed as percentages of apoptotic cells with those that resulted from the transfection of the control or Mcl-1 siRNA alone being subtracted. Columns, mean of three individual experiments; bars, SE.
Volume 13, Issue 16
