EGb761 regulates Bcl-2 family proteins expression in melanoma cells.

<p>(A) EGb761 alters the expression levels of anti- and pro-apoptotic Bcl-2 family proteins in melanoma cell lines. Whole cell lysates from Mel-RM and Mel-AT cells treated with EGb761 (400 μg/ml) for indicated time periods were subjected to Western blot analysis. The data shown are representative of three individual experiments. (B) 5% ethanol as control vehicle did not alter the expression levels of Mcl-1. Mel-AT cells with 5% ethanol for increasing periods. Whole cell lysates from Mel-AT cells treated were subjected to Western blot analysis. The data shown are representative of three individual experiments.(C) Mel-RM and Mel-AT cells were treated with EGb761 (400 μg/ml) or 5% ethanol for the indicated periods. Total RNA was isolated and subjected to real-time PCR analysis for Mcl-1. The relative abundance of mRNA expression treated with 5% ethanol was arbitrarily designated as 1. Columns, mean of three individual experiments; bars, SEM. * Present p<0.05 vs control.(D) Relative expression of anti-apoptosis Bcl-2 family proteins in melanoma cell lines Mel-RM and Mel-AT without treatment. Quantitative expression levels of Mcl-1, Bcl-2 and Bcl-X_L were normalized to GAPDH.(E) Relative expression of pro-apoptosis Bcl-2 family proteins in melanoma cell lines Mel-RM and Mel-AT without treatment. Quantitative expression levels of Bax, Bid, Noxa, PUMA, Bim, Bad and Bak were normalized to GAPDH.</p

EGb761 induces partially Caspase-Dependent Apoptosis in Melanoma Cells.

<p>(A) EGb761 induces caspase activation. Whole cell lysates from Mel-RM and Mel-AT cells treated with EGb761 (400 μg/ml) for the indicated time periods were subjected to Western blot analysis. The data shown are representative of three individual experiments. (B) Mel-RM and Mel-AT cells with or without treatment with EGb761 (400 μg/ml) for 16 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) Mel-RM and Mel-AT cells were pretreated with the pan-caspase inhibitor z-VAD-fmk (20 μmol/L), the caspase-3 inhibitor z-DEVD-fmk (30 μmol/L), or the caspase-9 inhibitor z-LEHD-fmk (30 μmol/L) 1h before adding EGb761 (400 μg/ml) for another 24h, respectively. Apoptosis was measured by the propidium iodide method using flow cytometry. Data are the mean ± SEM of three individual experiments. * Present p<0.05 vs control.</p

Mcl-1 plays critical roles in regulating the sensitivity of melanoma cells to apoptosis induced by EGb761.

<p>(A) Knockdown of Mcl-1by siRNA decreases the levels of Mcl-1expression. Mel-RM and Mel-AT cells were transfected with scramble SiRNA or Mcl-1siRNA. Whole cell lysates were then subjected to Western blot analysis.(B) Inhibition of Mcl-1 by siRNA induces apoptosis of melanoma and sensitizes melanoma cells to EGb761-induced apoptosis. Mel-RM and Mel-AT cells were transfected with scramble or Mcl-1siRNA. 24hours later, the cells were switched into normal culture medium for a further 48 h followed by treatment with 5% ethanol or EGb761. After another 24 hours, the cells were subjected to measurement of sub-G1content by the propidiumiodide method using flow cytometry. The data shown are the mean ± SEM of three individual experiments. * Present p<0.05 vs control.(C) Knockdown Mcl-1 can affect Bax and Bak activation status in Mel-RM cells. SiRNA inhibition of Mcl-1 resulted in a marked increase in the levels of conformational changed Bax and Bak, which detected by flow cytometry. The open and filled histograms were generated from control (5% ethanol) (C) and EGb761-treated (E) cells respectively. The numbers represent MFIs. The data shown are representative of three individual experiments.(D) Expression of Mcl-1 in a panel of melanoma cell lines after treatment with EGb761 or control (5% ethanol) for 24 hours. Whole cell lysates from melanoma cells were subjected to Western blot analysis. The data shown are representative of three individual experiments.</p

Apoptosis of melanoma induced by EGb761 involves activation of Bax and Bak and changes in mitochondria.

<p>(A) EGb761 induces reduction in ΔΨm. Mel-RM and Mel-AT cells treated with EGb761 (400 μg/ml) for the indicated intervals were subjected to measurement of ΔΨm by JC-1staining 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. (B) EGb761 induces activation of Bax and Bak. Mel-RM and Mel-AT cells treated with EGb761 (400 μg/ml) 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 (5% ethanol treated) (C) and EGb761-treated cells (E), respectively. The numbers represent MFIs. The data shown are representative of three individual experiments. (C) EGb761 induces mitochondrial release of cytochrome c and AIF. Mitochondrial and cytosolic fractions from Mel-RM and Mel-AT cells treated with EGb761 (400 μg/ml) for 16h were subjected to Western blot analysis. COX IV or GAPDH levels were included to show relative purity of the mitochondrial or cytosolic fractions. The data shown are representative of three individual experiments.</p

EGb761 inhibits cell growth and induces apoptosis in melanoma cells in vitro.

<p>(A) Effects of EGb761 on proliferation in Mel-RM and Mel-AT cells. After EGb761 treatment at indicated concentration (100-800ug/ml) or 5% ethanol treatment as the vehicle control, Mel-RM and Mel-AT cells proliferation was assessed in a 96-well plate at 72 hours by MTT assay. The data shown are the mean ± SEM of three individual experiments. (B) Representative dot plots showing fluorescence channel analyses of melanoma cells after dual staining with Annexin V and propidium iodide. Melanocytes, Mel-RM and Mel-AT cells were treated with EGb761 at 400 μg/ml for 24h (5% ethanol treatment as the vehicle control) and then stained with FITC-conjugated Annexin V (green fluorescence, horizontal axis) and PI(red fluorescence, vertical axis), and analyzed using flow cytometry. The data shown are representative of three individual experiments. (C) EGb761 induces apoptosis of melanoma cells. Mel-RM and Mel-AT cells treated with EGb761 (100–800 μg/ml) or 5% ethanol treatment as the vehicle control for indicated intervals were subjected to measurement of apoptosis by the Annexin V/PI staining method using flow cytometry. The data shown are the mean ± SEM of three individual experiments. (D) Induction of apoptosis by EGb761 in a panel of melanoma cell lines and melanocytes. Cells treated with EGb761 (400 μg/ml) or 5% ethanol for 24h were subjected to measurement of sub-G1content by the propidiumiodide method using flow cytometry. Columns, mean of three individual experiments; Bars, SEM.</p