Journal: Molecular carcinogenesis

Article Title: Eupafolin suppresses prostate cancer by targeting phosphatidylinositol 3-kinase-mediated Akt signaling

doi: 10.1002/mc.22139

Figure Lengend Snippet: Eupafolin inhibits proliferation and anchorage-independent growth of prostate cancer cells. A, left panel, the chemical structure of eupafolin. Right panel, eupafolin suppresses PC3 cell proliferation in a dose-dependent manner. PC3 cells (3 × 103 cells/well) were treated with the indicated doses of eupafolin for the specified times. The absorbance was measured as described in “Materials and Methods”. Data are shown as means ± S.D. and the asterisk (*) indicates a significant (p < 0.05) decrease in proliferation of cells treated with eupafolin compared to untreated control cells. B, eupafolin suppresses anchorage-independent growth of PC3 cells. Bar graphs depict the inhibitory effect of eupafolin on PC3 cells (left panel). Colony numbers are shown as means ± S.D. from 3 independent experiments. The asterisk (*) indicates a significant (p < 0.05) decrease in colony numbers in cells treated with eupafolin compared to the DMSO-treated group. Representative photographs of colony formation are shown in the right panels. C, eupafolin suppresses DU 145 (left panel) and LNCaP (right panel) prostate cancer cell proliferation. Cells (3 × 103 cells/well) were treated with the indicated doses of eupafolin for the specified times. The absorbance was measured as described in “Materials and Methods”. Data are shown as means ± S.D. and the asterisk (*) indicates a significant (p < 0.05) decrease in proliferation in cells treated with eupafolin compared to untreated control cells.

Article Snippet: Briefly, active PI3-K (100 ng, EMD Millipore, Billerica, MA) was incubated with DMSO or various concentrations of eupafolin (0, 5, 10, 15, or 20 μM) for 15 min and then mixed with phosphatidylinositol sodium salt (20 μl of 0.5 mg/ml MP Biomedical, Solon, OH) at 30 °C for 20 min.

Techniques:

Journal: Molecular carcinogenesis

Article Title: Eupafolin suppresses prostate cancer by targeting phosphatidylinositol 3-kinase-mediated Akt signaling

doi: 10.1002/mc.22139

Figure Lengend Snippet: Eupafolin suppresses PI3-K/Akt signaling. A, PC3 prostate cancer cells were treated with eupafolin for 24 h at the indicated concentration. The protein content of phosphorylated and total proteins associated with the PI3-K signaling pathway was visualized by Western blotting with specific primary and HRP-conjugated secondary antibodies. B, PC3 cells were treated as in A. The protein content of phosphorylated and total proteins associated with the ERKs signaling pathway was visualized by Western blotting with specific primary and HRP-conjugated secondary antibodies.

Article Snippet: Briefly, active PI3-K (100 ng, EMD Millipore, Billerica, MA) was incubated with DMSO or various concentrations of eupafolin (0, 5, 10, 15, or 20 μM) for 15 min and then mixed with phosphatidylinositol sodium salt (20 μl of 0.5 mg/ml MP Biomedical, Solon, OH) at 30 °C for 20 min.

Techniques: Concentration Assay, Western Blot

Journal: Molecular carcinogenesis

Article Title: Eupafolin suppresses prostate cancer by targeting phosphatidylinositol 3-kinase-mediated Akt signaling

doi: 10.1002/mc.22139

Figure Lengend Snippet: Eupafolin causes cell cycle arrest at G1. A, PC3 prostate cancer cells were serum-starved for 16 h to synchronize at G0 phase and then pretreated with eupafolin for 2 h followed by stimulation with 10% FBS including eupafolin for 16 h. The cell cycle distribution was measured by flow cytometry with propidium iodide. Data are expressed as the percentage of cells in G1/G0, S, or G2/M and are shown as means ± S.D. of values from triplicate experiments. B, eupafolin inhibits cyclin D1 transcriptional activity. The inhibitory effect of eupafolin on cyclin D1 activity was measured by introducing cyclin D1 reporter plasmids into PC3 cells followed by treatment with various concentrations of eupafolin. Luciferase activity was measured and data are shown as means ± S.D. of values from triplicate experiments (upper panel). The protein content of cyclin D1was determined by Western blotting (lower panel). The asterisk (*) indicates a significant (p < 0.05) difference in cell cycle distribution (A) or luciferase activity (B) in treated versus untreated cells.

Article Snippet: Briefly, active PI3-K (100 ng, EMD Millipore, Billerica, MA) was incubated with DMSO or various concentrations of eupafolin (0, 5, 10, 15, or 20 μM) for 15 min and then mixed with phosphatidylinositol sodium salt (20 μl of 0.5 mg/ml MP Biomedical, Solon, OH) at 30 °C for 20 min.

Techniques: Flow Cytometry, Activity Assay, Luciferase, Western Blot

Journal: Molecular carcinogenesis

Article Title: Eupafolin suppresses prostate cancer by targeting phosphatidylinositol 3-kinase-mediated Akt signaling

doi: 10.1002/mc.22139

Figure Lengend Snippet: Eupafolin binds to PI3-K. A, eupafolin binds to PI3-K in vitro. Active PI3-K (200 ng) was subjected to a pulldown assay with eupafolin conjugated with CNBr-Sepharose 4B beads. Eupafolin binding to PI3-K was visualized by Western blotting with a PI3-K antibody. B, eupafolin binds with PI3-K ex vivo. PC3 cell lysates (500 μg) were pulled down with CNBr or CNBr-eupafolin-conjugated beads. The pulled down proteins were visualized using antibodies to detect PI3-K. C, eupafolin binds with PI3-K in competition with ATP. Active PI3-K (200 ng) was subjected to a pulldown assay with eupafolin conjugated with CNBr-Sepharose 4B beads. Then the beads were incubated with different concentrations of ATP (0, 0.1, or 1 mM). Eupafolin binding to PI3-K was visualized by Western blotting with anti-PI3-K. D, upper panel, eupafolin (shown as surface representation with transparency of 50%) binds to the ATP binding pocket of PI3-K beta. Lower panel, LY294002 (shown as surface representation with transparency of 50%) binds to the ATP binding pocket of PI3-K beta.

Article Snippet: Briefly, active PI3-K (100 ng, EMD Millipore, Billerica, MA) was incubated with DMSO or various concentrations of eupafolin (0, 5, 10, 15, or 20 μM) for 15 min and then mixed with phosphatidylinositol sodium salt (20 μl of 0.5 mg/ml MP Biomedical, Solon, OH) at 30 °C for 20 min.

Techniques: In Vitro, Binding Assay, Western Blot, Ex Vivo, Incubation

Journal: Molecular carcinogenesis

Article Title: Eupafolin suppresses prostate cancer by targeting phosphatidylinositol 3-kinase-mediated Akt signaling

doi: 10.1002/mc.22139

Figure Lengend Snippet: Eupafolin inhibits PI3-K kinase activity in vitro. A, the effect of eupafolin on PI3-K activity was measured by an in vitro kinase assay using PIP2 as the PI3-K substrate. 32P-labeled PIP3 was visualized by autoradiography as described in “Materials and Methods”. B, quantification of the effect of eupafolin on PI3-K kinase activity (from A).

Article Snippet: Briefly, active PI3-K (100 ng, EMD Millipore, Billerica, MA) was incubated with DMSO or various concentrations of eupafolin (0, 5, 10, 15, or 20 μM) for 15 min and then mixed with phosphatidylinositol sodium salt (20 μl of 0.5 mg/ml MP Biomedical, Solon, OH) at 30 °C for 20 min.

Techniques: Activity Assay, In Vitro, Kinase Assay, Labeling, Autoradiography

Journal: Molecular carcinogenesis

Article Title: Eupafolin suppresses prostate cancer by targeting phosphatidylinositol 3-kinase-mediated Akt signaling

doi: 10.1002/mc.22139

Figure Lengend Snippet: Eupafolin suppresses tumor growth in vivo by inhibiting the PI3-K-related signaling pathway. A, the total average tumor weight in the eupafolin-treated groups is significantly less (*, p < 0.05) than that of the vehicle-treated group. Tumors were extracted and weighed after mice were sacrificed. Data are shown as means ± S.D. and significant differences were determined by one-way ANOVA. B, total average tumor volume in the eupafolin-treated group was significantly (*, p < 0.05) less than that of the vehicle-treated group. Tumor volume was measured and recorded 3 times a week throughout the study. Data are shown as means ± S.D. C, the protein levels of total and phosphorylated Akt, p70RSK, and GSK3β were assessed by Western blot analysis in vehicle- and eupafolin-treated tumor tissues. The relative density was compared with actin (lower panel). Data are shown as means ± S.E.

Article Snippet: Briefly, active PI3-K (100 ng, EMD Millipore, Billerica, MA) was incubated with DMSO or various concentrations of eupafolin (0, 5, 10, 15, or 20 μM) for 15 min and then mixed with phosphatidylinositol sodium salt (20 μl of 0.5 mg/ml MP Biomedical, Solon, OH) at 30 °C for 20 min.

Techniques: In Vivo, Western Blot