Combinatorial effects of corosolic acid and sorafenib on Huh7 cells.

<p>(A) Dose dependent migration inhibitory effects of corosolic acid and sorafenib on Huh7 cells. The control group was treated with 0.1% DMSO and the experimental groups were treated with indicated compounds. (n = 3, **P < 0.01, ***P < 0.001 compared with the DMSO treated group) (B) Transwell assay were performed to determine anti-migration effect of corosolic acid and sorafenib. The combination index (CI) values were examined at different levels of migration inhibition effect (fa), and the effective combination treatments between corosolic acid and sorafenib (CI < 1) were displayed. (C) Huh7 cells were treated with 0.1% DMSO (control), corosolic acid, sorafenib, or combination of corosolic acid and sorafenib for 30 min, and the lysates were analyzed by western blot. (D) For the <i>in vivo</i> combinatorial study, Huh7 cells (5 × 10⁶) were subcutaneously injected into each mouse. After 7 days, when the tumors reached 50 mm³, the mice were randomized into different groups. CA (2.5, 5 mg/kg), sorafenib (10, 20 mg/kg), or a combination of the two was administrated daily via intraperitoneal injection for 20 days the tumor volume was recorded every 3 to 4 days. (E) Weight of tumor mass (n = 5, **P < 0.01, ***P < 0.001 compared with the DMSO treated control group) and synergistic effects (CI < 1) between different combination group.</p

Migration activity of Huh7 cells is inhibited by corosolic acid without cytotoxicity.

<p>(A) Chemical structure of corosolic acid (B) Huh7 cells were treated with 0.1% DMSO (control) or various concentrations of corosolic acid for 24 h and cell viability was determined with an MTT assay. (C) The migration activity of Huh7 cells was inhibited by corosolic acid in a dose-dependent manner. (n = 3, **<i>P</i> < 0.01, ***<i>P</i> < 0.001 compared with the DMSO treated group)</p

Corosolic acid interacted with the ATP-binding site of VEGFR2 kinase domain by molecular docking analysis.

<p>(A) The three-dimensional diagram displays the interaction of corosolic acid to the ATP-binding site of VEGFR2 (PDB code: 1YWN). (B) The interaction of corosolic acid with the amino acid residues in the ATP-binding site; Glu883 significantly contributes to binding.</p

Corosolic acid exhibits significant anti-tumor effects on Huh7 cells <i>in vivo</i>.

<p>2 × 10⁶ of Huh7 cells were subcutaneously injected into the hind limb of NOD/SCID mice (n = 5). Corosolic acid (5 mg/kg/day) was administered by intraperitoneal injection for 21 days. (A-1) Representative appearance of excised tumor. (A-2) Tumor volume, measured every 5 days. (*<i>P</i> < 0.05, **<i>P</i> < 0.01, ***<i>P</i> < 0.001 compared with the DMSO treated control group) (B) Weight of tumor mass. (*<i>P</i> < 0.05, compared with the DMSO treated control group) (C) Body weight between mice treated with and without corosolic acid. (D) Immunostaining of Ki-67, p-VEGFR2 (Tyr951) and p-FAK (Tyr397) in excised tumor in mice. Single staining was done on several sections.</p

Corosolic acid reduces VEGFR2 kinase activity.

<p>(A) Huh7 cells were treated with 0.1% DMSO (control) or corosolic acid for 15 min and lysates were immunoprecipitated with anti-VEGFR1, VEGFR2, and VEGFR3 Ab, followed by blotting with anti-phospho-tyrosine Ab. (For VEGFR2, n = 3, ***<i>P</i> < 0.001 compared with the DMSO treated group) (B) ADP-Glo Kinase Assay (Promega, Madison, USA) was performed to assess the inhibitory effect of corosolic acid on VEGFR2 kinase activity. (n = 3, RLU data were normalized to the control group and shown as percentages) (C) Huh7 cells were transfected with 100 nM KDR siRNA or control siRNA, recovered for 24 h, and treated with 0.1% DMSO (control) or CA. Migration activity was assessed with a transwell assay. (n = 3, **<i>P</i> < 0.01, ***<i>P</i> < 0.001 compared with the DMSO treated cells in Huh7 control siRNA group)</p

Effect of corosolic acid on VEGFR2-mediated downstream signaling and actin rearrangement.

<p>(A) Huh7 cells were treated with 0.1% DMSO (control) or corosolic acid for 30 min, and the phosphorylation level of VEGFR2 (Tyr1058), Src (Tyr416), and FAK (Tyr397) were analyzed by western blot. (n = 3, *<i>P</i> < 0.05, **<i>P</i> < 0.01 compared with the DMSO treated group) (B) Huh7 cells were treated with 0.1% DMSO (control) or corosolic acid for 6 h, and Rho-GTPase activity was examined with a GST pull-down assay and western blot analysis. (For cdc42, n = 3, **<i>P</i> < 0.01 compared with the DMSO treated group) (C) Huh7 cells were treated with 0.1% DMSO (control) or corosolic acid for 6 h, and fractions containing either F-actin or G-actin were separated by procedures outlined in materials and methods. The ratio of F-actin and G-actin were then calculated. (n = 3, ***<i>P</i> < 0.001 compared with the DMSO treated group) (D) Huh7 cells were treated with 0.1% DMSO (control) or corosolic acid for 6 h followed by immunocytochemistry staining. The phalloidin-stained F-actin (red) and p-FAK (green) co-localized at the leading edge of control cells.</p

LPS-Induced G-CSF Expression in Macrophages Is Mediated by ERK2, but Not ERK1

<div><p>Granulocyte colony-stimulating factor (G-CSF) selectively stimulates proliferation and differentiation of neutrophil progenitors which play important roles in host defense against infectious agents. However, persistent G-CSF production often leads to neutrophilia and excessive inflammatory reactions. There is therefore a need to understand the mechanism regulating G-CSF expression. In this study, we showed that U0126, a MEK1/2 inhibitor, decreases lipopolysaccharide (LPS)-stimulated G-CSF promoter activity, mRNA expression and protein secretion. Using short hairpin RNA knockdown, we demonstrated that ERK2, and not ERK1, involves in LPS-induced G-CSF expression, but not LPS-regulated expression of TNF-α. Reporter assays showed that ERK2 and C/EBPβ synergistically activate G-CSF promoter activity. Further chromatin immunoprecipitation (ChIP) assays revealed that U0126 inhibits LPS-induced binding of NF-κB (p50/p65) and C/EBPβ to the G-CSF promoter, but not their nuclear protein levels. Knockdown of ERK2 inhibits LPS-induced accessibility of the G-CSF promoter region to DNase I, suggesting that chromatin remodeling may occur. These findings clarify that ERK2, rather than ERK1, mediates LPS-induced G-CSF expression in macrophages by remodeling chromatin, and stimulates C/EBPβ-dependent activation of the G-CSF promoter. This study provides a potential target for regulating G-CSF expression.</p></div

U0126 decreases the LPS-induced increased accessibility of the G-CSF promoter to DNase I.

<p>Raw264.7 macrophages were pretreated with DMSO or U0126 (10 μM), then incubated with LPS (100 ng/ml) or PBS for 4 h. Nuclei were then isolated and subjected to DNase I digestion for 2 min, then genomic DNA was isolated and quantitative real-time PCR was performed to measure the amount of undigested DNA in region -167/+12 of the G-CSF promoter (<b>A</b>) and in region -270/-4 of the TNF-α promoter (<b>B</b>), which is expressed as a percentage of that in the control cells. The results are the mean ± SD for three independent experiments. *<i>p</i><0.05 compared to the control.</p

ERK2 knockdown in THP-1 macrophages blocks LPS-induced G-CSF expression.

<p>THP-1 cells were infected with lentivirus carrying specific shRNA for ERK1 or ERK2 and induced to differentiate by incubation with PMA (160 nM) for 3 days; lentivirus carrying luciferase shRNA (Luc) was used as a control, then the following tests were performed. (<b>A</b>) Levels of ERK1/2 and β-actin in the cells were determined by Western blotting; the data shown are typical of the results of three experiments. (<b>B</b>) Cells were treated with LPS (100 ng/ml) for 8 h, then G-CSF levels in the medium were determined by ELISA; untreated Luc cells were used as the control. (<b>C</b> and <b>D</b>) Levels of G-CSF mRNA (<b>C</b>) and TNF-α mRNA (<b>D</b>) in cells were determined by RT-qPCR, normalized to the levels of GAPDH mRNA, and expressed relative to levels in the Luc control (relative value = 1). In (B-D), the results are the mean ± SD for five independent experiments. *<i>p</i><0.05 compared to the corresponding cells not treated with LPS.</p

Knockdown of ERK2, but not ERK1, decreases LPS-induced DNase I accessibility of the G-CSF promoter.

<p>THP-1 cells in which either ERK1 or ERK2 was knocked down using shRNA were induced to differentiate into macrophages. After incubation with DMSO or U0126 (10 μM), the cells were incubated with LPS (100 ng/ml) or PBS for 4 h, then nuclei were purified and subjected to DNase I digestion for 5 min. Genomic DNA was then isolated and the amount of undigested DNA within region -624/-450 of the G-CSF promoter (<b>A</b>) or within region -228/-71 of the TNF- α promoter (<b>B</b>) was determined by quantitative real-time PCR and expressed as a percentage of that in cells not treated with LPS and U0126. The results are the mean ± SD for three independent experiments. *<i>p</i><0.05 compared to the corresponding control.</p