Distributions of demographic characteristics in the 520 controls and 135 patients with hepatocellular carcinoma.

<p>Mann-Whitney U test or Fisher’s exact test was used between healthy controls and patients with HCC.</p>*<p><i>p</i><0.05 which was statistically significant.</p

Association of the nuclear factor (NF)-κB and inhibitor of NF-κB (IκB) genotypic frequencies with the hepatocellular carcinoma laboratory status.

<p>Association of the nuclear factor (NF)-κB and inhibitor of NF-κB (IκB) genotypic frequencies with the hepatocellular carcinoma laboratory status.</p

Clinical status and <i>NF-κB</i> genotypic frequencies in 135 hepatocellular carcinoma (HCC) patients.

<p>>T2: multiple tumor of >5 cm or tumor involving a major branch of the portal or hepatic veins.</p>a<p>The AORs with their 95% CIs were estimated by multiple logistic regression models after controlling for age, gender, smoking and drinking.</p>b<p>The AORs with their 95% CIs were estimated by multiple logistic regression models after controlling for age, gender, smoking and drinking.</p>*<p><i>P</i> value <0.05 as statistically significant.</p

Distribution frequency of the <i>IκB</i> haplotype in controls and hepatocellular carcinoma patients.

<p>OR, odds ratio; CI confidence interval.</p

BMP-7 Enhances Cell Migration and αvβ3 Integrin Expression via a c-Src-Dependent Pathway in Human Chondrosarcoma Cells

<div><p>Bone morphogenic protein (BMP)-7 is a member of the transforming growth factor (TGF)-beta superfamily, which is originally identified based on its ability to induce cartilage and bone formation. In recent years, BMP-7 is also defined as a potent promoter of cell motility, invasion, and metastasis. However, there is little knowledge of the role of BMP-7 and its cellular function in chondrosarcoma cells. In the present study, we investigated the biological impact of BMP-7 on cell motility using transwell assay. In addition, the intracellular signaling pathways were also investigated by pharmacological and genetic approaches. Our results demonstrated that treatment with exogenous BMP-7 markedly increased cell migration by activating c-Src/PI3K/Akt/IKK/NF-κB signaling pathway, resulting in the transactivation of αvβ3 integrin expression. Indeed, abrogation of signaling activation, by chemical inhibition or expression of a kinase dead form of the protein attenuated BMP-7-induced expression of integrin αvβ3 and cell migration. These findings may provide a useful tool for diagnostic/prognostic purposes and even therapeutically in late-stage chondrosarcoma as an anti-metastatic agent.</p></div

The c-Src is required for in BMP-7-induced cell migration and integrin αvβ3 expression in human chondrosarcoma cells.

<p>(A) JJ012 cells were incubated with BMP-7 for the indicated time intervals and the phosphorylation of the c-Src were determined by western blot. Data are representative of at least three independent experiments. (B–E) Cells were pretreated with PP2 (10 µM) for 30 min or co-transfected with c-Src mutant for 24 h followed by stimulation with BMP-7 for 24 h, and <i>in vitro</i> migration and integrin αvβ3 expression was measured by Transwell (n = 4) and qPCR (n = 4). (F) Cells were pretreated with PP2 for 30 min and then incubated with BMP-7 for 24 h. The protein levels of integrin αvβ3 were determined by flow cytometry analysis (n = 5). Results are expressed as the mean ± SEM. *<i>p</i><0.05, compared to basal expression levels. #<i>p</i><0.05, compared to expression levels in the BMP-7-treated group.</p

Akt is involved in BMP-7-mediated migration and up-regulation of integrin αvβ3 in human chondrosarcoma cells.

<p>(A) JJ012 cells were incubated with BMP-7 for indicated time intervals (upper panel) or pretreated with PP2, Ly294002, or wortmannin for 30 min followed by treatment with BMP-7 for 2 h (lower panel). The levels of p-Akt and Akt were measured by Western blot. Data are representative of at least three independent experiments. (B–E) Cells were pretreated with Akt inhibitor (10 µM) for 30 min or co-transfected with Akt mutant for 24 h, and then incubated with BMP-7 for 24 h. The <i>in vitro</i> migration and integrin αvβ3 expression was measured by Transwell (n = 4) and q-PCR (n = 4). (F) The effect of Akt inhibitor on BMP-7-induced up-regulation of integrin αvβ3 at protein level was determined by flow cytometry analysis (n = 5). Results are expressed as the mean ± SEM. *<i>p</i><0.05, compared to basal expression levels. #<i>p</i><0.05, compared to expression levels in the BMP-7-treated group.</p

BMP-7 enhanced cell migration through up-regulation of integrin αvβ3 expression.

<p>(A) JJ012 cells were incubated with BMP-7 (5–30 ng/ml) for 24 h, and <i>in vitro</i> migration was measured with the Transwell after 24 h (n = 6). (B) JJ012 cells were incubated with BMP-7 (30 ng/ml) for 24 h, and mRNA expression of αv, α5, α6, β1, β3, and β5 integrin were examined by q-PCR (n = 6). (C) Cells were incubated with or without BMP-7 for 24 h and the protein expression levels of integrin αvβ3 were examined by flow cytometry analysis (n = 5). (D) Cells were pretreated with αvβ3 monoclonal antibody (10 µg/ml) for 30 min followed by stimulation with BMP-7. The <i>in vitro</i> migration activity measured after 24 h (n = 5). Results are expressed as the mean ± SEM. *<i>p</i><0.05, compared to basal expression levels.</p

PI3K (p85α) is involved in BMP-7-induced migration and integrin αvβ3 expression.

<p>(A–B) JJ012 cells were incubated with BMP-7 for indicated time intervals or pretreated with PP2 for 30 min followed by treatment with BMP-7 for 2 h. The levels of p-p85α and p85α were measured by Western blot. Data are representative of at least three independent experiments. (C) Cells were pretreated with Ly294002 (10 µM) or wortmannin (10 µM) followed by stimulation with BMP-7 for 24 h, and <i>in vitro</i> migration was measured by Transwell (n = 4). (D–F) Cells were pretreated with PI3K inhibitor Ly294002 (10 µM) or wortmannin (10 µM) for 30 min or co-transfected with p85 mutant for 24 h followed by incubation with BMP-7 for 24 h. The expression of integrin αvβ3 was measured by q-PCR (n = 4) and flow cytometry (n = 5). Results are expressed as the mean ± SEM. *<i>p</i><0.05, compared to basal expression levels. #<i>p</i><0.05, compared to expression levels in the BMP-7-treated group.</p

BMP-7 induced activation of NF-κB through c-Src/PI3K/Akt pathway.

<p>(A) JJ012 cells were transfected with NF-κB-luciferase reporter for 24 h and then treated with BMP-7 in a dose-dependent manner for 24 h (n = 5). (B) The transfected JJ012 cells pretreated with PP2, Ly294002, wortmannin, Akt inhibitor, PDTC, or TPCK, followed by stimulation with BMP-7 for 24 h. Equal amounts of cell extract were assayed for dual-luciferase activity (n = 5). (C) Proposed scheme for BMP-7-stimulated signaling involved in up-regulation of integrin αvβ3 expression, leading to enhanced cell migration. Results are expressed as the mean ± SEM. *<i>p</i><0.05, compared to basal expression levels. #<i>p</i><0.05, compared to expression levels in the BMP-7-treated group.</p