Des-␥-carboxyl prothrombin (DCP) is a well recognized tumor marker for hepatocellular carcinoma. Previously, we have demonstrated that DCP stimulates cell proliferation in hepatocellular carcinoma cell lines through Met-Janus kinase 1 signal transducer and activator of transcription 3 signaling pathway. In the present study, we demonstrated that DCP induces both cell proliferation and migration in human umbilical vein endothelial cells. DCP was found to bind with the kinase insert domain receptor (KDR), alternatively referred to as vascular endothelial growth factor receptor-2. Furthermore, DCP induced autophosphorylation of KDR and its downstream effector phospholipase C-␥ and mitogen-activated protein kinase (MAPK). To support these results, we showed that DCP-induced cell proliferation and cell migration were inhibited by KDR short interfering RNA, KDR kinase inhibitor, or MAPK inhibitor. In conclusion, these results indicate that DCP is a novel type of vascular endothelial growth factor that possesses potent mitogenic and migrative activities.
Loss or decreased expression of runt-related transcription factor 3 (RUNX3), a tumor suppressor gene involved in gastric and other cancers, has been frequently observed in hepatocellular carcinoma (HCC). The objective of this study was to identify the regulatory mechanism of the epithelial-mesenchymal transition (EMT) by RUNX3 in HCC. Human HCC cell lines, Hep3B, Huh7, HLF and SK-Hep1, were divided into low-and high-EMT lines, based on their expression of TWIST1 and SNAI2, and were used in this in vitro study. Ectopic RUNX3 expression had an anti-EMT effect in low-EMT HCC cell lines characterized by increased Ecadherin expression and decreased N-cadherin and vimentin expression. RUNX3 expression has previously been reported to reduce jagged-1 (JAG1) expression; therefore, JAG1 ligand peptide was used to reinduce EMT in RUNX3-expressing low-EMT HCC cells. Immunohistochemical analyses were performed for RUNX3, E-cadherin, N-cadherin and TWIST1 in 33 human HCC tissues, also divided into low-and high-EMT HCC, based on TWIST1 expression. E-cadherin expression was correlated positively and N-cadherin expression was correlated negatively with RUNX3 expression in low-EMT HCC tissues. Correlations between EMT markers and RUNX3 mRNA expression were analyzed using Oncomine datasets. Similarly, mRNA expression of E-cadherin was also significantly correlated with that of RUNX3 in low-EMT HCC, while mRNA expression of JAG1 was negatively correlated with that of RUNX3. These results suggest a novel mechanism by which loss or decreased expression of RUNX3 induces EMT via induction of JAG1 expression in low-EMT HCC.
Runt-related transcription factor 3 (RUNX3) is reported as a tumor suppressor gene for gastric cancer, and may be important in the development of hepatocellular carcinoma (HCC). RUNX3 expression is frequently lost or decreased by hemizygous deletion or hypermethylation of its promoter lesion in HCC. The significance of decreased expression of RUNX3 in HCC has not been fully elucidated, but is likely related to dysfunction of cell cycle regulation, decrement of apoptosis, enhancement of angiogenesis, and development of epithelial-mesenchymal transition. RUNX3 is a promising candidate as a tumor suppressor gene for HCC.
Abstract. runt-related transcription factor 3 (rUnX3) is a candidate tumor suppressor gene that is downregulated in various cancers. In the present study, we analyzed the regulatory function of rUnX3 on Jagged-1 (JAg1) expression and cancer stem cell (csc) signaling in hepatocellular carcinoma (Hcc). eleven Hcc cell lines and 30 human Hcc tissues were used. rUnX3 and JAg1 expression levels were analyzed by immunoblotting and immunohistochemistry. ectopic rUnX3 expression was induced by introducing rUnX3 cDnA into the rUnX3-negative Hcc cell line Hep3B and Huh7 cells. Furthermore endogenous rUnX3 expression was knocked down by rUnX3 sirnA in sK-Hep-1 cells. In order to analyze JAg1 transcriptional regulation, we conducted reporter assays, chromatin immunoprecipitation (chIp) assays and electrophoretic mobility shift assays (eMsAs). tumorigenicity was analyzed using a scID mouse liver injection model. An inverse correlation was observed between rUnX3 expression and JAg1 expression in most Hcc cell lines and tissues. restoring rUnX3 expression decreased the expression of JAg1 in Hep3B and Huh7 cells, whereas JAg1 expression was upregulated in rUnX3 sirnA-treated sK-Hep-1 cells. reporter assays, chIp assays and eMsAs revealed that rUnX3 directly bound to the transcriptional regulatory region of JAg1 and suppressed JAg1 transcription. Moreover, rUnX3 restoration downregulated cscs by suppressing JAg1-mediated notch signaling. the tumorigenic capacity of rUnX3-expressing Hep3B cells was lower compared to that of control Hep3B cells. rUnX3 expression suppressed JAg1 expression and resulted in downregulation of tumorigenesis by suppression of JAg1-mediated cscs. IntroductionHepatocellular carcinoma (Hcc) is the third most frequent cause of cancer-related death worldwide, and its incidence rate is increasing (1-3). patients diagnosed with Hcc have a poor prognosis because of the aggressive nature of the disease (1,4). Although various therapeutic strategies have been developed, such as operation, ablation and chemotherapy, therapies to treat patients with advanced Hcc are lacking. there is an urgent need to elucidate the pathologic mechanism underlying molecular-targeted therapies in Hcc. Many studies have considered that the dysfunction of oncogenes and tumor suppressor genes, reactivation of developmental pathways, and activation of growth factors and their receptors play major roles in the development and progression of Hcc (1,5). Further, the expression of a number of tumor suppressor genes is attenuated by a combination of genetic and epigenetic events, including loss of an allele, mutation, or promoter methylation (6,7). the loss of heterozygosity (loH) on chromosome 1p36 was reported as one of the most frequent loH in Hcc (7,8). loH on chromosome 1p36 suggests the presence of important tumor suppressor genes on that chromosome. It was reported that runt-related transcription factor 3 (rUnX3), located on chromosome 1p36, correlates with tumorigenesis and progression of gastric cancer (9). rUnX3 is an apoptotic fact...
BackgroundRunt-related transcription factor 3 (RUNX3) is known as a tumor suppressor gene for gastric cancer and other cancers, this gene may be involved in the development of hepatocellular carcinoma (HCC).MethodsRUNX3 expression was analyzed by immunoblot and immunohistochemistry in HCC cells and tissues, respectively. Hep3B cells, lacking endogenous RUNX3, were introduced with RUNX3 constructs. Cell proliferation was measured using the MTT assay and apoptosis was evaluated using DAPI staining. Apoptosis signaling was assessed by immunoblot analysis.ResultsRUNX3 protein expression was frequently inactivated in the HCC cell lines (91%) and tissues (90%). RUNX3 expression inhibited 90 ± 8% of cell growth at 72 h in serum starved Hep3B cells. Forty-eight hour serum starvation-induced apoptosis and the percentage of apoptotic cells reached 31 ± 4% and 4 ± 1% in RUNX3-expressing Hep3B and control cells, respectively. Apoptotic activity was increased by Bim expression and caspase-3 and caspase-9 activation.ConclusionRUNX3 expression enhanced serum starvation-induced apoptosis in HCC cell lines. RUNX3 is deleted or weakly expressed in HCC, which leads to tumorigenesis by escaping apoptosis.
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