Downregulation of discoidin domain receptor 2 decreases tumor growth of hepatocellular carcinoma.

PURPOSE: Discoidin domain receptors (DDRs) have been identified as tyrosine kinase receptors for collagen, and the overexpression of DDR1 was correlated with hepatocellular carcinoma (HCC) progression in vitro. Little is known about DDR2 on HCC cells, and we investigated the expression and function of DDR2 in human HCC cells. METHODS: Expression of DDR2 in human HCC cell lines and patient HCC tissues was observed. The suppression of DDR2 by siRNA against DDR2 was performed in vitro and in vivo study. RESULTS: All of HCC cell lines expressed DDR2 mRNA, and all HCC tissues from the ten patients with HCC demonstrated DDR2 mRNA expression. Transfection of DDR2 siRNA significantly inhibits cell growth compared to cells with nontarget siRNA transfection in vitro (P < 0.001). In SNU182, Hep3B, and HeLa cell xenograft models, there was a significant difference in average tumor volumes after 12 days of the DDR2 siRNA injection (P < 0.05) in SNU182 xenograft mice. DDR2 siRNA injection decreased the mean tumor volume by 65.6 % compared to that of the control. The apoptosis analysis demonstrated that DDR2 siRNA treatment significantly increased apoptotic cells (P < 0.01). Cell migration (P < 0.05) and cell invasion (P < 0.01) were significantly decreased by DDR2 siRNA treatment. CONCLUSIONS: The inhibition of DDR2 by RNA interference suppressed in vivo and in vitro growth of human HCC cells. Our results may support that the use of DDR2 as a novel target of HCC treatment through control of tumor apoptosis, migration, and invasion.

Dual action of a selective cyclooxygenase-2 inhibitor on vascular endothelial growth factor expression in human hepatocellular carcinoma cells: novel involvement of discoidin domain receptor 2.

PURPOSE: Vascular endothelial growth factor (VEGF) greatly contributes to the progression of hepatocellular carcinoma (HCC). It is reported that a selective cyclooxygenase-2 (COX-2) inhibitor inhibits cellular proliferation and may attenuate VEGF expression in HCC. We propose that different cascades in the VEGF pathway respond to COX-2 inhibition, depending on the cell types. METHODS: The six human HCC cell lines--Hep3B, SNU387, SNU182, SNU423, SNU449, and PLC/PRF5--were cultured under normoxic and hypoxic conditions. Cells were treated with a selective COX-2 inhibitor (NS-398) and discoidin domain receptor 2 (DDR2) siRNA, and microarray analysis was performed. RESULTS: NS-398 inhibited HCC proliferation and decreased the expression level of VEGF in HCC cells only under normoxia conditions. In hypoxia conditions, VEGF expression level in Hep3B cell was suppressed, while that in SNU387 cell was increased by NS-398 (P < 0.001). The NS-398-induced increase in VEGF expression in SNU387 cell was associated with the up-regulation of the DDR2 gene. NS-398-treated SNU series cells and PLC/PRF5 cells displayed a robust increase in DDR2 mRNA expression. Also, transfection with DDR2 siRNA decreased the VEGF expression level of SNU387, 423, 449 cells under hypoxia conditions (P < 0.05). In vivo chromatin immunoprecipitation assay demonstrated that NS-398 induces the enhancement of HIF-1α binding on VEGF promoter, leading to the increase in VEGF gene expression in hypoxic conditions. There is strong evidence that it is related to the DDR2 gene expression in SNU387 cells. CONCLUSION: These findings disclose a novel cell-dependent regulatory mechanism of VEGF involving DDR2 gene in HCC cells.

Alpha-fetoprotein and human telomerase reverse transcriptase mRNA levels in peripheral blood of patients with hepatocellular carcinoma.

PURPOSE: Tumor-associated gene expression in peripheral blood reflects the presence of circulating hepatocellular carcinoma (HCC) cells and might be associated with aggressive features of HCC. We assessed the prognostic significance of alpha-fetoprotein (AFP) and human telomerase reverse transcriptase protein (hTERT) mRNA expression in the peripheral blood of HCC patients. METHODS: About 343 HCC patients, treated at the National Cancer Center, Korea, were included in this study. We measured AFP and hTERT mRNA levels in peripheral blood using quantitative real-time reverse transcription polymerase chain reaction. The association between the expression of each gene and survival was analyzed. RESULTS: AFP and hTERT mRNA were detected in 204 (59.5%) and 48 (14.0%) patients with HCC, respectively. The mean AFP copy number was 203 copies/microL (range 0-19992 copies/microL) and that of hTERT was 26 copies/microL (0-1711 copies/microL). AFP mRNA expression was correlated with the number of tumors (P = 0.05), but there were no significant association between hTERT expression and clinical features. There was also no relationship between overall survival and AFP (P = 0.08) or hTERT (P = 0.67) mRNA expression. CONCLUSIONS: This is the first report to evaluate the association between peripheral blood hTERT mRNA expression, and survival of HCC patients. The results indicate that AFP and hTERT mRNA expression in peripheral blood may not be useful HCC prognostic markers.

TTF-1 regulates growth hormone and prolactin transcription in the anterior pituitary gland.

Thyroid transcription factor 1 (TTF-1) is required for morphogenesis of the fetal diencephalon. Previous reports showed that mice carrying a TTF-1 null mutation lacked normal development of the pituitary gland. In this study, a role for TTF-1 in the regulation of growth hormone and prolactin transcription was identified. In-situ hybridization analysis demonstrated TTF-1 mRNA in the growth hormone-producing cells and prolactin-producing cells of the rat anterior pituitary gland. In the GH3 pituitary cell line, we identified TTF-1 as a factor functionally regulating growth hormone and prolactin transcription. TTF-1 activated prolactin transcription, but inhibited growth hormone transcription. Inhibition and activation of growth hormone and prolactin transcription, respectively, by TTF-1 disappeared upon deletion of the TTF-1 binding motifs within the promoters of these genes. These data suggest that TTF-1 plays a regulatory role in the transcription of growth hormone and prolactin genes and may regulate transdifferentiation of cells expressing these two hormones.