Ming-Hong Tai

Propofol pretreatment attenuates LPS-induced granulocyte–macrophage colony-stimulating factor production in cultured hepatocytes by suppressing MAPK/ERK activity and NF-κB translocation

Propofol (PPF), a widely used intravenous anesthetic for induction and maintenance of anesthesia during surgeries, was found to possess suppressive effect on host immunity. This study aimed at investigating whether PPF plays a modulatory role in the lipopolysaccharide (LPS)-induced inflammatory cytokine expression in a cell line of rat hepatocytes. Morphological observation and viability assay showed that PPF exhibits no cytotoxicity at concentrations up to 300 microM after 48 h incubation. Pretreatment with 100 microM PPF for 24 h prior to LPS stimulation was performed to investigate the modulatory effect on LPS-induced inflammatory gene production. The results of semi-quantitative RT-PCR demonstrated that PPF pretreatment significantly suppressed the LPS-induced toll-like receptor (TLR)-4, CD14, tumor necrosis factor (TNF)-alpha, and granulocyte-macrophage colony-stimulating factor (GM-CSF) gene expression. Western blotting analysis showed that PPF pretreatment potentiated the LPS-induced TLR-4 downregulation. Flow cytometrical analysis revealed that PPF pretreatment showed no modulatory effect on the LPS-upregulated CD14 expression on hepatocytes. In addition, PPF pretreatment attenuated the phosphorylation of mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) and IkappaBalpha, as well as the nuclear translocation of NF-kappaB primed by LPS. Moreover, addition of PD98059, a MAPK kinase inhibitor, significantly suppressed the LPS-induced NF-kappaB nuclear translocation and GM-CSF production, suggesting that the PPF-attenuated GM-CSF production in hepatocytes may be attributed to its suppressive effect on MAPK/ERK signaling pathway. In conclusion, PPF as an anesthetic may clinically benefit those patients who are vulnerable to sepsis by alleviating sepsis-related inflammatory response in livers.

Hepatoma-derived growth factor is a novel prognostic factor for gastrointestinal stromal tumors

Proliferating activity as mitotic count is generally accepted as a major prognostic indicator for gastrointestinal stromal tumors (GISTs). Hepatoma‐derived growth factor (HDGF) is a novel growth factor and elevated in several types of cancer. Our study was designed to elucidate the expression and prognostic role of HDGF in GISTs. A total 178 surgically resected CD117‐positive GISTs specimens were collected for immunohistochemical analysis using antibodies against HDGF. The immunoreactivities were scored as labeling index (LI) and correlated with clinicopathologic parameters of GIST patients. The HDGF immunoreactivities were detected in both nucleus and cytoplasm of GISTs tissues. Besides, the nuclear and cytoplasmic HDGF was parallely upregulated in GISTs (p < 0.001). The nuclear HDGF LI were positively correlated with that of PCNA (p < 0.001) and Ki‐67 (p < 0.001), tumor mitosis (p < 0.001), tumor sizes (p = 0.007) and NIH risk categories (p < 0.001). In addition, the cytoplasmic HDGF LI were also positively correlated with that of PCNA (p = 0.031) and Ki‐67 (p = 0.038), tumor sizes (p = 0.003) and tumor mitosis (p = 0.015). Patients with higher HDGF levels had earlier tumor recurrence and unfavorable outcome (p < 0.05). In addition to standard prognostic factors (NIH risk categories), the nuclear HDGF LI is an independent prognostic factor for disease free and overall survivals of GIST patients after operation. We conclude that HDGF is a novel prognostic factor for GIST patients.

Electroporation for direct spinal gene transfer in rats

We investigated the feasibility of delivering exogenous genes into spinal cord using direct in vivo electrotransfection. Gene transfer to the spinal cord was accomplished via direct intrathecal injection of pE-GFP C1 vector, followed by five electric pulses for 50 ms at 200 V delivered intrathecally. The spinal cords were retrieved and analyzed with fluorescence microscopy, reverse transcription polymerase chain reaction (RT-PCR), and Western blotting. At day 1, 3 or 7 following electroporation a clear GFP expression in spinal cord tissue was detected. The most prominent transfection occurred in the meningeal cells and superficial layer of the spinal cord. Successful transfection was also confirmed with RT-PCR and Western blotting. The expression of GFP protein was peaked between 3 and 7 days after electroporation and significantly decreased at 14 days. No behavioral or spinal neurodegenerative changes were detected at any time point. This study demonstrates that direct in vivo electrotransfection represents an effective and simple method for spinal gene delivery and have a potential to be used clinically, especially, acute or chronic pain.

Overexpression of VEGF is associated with positive p53 immunostaining in hepatocellular carcinoma (HCC) and adverse outcome of HCC patients.

To elucidate the clinicopathological correlations among vascular endothelial growth factor (VEGF), microvessel density (MVD) and tumor suppressor gene p53 in hepatocellular carcinomas (HCCs), we adopted a new definition of “VEGF overexpression.”

Hepatoma-derived growth factor regulates breast cancer cell invasion by modulating epithelial--mesenchymal transition.

Hepatoma‐derived growth factor (HDGF) participates in tumourigenesis but its role in breast cancer is unclear. We set out to elucidate the expression profile and function of HDGF during breast carcinogenesis. Immunoblot and immunohistochemical studies revealed elevated HDGF expression in human breast cancer cell lines and tissues. Nuclear HDGF labelling index was positively correlated with tumour grade, stage and proliferation index, but negatively correlated with survival rate in breast cancer patients. HDGF over‐expression was associated with lymph node metastasis and represented an independent prognostic factor for tumour recurrence. Gene transfer studies were performed to elucidate the influence of cellular HDGF level on the malignant behaviour and epithelial‐mesenchymal transition (EMT) of breast cancer cells. Adenovirus‐mediated HDGF over‐expression stimulated the invasiveness and colony formation of MCF‐7 cells. Moreover, HDGF over‐expression promoted breast cancer cell EMT by E‐cadherin down‐regulation and vimentin up‐regulation. Conversely, HDGF knockdown by RNA interference in MDA‐MB‐231 cells attenuated the malignant behaviour and elicited EMT reversal by enhancing E‐cadherin expression while depleting vimentin expression. Because HDGF is a secreted protein, we evaluated the cellular function of recombinant HDGF and found that exogenously supplied HDGF enhanced the invasiveness of breast cancer cells by down‐regulating E‐cadherin and up‐regulating vimentin at transcriptional and translational levels. In contrast, blockade of HDGF secretion with an HDGF antibody inhibited the malignant behaviours and EMT. Finally, exogenous HDGF partially reversed benzyl isothiocyanate (BITC)‐induced EMT suppression. HDGF over‐expression may exert a prognostic role for tumour metastasis and recurrence in breast cancer by modulating EMT. Copyright

Amelioration of collagen-induced arthritis in rats by adenovirus-mediated PTEN gene transfer.

OBJECTIVE The phosphatidylinositol 3-kinase (PI 3-kinase)/Akt pathway is known to be activated in rheumatoid arthritis (RA) synovial tissue, which impacts cell growth, proliferation, survival, and migration. Phosphatase and tensin homolog deleted from chromosome 10 (PTEN) functions as a negative regulator of PI 3-kinase signaling, thus blocking Akt activation. The aim of this study was to examine the effect of PTEN gene transfer in rats with collagen-induced arthritis (CIA). METHODS Adenoviral vectors encoding human PTEN (AdPTEN) or beta-galactosidase (AdLacZ) were injected intraarticularly into rats with CIA, and their treatment responses were monitored by measures of clinical, radiographic, and histologic changes. The expression of phosphorylated Akt, total Akt, vascular endothelial growth factor (VEGF), proinflammatory cytokines, and chemokines, as well as the extent of microvessel density in the ankle joints were determined. RESULTS AdPTEN treatment reduced Akt phosphorylation and decreased VEGF production in human RA synovial fibroblasts. Compared with AdLacZ treatment of the rats with CIA, AdPTEN treatment significantly reduced ankle circumference, articular index scores, radiography scores, and histology scores, and also decreased microvessel density and levels of VEGF and interleukin-1beta. Furthermore, PTEN gene transfer led to down-regulation of Akt activation and increased apoptosis in the ankle joints. CONCLUSION This study is the first to demonstrate the in vivo effect of intraarticular gene delivery of PTEN on amelioration of arthritis symptoms in rats with CIA, which involved antiangiogenic, antiproliferative, and antiinflammatory effects of PTEN via inhibition of the PI 3-kinase/Akt signaling pathway. Our findings also implicate the PI 3-kinase/Akt pathway as a therapeutic target for the treatment of RA or other inflammatory diseases.

Matrilysin (MMP-7) is a major matrix metalloproteinase upregulated in biliary atresia-associated liver fibrosis

Matrix metalloproteinases (MMPs) are the proteases responsible for tissue remodeling during liver fibrosis caused by various disorders including biliary atresia. However, information regarding the relative contribution of these proteases to liver fibrosis is still limited. We studied matrix metalloproteinase-2 (MMP-2), -7, -9 and -13 mRNA expressions in the liver tissue of early-stage biliary atresia at the time of Kasais procedure, late-stage biliary atresia at the time of liver transplantation with advanced fibrosis and nondiseased control without liver fibrosis. The results of real-time quantitative reverse transcriptase-PCR analysis revealed that only MMP-2 and -7 expressions were significantly different between groups. MMP-2 was significantly higher in Liver Transplantation group than both in Control (P=0.010) and in Kasais Procedure (P=0.001) groups, whereas the difference of MMP-2 expression between Control and Kasais Procedure was not significant. However, the relative expression level of MMP-7 was sequentially elevated when comparing Control, Kasais Procedure and Liver Transplantation groups, and there was significant (P=0.019) difference when comparing Control and Liver Transplantation groups. Moreover, the fold difference in MMP-7 mRNA was much higher than that in MMP-2 mRNA between groups. The expressions of MMP-7 were further confirmed by agarose gel electrophoresis and Western blotting. Immunohistochemical analysis revealed a significant positive correlation of the scores of MMP-7 immunostaining with the stages of liver fibrosis. In situ hybridization demonstrated that the bile ductular epithelial cells, Kupffer cells and hepatocytes were the major producers of matrix metalloproteinase-7 in the liver. Our results imply that MMP-7 is a major MMP associated with the tissue remodeling during the progression of liver fibrosis in biliary atresia.

The Expression and Prognostic Role of Hepatoma-Derived Growth Factor in Colorectal Stromal Tumors

PURPOSE: This study investigated the expression and prognostic role of hepatoma-derived growth factor (HDGF) in colorectal stromal tumor. METHODS: Fifty-two surgically resected colorectal stromal tumors were collected from 1986 to 2006. Immunohistochemical studies were performed with antibodies of HDGF, proliferating cell nuclear antigen (PCNA) and Ki-67. RESULTS: Sixteen patients (30.7 percent) had positive Ki-67 immunostaining. Immunoreactivity to PCNA ranged from 10 to 93 percent. HDGF immunostaining was found in the nucleus (20-95 percent) as well as in the cytoplasm (weak: 16; intermediate: 17; strong: 19). Upregulation of nuclear HDGF levels existed in increased cytoplasmic HDGF levels (P < 0.001). Nuclear HDGF levels were positively correlated with tumor mitotic count (P < 0.001), tumor size (P = 0.002), PCNA (P < 0.001), Ki-67 (P = 0.049), cellular pleomorphism (P = 0.029), and increased National Institutes of Health risk level (P = 0.037). Cytoplasmic HDGF levels were also correlated with PCNA (P = 0.001), tumor mitotic count (P = 0.001), high cellular pleomorphism (P = 0.001), and increased NIH risk (P = 0.043). Kaplan-Meier analyses revealed that patients with high nuclear HDGF (P < 0.001) or cytoplasmic levels (P = 0.001) had shorter disease-free survival than patients with low HDGF levels. Like tumor mitotic count, nuclear HDGF was an independent prognostic factor for patients with colorectal stromal tumors. CONCLUSIONS: This study provides clinicopathologic correlations and prognostic prediction of HDGF expression for the relatively rare colorectal stromal tumors.

Upregulation of hepatoma-derived growth factor is involved in murine hepatic fibrogenesis

BACKGROUND & AIMS Hepatoma-derived growth factor (HDGF) expression is correlated with progression of hepatocellular carcinoma. Since liver fibrosis frequently occurs before hepatoma development, this study investigated the expression profile of HDGF and its relationship with transforming growth factor-beta (TGF-beta) signaling in experimental models of hepatofibrogenesis. METHODS Liver fibrosis was induced in mice receiving bile duct ligation (BDL) or carbon tetrachloride (CCl(4)) administration. The expression levels of HDGF and other fibrosis-related markers were measured using quantitative RT-PCR, Western blotting, and enzyme-linked immunosorbent assays. Hepatic HDGF overexpression was achieved by adenovirus gene delivery. Rat hepatocytes were used to study the interplay between HDGF and TGF-beta1. RESULTS In both liver fibrosis models, HDGF de novo synthesis significantly increased during the progression of fibrosis. The HDGF upregulation was observed mainly in hepatocytes and correlated with the expression of TGF-beta1 and collagen COL1A1 and COL1A2 proteins. Hepatic HDGF overexpression itself deteriorated hepatocellular structure and integrity, and aggravated the extents of BDL- and CCl(4)-induced liver fibrosis with concomitant upregulation of TGF-beta1 and COL1A1. Exogenous TGF-beta1 stimulated HDGF expression only in cultured primary hepatocytes grown on collagen matrix, whereas exogenous HDGF also increased TGF-beta1 production in hepatocytes in a collagen-dependent manner. Moreover, HDGF enhanced Smad2 phosphorylation dose-dependently and the TGF-beta1-driven luciferase activities. CONCLUSION HDGF plays a pro-fibrogenic role during liver fibrosis in mice through activation of TGF-beta pathway. The mutual regulation between TGF-beta1 and HDGF may facilitate a vicious cycle to promote the progression of hepatic fibrogenesis.

Tumorigenesis and prognostic role of hepatoma-derived growth factor in human gliomas

Hepatoma-derived growth factor (HDGF) is a neurotrophic factor found in mouse spinal cord and hippocampal neurons. In various malignant tumors, the role of HDGF in tumor progression and its use as a diagnostic biomarker or therapeutic target have been extensively explored. However, the prognostic function and mitogenic role of HDGF in gliomagenesis are yet to be verified. In this study, we found a significant incidence of HDGF prevalence between the different pathological types and stages of glioma in 105 patients. We also found a prognostic significance in 41 glioblastoma multiforme (GBM) patients, with prevalence of nuclear HDGF predicting short survival of patients with GBM after surgery. To delineate the mitogenic role of HDGF in gliomagenesis, an adenoviral-expressed HDGF small interfering RNA (Ad-HDGF siRNA) was used to knock down expression of nuclear HDGF. After knocking down nuclear HDGF expression in human GBM cells, cell growth and cell invasion and induction on apoptosis by caspase-3 activation were significantly inhibited. We conclude that HDGF is a mitogenic growth factor in glioma progression and can be a useful prognostic marker for GBM and therapeutic target for clinical management of glioma in the future.