Yung-Yu Hsieh

Resistin-induced stromal cell-derived factor-1 expression through Toll-like receptor 4 and activation of p38 MAPK/ NFκB signaling pathway in gastric cancer cells

BackgroundStromal cell-derived factor-1 (SDF-1) (CXC chemokine ligand-12)/CXC chemokine receptor 4 (CXCR4) is involved in the carcinogenesis of human gastric cancer, where it stimulates angiogenesis and favors metastasis of tumor cells to distant organs. In addition, resistin is suggested to be an important link between obesity and the development of gastric cancer. Resistin has identified as an important player in inflammatory responses, and emerged as a mediator in inflammation-associated cancer. A limited number of studies have investigated the association of resistin and SDF-1 with gastric cancer. Herein, we investigated the molecular mechanisms by which resistin influences the expression of SDF-1 in gastric carcinoma cells.ResultsHuman gastric cancer cell lines were exposed to doses of resistin; SDF-1 expression and secretion levels were then determined. Real-time polymerase chain reaction and western blotting analyses were performed to clarify molecular changes. Inhibition of Toll-like receptor 4 (TLR4) by a competitive antagonist inhibited resistin-induced SDF-1 expression. Pharmacological inhibitors and small interfering RNA (siRNA) demonstrated that activation of the p38 mitogen-activated protein kinase (MAPK) pathway is critical for resistin-induced SDF-1 expression mediated by TLR4. The promoter activity and transcription factor enzyme-linked immunosorbent assay revealed that resistin induced expression of SDF-1 mediated by NF-κB in gastric cancer cells. Inhibition of p38 MARK activation blocked the SDF-1-induced expression and the SDF-1 promoter activity in the cancer gastric cells. Chromatin immunoprecipitation assay revealed that inhibition of p38 MARK activation also blocked the resistin-increased NF-κB-DNA-binding activity.ConclusionsResistin-induced SDF-1 upregulation by activation of TLR4, p38 MARK and NF-κB may explain a new role of resistin in the link of obesity and gastric cancer.

Routine blood tests to predict liver fibrosis in chronic hepatitis C.

AIM To verify the usefulness of FibroQ for predicting fibrosis in patients with chronic hepatitis C, compared with other noninvasive tests. METHODS This retrospective cohort study included 237 consecutive patients with chronic hepatitis C who had undergone percutaneous liver biopsy before treatment. FibroQ, aspartate aminotransferase (AST)/alanine aminotransferase ratio (AAR), AST to platelet ratio index, cirrhosis discriminant score, age-platelet index (API), Pohl score, FIB-4 index, and Loks model were calculated and compared. RESULTS FibroQ, FIB-4, AAR, API and Loks model results increased significantly as fibrosis advanced (analysis of variance test: P < 0.001). FibroQ trended to be superior in predicting significant fibrosis score in chronic hepatitis C compared with other noninvasive tests. CONCLUSION FibroQ is a simple and useful test for predicting significant fibrosis in patients with chronic hepatitis C.

Stromal cell‐derived factor‐1/CXC receptor 4 and β1 integrin interaction regulates urokinase‐type plasminogen activator expression in human colorectal cancer cells

The stromal cell‐derived factor‐1 (SDF‐1)/CXC receptor 4 (CXCR4) axis has been shown to play a role in colorectal cancer progression. In addition, the protease urokinase‐type plasminogen activator (uPA) is an important factor in tumor cell invasion and metastasis. However, the mechanism by which SDF‐1 mediates uPA expression in human colorectal cancer cells remains unknown. We investigated the molecular mechanism governing the interaction between SDF‐1 stimulation and uPA expression in three human colon cancer cell lines (DLD‐1, SW48, and COLO 205). We found that SDF‐1 stimulation led to an increase in the expression and secretion of uPA in these cells. Experiments involving specific inhibitors and small interfering RNA demonstrated that the activation of p38 mitogen‐activated protein kinase (MAPK) and phosphatidylinositol 3‐kinase (PI3K)/Akt pathways are critical for SDF‐1‐induced uPA expression. Analysis of transcription factor binding using ELISA and chromatin immunoprecipitation assays revealed that SDF‐1 increased Sp1‐ and AP‐1‐DNA‐binding activities in DLD‐1 cells. Inhibition of Sp1 and AP‐1 activation blocked the SDF‐1‐induced expression and activity of the uPA promoter. The effect of SDF‐1 on DLD‐1 signaling and uPA expression was mediated by the CXCR4/β1 integrin axis. In summary, our findings elucidate the mechanisms of SDF‐1/CXCR4 downstream signaling and provide insights into the function of SDF‐1 in colon cancer cells. J. Cell. Physiol. 227: 1114–1122, 2012.

Exploring the effects of tert-butylhydroperoxide induced liver injury using proteomic approach

Tert-butyl hydroperoxide (t-BHP), an organic lipid hydroperoxide analog, has been demonstrated to exert pro-oxidant effects to evaluate mechanisms involving oxidative stress in hepatocyte cells and rat liver. Herein, we present an investigation of the event of molecular mechanism of t-BHP related acute liver injury. A proteomic approach was used to identify proteins which are differentially expressed in liver cells following t-BHP treatment and the mechanism of its action in apoptotic and endoplasmic reticulum stress pathways. Our results demonstrate that the t-BHP treatment of liver cells increased cell cytoxicity and apoptosis. t-BHP dose-dependent induction of cell apoptosis and stained liver sections relieved the acute rat liver injury were accompanied by sustained phosphorylation of JNK1/2 and p65. In addition, there were 13 differentially displayed proteins between the t-BHP-induced and untreated were assayed and validated in vivo. Furthermore, we demonstrated that t-BHP induced human Chang liver cell viability and apoptosis properties by up-regulating the levels of ETFA (electron transfer flavoprotein subunit alpha). This study demonstrated that there was an increase in the cellular levels of ETFA in the t-BHP induction in viability and apoptosis via the activation of JNK1/2 and NFκB signaling modules. NAC administration and shRNA ETFA conferred resistance to t-BHP-increased ETFA and CHOP expression via IRE1-alpha/TRAF2 complex formation, activation of JNK1/2 and p50. We concluded that the mechanism of t-BHP-induced an apoptosis cascade and endoplasmic reticulum stress in hepatocyte cells by up-regulation of ETFA, providing a new mechanism for liver injury.

Increased Abundance of Clostridium and Fusobacterium in Gastric Microbiota of Patients with Gastric Cancer in Taiwan

Helicobacter pylori is recognised as a main risk factor for gastric cancer. However, approximately half of the patients with gastritis are negative for H. pylori infection, and the abundance of H. pylori decreases in patients with cancer. In the current study, we profiled gastric epithelium-associated bacterial species in patients with gastritis, intestinal metaplasia, and gastric cancer to identify additional potential pathogenic bacteria. The overall composition of the microbiota was similar between the patients with gastritis and those with intestinal metaplasia. H. pylori was present in half of the non-cancer group, and the dominant bacterial species in the H. pylori-negative patients were Burkholderia, Enterobacter, and Leclercia. The abundance of those bacteria was similar between the cancer and non-cancer groups, whereas the frequency and abundance of H. pylori were significantly lower in the cancer group. Instead, Clostridium, Fusobacterium, and Lactobacillus species were frequently abundant in patients with gastric cancer, demonstrating a gastric cancer-specific bacterial signature. A receiver operating characteristic curve analysis showed that Clostridium colicanis and Fusobacterium nucleatum exhibited a diagnostic ability for gastric cancer. Our findings indicate that the gastric microenvironment is frequently colonised by Clostridium and Fusobacterium in patients with gastric cancer.

The Association of CXC Receptor 4 Mediated Signaling Pathway with Oxaliplatin-Resistant Human Colorectal Cancer Cells.

The stromal cell–derived factor-1 (SDF-1)/CXC receptor 4 (CXCR4) axis plays an important role in tumor angiogenesis and invasiveness in colorectal cancer (CRC) progression. In addition, metastatic CRC remains one of the most difficult human malignancies to treat because of its chemoresistant behavior. However, the mechanism by which correlation occurs between CXCR4 and the clinical response of CRC to chemotherapy remains unknown. We generated chemoresistant cells with increasing doses of oxaliplatin (OXA) and 5-Fluorouracil (5FU) to develop resistance at a clinical dose. We found that the putative markers did not change in the parental cells, but HCT-116/OxR and HCT-116/5-FUR were more aggressive and had higher tumor growth (demonstrated by wound healing, chemotaxis assay, and a nude mice xenograft model) with the use of oxaliplatin. Apoptosis induced by oxaliplatin treatment was significantly decreased in HCT-116/OxR compared to the parental cells. Moreover, HCT-116/OxR cells displayed increased levels of p-gp, p-Akt p-ERK, p-IKBβ, CXCR4, and Bcl-2, but they also significantly inhibited the apoptotic pathways when compared to the parental strain. We evaluated the molecular mechanism governing the signaling pathway associated with anti-apoptosis activity and the aggressive status of chemoresistant cells. Experiments involving specific inhibitors demonstrated that the activation of the pathways associated with CXCR4, ERK1/2 mitogen-activated protein kinase (MAPK), and phosphatidylinositol 3-kinase (PI3K)/Akt is critical to the functioning of the HCT-116/OxR and HCT-116/5-FUR characteristics of chemosensitivity. These findings elucidate the mechanism of CXCR4/PI3K/Akt downstream signaling and provide strategies to inhibit CXCR4 mediated signaling pathway in order to overcome CRC’s resistance to chemotherapy.

A proteomics approach to identifying novel protein targets involved in erinacine A–mediated inhibition of colorectal cancer cells’ aggressiveness

Erinacine A, a major active component of a diterpenoid derivative isolated from Hericium erinaceus mycelium, has been demonstrated to exert anticancer effects. Herein, we present an investigation of the molecular mechanism of erinacine A induction associated with cancer cells’ aggressive status and death. A proteomic approach was used to purify and identify the differentially expressed proteins following erinacine A treatment and the mechanism of its action in apoptotic and the targets of erinacine A. Our results demonstrate that erinacine A treatment of HCT‐116 and DLD‐1 cells increased cell cytotoxicity and reactive oxygen species (ROS) production as well as decreased cell proliferation and invasiveness. Ten differentially displayed proteins were determined and validated in vitro and in vivo between the erinacine A‐treated and untreated groups. In addition, erinacine A time‐dependent induction of cell death and inhibitory invasiveness was associated with sustained phosphorylation of the PI3K/mTOR/p70S6K and ROCK1/LIMK2/Cofilin pathways. Furthermore, we demonstrated that erinacine A–induced HCT‐116 and DLD‐1 cells viability and anti‐invasion properties by up‐regulating the activation of PI3K/mTOR/p70S6K and production of ROS. Experiments involving specific inhibitors demonstrated that the differential expression of cofilin‐1 (COFL1) and profilin‐1 (PROF1) during erinacine A treatment could be involved in the mechanisms of HCT‐116 and DLD‐1 cells death and decreased aggressiveness, which occurred via ROCK1/LIMK2/Cofilin expression, with activation of the PI3K/mTOR/p70S6K signalling pathway. These findings elucidate the mechanism of erinacine A inhibiting the aggressive status of cells by activating PI3K/mTOR/p70S6K downstream signalling and the novel protein targets COF1 and PROF1; this could be a good molecular strategy to limit the aggressiveness of CRC cells.

Outcomes of endoscopic submucosal dissection for early gastric cancer and precancer lesion: Experience from a center in Southern Taiwan

Limited data are available on the interval of disease‐free status after endoscopic submucosal dissection (ESD) for early gastric cancer and precancer lesion in Taiwan. In this long‐term (2–105 months) follow‐up study, we analyzed the risk factors that affect the local recurrence and noncurative resection (non‐CR) of these lesions.

Comparative Proteomic Identification of Protein Disulphide Isomerase A6 Associated with Tert-Butylhydroperoxide-Induced Liver Injury in Rat Hepatocytes.

Background/Aims: Oxidants are important human toxicants. They have been implicated in the occurrence and development of liver diseases. Increased intracellular tert-butylhydroperoxide (t-BHP) may be critical for oxidant toxicity, and is commonly used for evaluating mechanisms involving oxidative stress, but the method remains controversial. Methods: Primary cultures of hepatocytes as well as human Hep G2 and mouse FL83B liver cells were obtained. Cell viability was measured by annexin V–FITC/propidium iodide and DAPI staining to determine the effects of t-BHP treatment on acute liver injury. A proteomic assay provided information that was used to identify the differentially expressed proteins following t-BHP treatment; immunohistochemistry and western blotting were performed to detect the expression of PDIA6 activity in apoptotic and endoplasmic reticulum (ER) stress pathways. Results: Our results demonstrate that t-BHP treatment of liver cells increased cell cytotoxicity and the generation of reactive oxygen species. This treatment also increased the level of PDIA6; this was validated in vitro and in vivo based on a comparison of t-BHP-treated and -untreated groups. Treatment of mouse liver FL83B cells with t-BHP activated caspase 3, increased the expression of apoptotic molecules, caused cytochrome c release, and induced Bcl-2, Bax and IRE1α/TRAF2 complex formation. t-BHP-dependent induction of apoptosis was accompanied by sustained phosphorylation of the IRE1α/ASK1/JNK1/2/p38 pathways and PDIA6 expression. Furthermore, t-BHP induced liver FL83B cell viability and apoptosis by upregulating the levels of PDIA6; this process could be involved in the activation of the IRE1α/ASK1/JNK1/2/p38 signalling pathways. Conclusions: We conclude that t-BHP induced an apoptosis cascade and ER stress in hepatocytes by upregulation of PDIA6, providing a new mechanism underlying the effects of t-BHP on liver injury.

Upregulation of bone morphogenetic protein 1 is associated with poor prognosis of late-stage gastric Cancer patients

BackgroundGastric cancer is the eighth most common cancer in Taiwan, with a 40% 5-year survival rate. Approximately 40% of patients are refractory to chemotherapy. Currently, the anti-HER2 therapy is the only clinically employed targeted therapy. However, only 7% patients in Taiwan are HER2-positive. Identifying candidate target genes will facilitate the development of adjuvant targeted therapy to increase the efficacy of gastric cancer treatment.MethodsClinical specimens were analyzed by targeted RNA sequencing to assess the expression levels of target genes. Statistical significance of differential expression and correlation between specimens was evaluated. The correlation with patient survival was analyzed as well. In vitro cell mobility was determined using wound-healing and transwell mobility assays.ResultsExpression of BMP1, COL1A1, STAT3, SOX2, FOXA2, and GATA6 was progressively dysregulated through the stages of gastric oncogenesis. The expression profile of these six genes forms an ubiquitously biomarker signature that is sufficient to differentiate cancer from non-cancerous specimens. High expression status of BMP1 correlates with poor long-term survival of late-stage patients. In vitro, suppression of BMP1 inhibits the mobility of the gastric cancer cell lines, indicating a role of BMP1 in metastasis.ConclusionsBMP1 is upregulated in gastric cancer and is correlated with poor patient survival. Suppression of BMP1 reduced gastric cancer mobility in vitro. Our finding suggests that anti-BMP1 therapy will likely augment the efficacy of standard chemotherapy and improve the treatment outcome.