Yu-Lueng Shih

SOX1 functions as a tumor suppressor by antagonizing the WNT/β-catenin signaling pathway in hepatocellular carcinoma†‡§

Oncogenic activation of the Wnt/β‐catenin signaling pathway is common in hepatocellular carcinoma (HCC). Our recent studies have demonstrated that SRY (sex determining region Y)‐box 1 (SOX1) and secreted frizzled‐related proteins are concomitantly promoter‐hypermethylated, and this might lead to abnormal activation of the Wnt signaling pathway in HCC. SOX1 encodes a transcription factor involved in the regulation of embryonic development and cell fate determination. However, the expression and functional role of SOX1 in HCC remains unclear. In this study, we confirmed via quantitative methylation‐specific polymerase chain reaction that SOX1 was frequently downregulated through promoter hypermethylation in HCC cells and tissues. Overexpression of SOX1 by a constitutive or inducible approach could suppress cell proliferation, colony formation, and invasion ability in HCC cell lines, as well as tumor growth in nonobese diabetic/severe combined immunodeficiency mice. Conversely, knockdown of SOX1 by withdrawal of doxycycline could partially restore cell proliferation and colony formation in HCC cells. We used a T cell factor (TCF)‐responsive luciferase reporter assay and western blot analysis to prove that SOX1 could regulate TCF‐responsive transcriptional activity and inhibit the expression of Wnt downstream genes. Furthermore, we used glutathione S‐transferase pull‐down, co‐immunoprecipitation, and confocal microscopy to demonstrate that SOX1 could interact with β‐catenin but not with the β‐catenin/TCF complex. Moreover, restoration of the expression of SOX1 induces significant cellular senescence in Hep3B cells. Conclusion: Our data show that a developmental gene, SOX1, may function as a tumor suppressor by interfering with Wnt/β‐catenin signaling in the development of HCC. (HEPATOLOGY 2012;56:2142–2153)

Spontaneous intracranial hemorrhage in cirrhotic patients.

OBJECTIVE The major characteristics of spontaneous intracranial hemorrhage (SICH) in cirrhotic patients have not been completely defined. Cirrhotic patients with SICH were thus analyzed in an effort to better understand the risk factors for SICH and predict patient outcomes. PATIENTS AND METHODS From 1997 to 2006, 4515 hospitalized cirrhotic patients were recruited, with a focus on 36 cirrhotic patients with SICH who had no history of cerebral vascular accidents, head injuries, or cerebral arteriovenous malformations. The patient characteristics, severity of cirrhosis, location of the hematoma, and prognosis were analyzed. RESULTS Of the patients, 78% were males, 72% consumed alcohol, and 81% had a mild-to-moderate degree of cirrhosis. The overall incidence of SICH was related to the etiology of cirrhosis as follows: virus-related cirrhosis (0.3%), alcohol-related cirrhosis (1.9%), and combined virus- and alcohol-related cirrhosis (3%). The outcome of patients with SICH was associated with the size of the hematoma (P<0.005), the initial Glasgow Coma Scale score (P<0.05), the Child-Pugh classification (P=0.05), and the serum total bilirubin level (P<0.05). CONCLUSION SICH occurs primarily in young males with mild-to-moderate alcoholic cirrhosis of the liver. The etiology of cirrhosis is related to the incidence of SICH, but not to the patient outcome. The severity of liver cirrhosis is associated with patient outcome, but not the incidence of SICH.

Hepatitis C Virus Core Protein Down-Regulates p21Waf1/Cip1 and Inhibits Curcumin-Induced Apoptosis through MicroRNA-345 Targeting in Human Hepatoma Cells

Background Hepatitis C virus (HCV) has been reported to regulate cellular microRNAs. The HCV core protein is considered to be a potential oncoprotein in HCV-related hepatocellular carcinoma, but HCV core-modulated cellular microRNAs are unknown. The HCV core protein regulates p21Waf1/Cip1 expression. However, the mechanism of HCV core-associated p21Waf1/Cip1 regulation remains to be further clarified. Therefore, we attempted to determine whether HCV core-modulated cellular microRNAs play an important role in regulating p21Waf1/Cip1 expression in human hepatoma cells. Methods Cellular microRNA profiling was investigated in core-overexpressing hepatoma cells using TaqMan low density array. Array data were further confirmed by TaqMan real-time qPCR for single microRNA in core-overexpressing and full-length HCV replicon-expressing cells. The target gene of microRNA was examined by reporter assay. The gene expression was determined by real-time qPCR and Western blotting. Apoptosis was examined by annexin V-FITC apoptosis assay. Cell cycle analysis was performed by propidium iodide staining. Cell proliferation was analyzed by MTT assay. Results HCV core protein up- or down-regulated some cellular microRNAs in Huh7 cells. HCV core-induced microRNA-345 suppressed p21Waf1/Cip1 gene expression through targeting its 3′ untranslated region in human hepatoma cells. Moreover, the core protein inhibited curcumin-induced apoptosis through p21Waf1/Cip1-targeting microRNA-345 in Huh7 cells. Conclusion and Significance HCV core protein enhances the expression of microRNA-345 which then down-regulates p21Waf1/Cip1 expression. It is the first time that HCV core protein has ever been shown to suppress p21Waf1/Cip1 gene expression through miR-345 targeting.

Frequent concomitant epigenetic silencing of SOX1 and secreted frizzled-related proteins (SFRPs) in human hepatocellular carcinoma

Except for genetic mutations, epigenetic changes are also involved in the development of human cancers. Recently, we have identified SOX1, SRY (sex determining region Y)‐box 1, is hypermethylated in cervical cancer and ovarian cancer. Therefore, we investigated whether promoter hypermethylation of SOX1 is common in hepatocellular carcinoma (HCC).

SOX1 suppresses cell growth and invasion in cervical cancer

OBJECTIVE Abnormal activation of the Wnt/β-catenin signaling pathway is common in human cancers, including cervical cancer. Many papers have shown that SRY (sex-determining region Y)-box (SOX) family genes serve as either tumor suppressor genes (TSGs) or oncogenes by regulating the Wnt signaling pathway in different cancers. We have demonstrated recently that epigenetic silencing of SOX1 gene occurs frequently in cervical cancer. However, the possible role of SOX1 in cervical cancer remains unclear. This study aimed to explore whether SOX1 functions as a TSG in cervical cancer. METHODS We established a constitutive and an inducible system that overexpressed SOX1 and monitored its function by in vitro experiments. To confirm SOX1 function, we manipulated SOX1 using an inducible expression approach in cell lines. The effect of SOX1 on tumorigenesis was also analyzed in animal models. RESULTS Overexpression of SOX1 inhibited cell proliferation, anchorage independency, and invasion in vitro. SOX1 suppressed tumor growth in nonobese diabetic/severe combined immunodeficiency mice. After induction of SOX1 by doxycycline (DOX), SOX1 inhibited cell growth and invasion in the inducible system. Repression of SOX1 by withdrawal of DOX partially reversed the malignant phenotype in cervical cells. SOX1 inhibited TCF-dependent transcriptional activity and the Wnt target genes. SOX1 also repressed the invasive phenotype by regulating the expression of invasion-related genes. CONCLUSIONS Taken together, these data suggest that SOX1 can function as a tumor suppressor partly by interfering with Wnt/β-catenin signaling in cervical cancer.

Carbon dioxide insufflation does not reduce pain scores during colonoscope insertion in unsedated patients: a randomized, controlled trial.

BACKGROUND CO(2) is rapidly absorbed from the colon and eliminated via the lung. Insufflation of CO(2) instead of air during colonoscopy can reduce distention-induced pain. OBJECTIVE This study aimed to evaluate the effects of CO(2) insufflation on pain during intubation and extubation and to identify predictors of pain and discomfort during colonoscope insertion. DESIGN Prospective, randomized, controlled trial. SETTING Single tertiary medical center in Taiwan. PATIENTS A total of 193 patients enrolled from September 2010 through June 2011. INTERVENTIONS Colonoscope insertion with either air or CO(2) insufflation. CO(2) was used for extubation in both groups. MAIN OUTCOME MEASUREMENTS The main outcome measurement was pain, recorded on a 10-point visual analog scale (VAS) for left-sided colonoscope insertion and right-sided colonoscope insertion and at 1, 3, 6, and 24 hours post-procedure. Colonoscope cecal intubation time and extubation time, completeness of intubation, and loop formation were also assessed. RESULTS CO(2) insufflation during colonoscope intubation was used in 98 patients and air in 97 patients. The mean pain scores during intubation were low (2-3) for patients undergoing air insufflation and were not reduced further in patients receiving CO(2). A mean pain score of 0 was reported by both groups for all postprocedure time points. Multivariate analysis identified sex, loop formation of the sigmoid colon, time to reach the transverse colon, and requested sedation as factors that significantly affect VAS pain scores. LIMITATIONS This study was limited in scope to a single medical center with experienced endoscopists. CONCLUSIONS We detected no significant benefit to the use of CO(2) insufflation compared with air insufflation during intubation for colonoscopy performed by experienced colonoscopists. The absence of postprocedure pain in both groups supports previous observations that CO(2) insufflation during extubation is effective in reducing postprocedure pain. Female sex and loop formation were identified as key factors influencing pain scores on colonoscope insertion. ( CLINICAL TRIAL REGISTRATION NUMBER TSGHIRB-099-05-081.).

Modified mallampati classification as a clinical predictor of peroral esophagogastroduodenoscopy tolerance

BackgroundUnsedated esophagogastroduodenoscopy (EGD) is simpler and safer than sedated EGD; however, approximately 40% of patients cannot tolerate it. Early identification of patients likely to poorly tolerate unsedated EGD is valuable for improving compliance. The modified Mallampati classification (MMC) has been used to evaluate difficult tracheal intubation and laryngoscope insertion. We tried to assess the efficacy of MMC to predict the tolerance of EGD in unsedated patients.MethodsTwo hundred patients who underwent an unsedated diagnostic EGD were recruited. They were stratified according to the view of the oropharynx as either MMC class I + II (good view) or class III + IV (poor view). EGD tolerance was assessed in three ways: gag reflex by endoscopist assessment, patient satisfaction by interview, and the degree of change in vital signs.ResultsMMC was significantly correlated to gag reflex (P < 0.001), patient satisfaction (P = 0.028), and a change of vital signs (P = 0.024). Patients in the poor view group had a 3.87-fold increased risk of gag reflex (P < 0.001), a 1.78-fold increased risk of unsatisfaction (P = 0.067), and a 1.96-fold increased risk of a change in vital signs (P = 0.025) compared to those in the good view group.ConclusionsMMC appears to be a clinically useful predictor of EGD tolerance. Patients with poor view of oropharynx by MMC criteria may be candidates for sedated or transnasal EGD.

Isolated gastric variceal bleeding caused by splenic lymphoma-associated splenic vein occlusion

Isolated gastric varices (IGV) can occur in patients with left-sided portal hypertension resulting from splenic vein occlusion caused by thrombosis or stenosis. In left-sided portal hypertension, blood flows retrogradely through the short and posterior gastric veins and the gastroepiploic veins, leading to the formation of an IGV. The most common causes of splenic vein occlusion are pancreatic diseases, such as pancreatic cancer, pancreatitis, or a pseudocyst. However, various other cancers, such as colon, gastric, or renal cancers, have also been known to cause splenic vein occlusion. Our patient presented with a rare case of IGV bleeding induced by splenic lymphoma-associated splenic vein occlusion. Splenectomy, splenic artery embolization, and stenting of the splenic vein are the current treatment choices. Chemotherapy, however, is an alternative effective treatment for splenic vein occlusion caused by chemotherapy-sensitive tumors. Our patient responded well to chemotherapy with a cyclophosphamide, hydroxydaunorubicin, oncovin, and prednisolone regimen, and the splenic vein occlusion resolved after the lymphoma regressed.

The basal body gene, RPGRIP1L, is a candidate tumour suppressor gene in human hepatocellular carcinoma

Loss of heterozygosity (LOH) on chromosome 16q is one of the most frequent genetic alterations in hepatocellular carcinoma (HCC). Our previous data showed that the smallest common deleted region was between D16S415 and D16S419, encompassed approximately by a 0.75cM region on 16q12.2, suggesting that the putative tumour suppressor genes (TSGs) at this locus might be involved in the development of HCC. Of the four genes (CHD9, RBL2, AKTIP and RPGRIP1L) located in this region, only RPGRIP1L was downregulated in HCCs. Downregulation of RPGRIP1L was found in 91% (10/11) HCC cell lines and in 35% (14/40) HCCs, respectively. To investigate the role of RPGRIP1L in HCCs, we used the overexpression of RPGRIP1L in four HCC cell lines (HepG2, Huh6, Huh7 and Hep3B). Overexpression of RPGRIP1L suppressed colony formation of tumour cells. Conversely, expression of RPGRIP1LM (dominant negative form) in HCC cells enhanced colony formation. Furthermore, knockdown RPGRIP1L by RNA interference in SK-HepI cells promoted colony formation. Taken together, these data strongly suggest that RPGRIP1L might be the putative TSG in HCC. Moreover, we showed that Mad2, Survivin and Securin were elevated in RPGRIP1LM-HepG2 transfectants and RPGRIP1L-shRNA-SK-HepI transfectants. After knockdown of MAD2 in RPGRIP1L-shRNA-SK-HepI transfectants partly reverse cellular colony formation capability. These data suggest that RPGRIP1L suppresses anchorage-independent growth partly through the mitotic checkpoint protein Mad2.

The Influence of Pacifier Sucking on Mesenteric Blood Flow in Infants

Thirty-six neonates were randomly assigned to 2 groups of 18 neonates each. In the pacifier group, infants were given a pacifier before milk feeding. Newborns in the control group were fed without preprandial use of a pacifier. Doppler measurement of peak systolic velocity, enddiastolic velocity, and Pourcelot resistance index in the superior mesenteric artery was performed in both groups. Peak systolic and end-diastolic velocities were significantly increased after pacifier sucking in the preprandial stage. The resistance index decreased significantly after milk feeding but not after pacifier sucking. The feeding volume was not affected by preprandial pacifier sucking.