George Ou

Trichostatin A and sirtinol suppressed survivin expression through AMPK and p38MAPK in HT29 colon cancer cells

BACKGROUND Elevated levels of survivin and histone deacetylases (HDACs) are often found over-expressed in human cancers, including colorectal cancer, and have been implicated in tumorigenesis. HDAC inhibition induces growth arrest and cell death in various transformed cell; however, the mechanisms by which this reduces cell viability in colorectal cancer cells remain unexplained. METHODS We explored the actions of two HDAC inhibitors, trichostatin A (TSA) and sirtinol, in HT29 colon cancer cells. RESULTS TSA and sirtinol induced apoptosis and inhibited cell proliferation in HT29 cells. These results are associated with the modulation of survivin. Survivin promoter luciferase activity and Sp1, a transcription factor that contributes to survivin expression, were suppressed in cells exposed to TSA or sirtinol. TSA and sirtinol also activated p38 mitogen-activated protein kinase (p38MAPK) and AMP-activated protein kinase (AMPK). Inhibitors of p38MAPK or AMPK signaling abrogated TSA and sirtinols effects of decreasing cell viability. Survivin promoter luciferase activity in the presence of TSA or sirtinol was restored by AMPK dominant negative mutant or p38MAPK inhibitor. Furthermore, Sp1 binding to the survivin promoter region decreased while p63 binding to the promoter region increased after TSA or sirtinol exposure. CONCLUSIONS We report a p38MAPK- and AMPK-mediated downregulation of survivin, and its functional correlation with decreased colon cancer cell viability in the presence of HDAC inhibitor. p63 and Sp1 may also contribute to TSA and sirtinol actions. GENERAL SIGNIFICANCE This study delineates, in part, the underlying mechanisms of TSA and sirtinol in decreasing survivin expression and subsequent colon cancer cell viability.

Factors Associated With Positive Findings From Capsule Endoscopy in Patients With Obscure Gastrointestinal Bleeding

BACKGROUND & AIMS Capsule endoscopy (CE) is used most frequently to identify causes of obscure gastrointestinal bleeding (OGIB). Identifying factors associated with the detection of lesions by CE could improve resource utilization and thereby improve patient selection for CE examination. We sought to identify clinical factors associated with positive findings from CE in patients with OGIB. METHODS We analyzed data from 698 CE procedures performed between December 2001 and April 2011 at St Pauls Hospital, Vancouver, Canada (50.3% of patients were female; mean age, 63.4 years). A positive finding was defined as a lesion that was believed to be the source of the bleeding (ulceration, mass lesion, vascular lesion, or visible blood). Univariate and multivariate logistic regression analyses were used to correlate demographic and clinical parameters with positive findings. RESULTS A lesion believed to be the cause of bleeding was identified in 42% of cases. In univariate analysis, the number of esophagogastroduodenoscopies (EGDs), the presence of connective tissue disease or diabetes with end-organ damage, Charlson comorbidity index scores, and increasing transfusion requirements were significantly associated with identification of causative pathology from CE (all P < .027). In multivariate analysis, increasing number of EGDs (odds ratio [OR], 1.17; 95% confidence interval [CI], 1.00-1.37), increasing transfusion requirements (3-9 units: OR, 1.70; 95% CI, 1.08-2.66, and ≥10 units: OR, 2.72; 95% CI, 1.69-4.37), and connective tissue disease (OR, 2.24; 95% CI, 1.14-4.41) were all significantly associated with identification of positive findings by using CE (all P < .045). CONCLUSIONS Patients with a higher number of precapsule EGDs or transfusions, or connective tissue disease, are superior candidates for analysis of OGIB by CE.

A randomized controlled trial assessing the effect of prescribed patient position changes during colonoscope withdrawal on adenoma detection

BACKGROUND High-quality colonoscope withdrawal technique is associated with a higher adenoma detection rate. Position change is routinely used in barium enema and CT colonography to facilitate adequate distension of the colon and promote movement of fluid from the segment of the colon being assessed. OBJECTIVE To determine whether prescribed position changes during colonoscope withdrawal affect the adenoma detection rate compared with the usual care per endoscopist. DESIGN Prospective, randomized, controlled trial. SETTING Tertiary-care, university-affiliated hospital. PATIENTS Patients referred for outpatient colonoscopy between July 2011 and July 2012 were evaluated for eligibility. Inclusion criteria were outpatient status and age ≥40 years. Exclusion criteria were (1) complete colonoscopy within 1 year before the procedure, (2) inability to provide informed consent, (3) incomplete colonoscopy to the cecum, (4) previous bowel resection, (5) inflammatory bowel disease, (6) colonic polyposis syndrome, (7) inadequate bowel preparation, and (8) musculoskeletal disorder or other mobility issues limiting effective patient position changes during colonoscopy. INTERVENTIONS Prescribed position changes during colonoscope withdrawal. MAIN OUTCOME MEASUREMENTS Polyp detection rate (PDR) and adenoma detection rate (ADR). RESULTS A total of 776 patients were enrolled, with 388 in the dynamic group. There was no difference in PDR (odds ratio [OR] 0.99; P = .93) or ADR (OR 1.17; P = .28). Colonoscope withdrawal time was longer in the dynamic group (median time 466.5 vs 422.5 seconds; P < .0001). LIMITATIONS Single-center study. Indication for procedure not controlled. Lack of standardized bowel preparation and blinding. CONCLUSION Prescribed position changes during colonoscope withdrawal do not affect polyp/adenoma detection compared with the usual practice when the baseline ADR is above the recommended standard. ( CLINICAL TRIAL REGISTRATION NUMBER NCT01395173.).

Simvastatin induced HCT116 colorectal cancer cell apoptosis through p38MAPK-p53-survivin signaling cascade

BACKGROUND Statins, the 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors with cholesterol-lowering properties, were recently shown to exhibit anti-cancer effects. However, the molecular mechanism underlying statin-induced cancer cell death remains to be elucidated. Elevated level of survivin is often found over-expressed in human cancers and has been implicated in the progression of tumorigenesis. Given its central role in cell division and action as an apoptosis suppressor, survivin represents a potential molecular target in cancer management. METHODS In this study, we explored the underlying mechanisms in simvastatin-induced HCT116 colorectal cancer cell apoptosis. RESULTS Simvastatin decreased cell viability and induced cell apoptosis in HCT116 cells. These results are associated with the modulation of p21(cip/Waf1) and survivin. Survivin knockdown using survivin siRNAs also decreased cell viability and induced cell apoptosis. Simvastatins actions on p21(cip/Waf1), survivin and apoptosis were reduced in p53 null HCT116 cells. Simvastatin caused an increase in p53 phosphorylation and acetylation. In addition, simvastatin activated p38 mitogen-activated protein kinase (p38MAPK), whereas an inhibitor of p38MAPK signaling abrogated simvastatins effects of increasing p53 and p21(cip/Waf1) promoter luciferase activity. Cell viability and survivin promoter luciferase activity in the presence of simvastatin were also restored by p38MAPK inhibitor. Furthermore, Sp1 binding to the survivin promoter region decreased while p53 and p63 binding to the promoter region increased after simvastatin exposure. CONCLUSIONS Simvastatin activates the p38MAPK-p53-survivin cascade to cause HCT116 colorectal cancer cell apoptosis. GENERAL SIGNIFICANCE This study delineates, in part, the underlying mechanisms of simvastatin in decreasing survivin and subsequent colorectal cancer cell apoptosis.

Valproic acid suppresses lipopolysaccharide-induced cyclooxygenase-2 expression via MKP-1 in murine brain microvascular endothelial cells

Inflammation and vascular perturbations are increasingly implicated in the pathogenesis of neurodegenerative diseases. Prevailing evidence suggests that valproic acid (VPA), an antiepileptic and mood stabilizer, exhibits not only neuro-protective effects, but also anti-inflammatory effects in neurodegenerative diseases. However, the underlying mechanism contributing to VPAs suppression of inflammatory responses remains unclear. In this study, we explored the inhibitory action of VPA on cyclooxygenase (COX)-2 expression in bEnd.3 mouse brain microvascular endothelial cells exposed to lipopolysaccharide (LPS), a pro-inflammatory stimulus. The LPS-induced increases in COX-2 protein level and COX-2 promoter-luciferase activity were significantly suppressed by VPA. VPA inhibited p38MAPK and JNK phosphorylation in LPS-stimulated bEnd.3 cells. Treatment of cells with a p38MAPK inhibitor (p38MAPK inhibitor III) or a JNK signaling inhibitor (JNK inhibitor II) significantly inhibited LPS-induced COX-2 expression. VPA inhibited LPS-induced NF-κB subunit p65 phosphorylation and κB-luciferase activity. LPS-increased p65 and C/EBPβ binding to the COX-2 promoter region was attenuated in the presence of VPA. In addition, VPA suppression of p38MAPK, JNK and p65 phosphorylation, and subsequent COX-2 expression was restored in cells transfected with mitogen-activated protein kinase phosphatase-1 (MKP-1) dominant negative (DN) mutant. VPA also caused increases in MKP-1 acetylation and MKP-1 phosphatase activity in bEnd.3 cells. In conclusion, VPA may cause MKP-1 activation to dephosphorylate p38MAPK and JNK, leading to decrease in p65 and C/EBPβ binding to the COX-2 promoter region and COX-2 down-regulation in LPS-stimulated bEnd.3 cells. The present study therefore supports the therapeutic value of VPA in alleviating brain inflammatory processes.

Establishing the learning curve for achieving competency in performing colonoscopy: a systematic review.

Colonoscopy (CSPY) allows competent endoscopists to safely, tolerably, and accurately examine the entire colon, thus facilitating the diagnosis of colonic diseases as well as the performance of therapeutic interventions. It can be technically demanding and requires significant time and practice to master the psychomotor and cognitive aspects of the procedure. Therefore, with a significant number of graduating gastroenterology and surgical trainees expected to perform this procedure as a core component of their future practice, the need for appropriate training to allow for the acquisition of competence is critical. With this in mind, 2 questions arise: (1) what is procedural competence in this setting and (2) at what point do trainees become competent? Unfortunately, even though CSPY allows for objective assessment, the definition of competence remains difficult to delineate. Arguably the most frequently referenced CSPY performance marker is the cecal intubation rate (CIR). Because cecal intubation is a critical component to defining a complete CSPY, it is often reported as a prerequisite for determining competence. The American Society for Gastrointestinal Endoscopy (ASGE) in conjunction with the American College of Gastroenterology (ACG) published quality indicators to help define competence. These, alongside other recommendations, suggest at least a 90% CIR in all cases. However, cecal intubation is only 1 component of a complete CSPY and alone does not sufficiently define competency. Regarding the number of procedures required to become competent, the first guidelines were based on expert opinion. Subsequently, with the emergence of a pivotal study by Cass et al, the ASGE recommended that trainees complete a minimum of 140 CSPYs before competence can be assessed. However, both references above are now relatively outdated, and as additional studies

Effect of longer battery life on small bowel capsule endoscopy

AIM To determine if longer battery life improves capsule endoscopy (CE) completion rates. METHODS A retrospective study was performed at a tertiary, university-affiliated hospital in Vancouver, Canada. Patients who underwent CE with either PillCam™ SB2 or SB2U between 01/2010 and 12/2013 were considered for inclusion. SB2 and SB2U share identical physical dimensions but differ in their battery lives (8 h vs 12 h). Exclusion criteria included history of gastric or small bowel surgery, endoscopic placement of CE, interrupted view of major landmarks due to technical difficulty or significant amount of debris, and repeat CE using same system. Basic demographics, comorbidities, medications, baseline bowel habits, and previous surgeries were reviewed. Timing of major landmarks in CE were recorded, and used to calculate gastric transit time, small bowel transit time, and total recording time. A complete CE study was defined as visualization of cecum. Transit times and completion rates were compared. RESULTS Four hundred and eight patients, including 208 (51.0%) males, were included for analysis. The mean age was 55.5 ± 19.3 years. The most common indication for CE was gastrointestinal bleeding (n = 254, 62.3%), followed by inflammatory bowel disease (n = 86, 21.1%). There was no difference in gastric transit times (group difference 0.90, 95%CI: 0.72-1.13, P = 0.352) and small bowel transit times (group difference 1.07, 95%CI: 0.95-1.19, P = 0.261) between SB2U and SB2, but total recording time was about 14% longer in the SB2U group (95%CI: 10%-18%, P < 0.001) and there was a corresponding trend toward higher completion rate (88.2% vs 93.2%, OR = 1.78, 95%CI 0.88-3.63, P = 0.111). There was no statistically significant difference in the rates of positive findings (OR = 0.98, 95%CI: 0.64-1.51, P = 0.918). CONCLUSION Extending the operating time of CE may be a simple method to improve completion rate although it does not affect the rate of positive findings.

The effect of chewing gum on small-bowel transit time in capsule endoscopy: a prospective, randomized trial.

BACKGROUND Approximately 1 in 6 capsule endoscopies (CEs) does not visualize the entire small bowel at completion of the examination because of limited battery life. OBJECTIVE To determine whether chewing gum can reduce the small-bowel transit time and increase CE completion rates. DESIGN Prospective, single-blind, randomized, controlled trial. SETTING A tertiary university-affiliated hospital. PATIENTS Consecutive patients 19 years of age and older undergoing outpatient small-bowel CE from October 2010 to July 2012 were assessed for eligibility. Those with previous gastric or small-bowel surgery or ileostomy, dysphagia prohibiting capsule ingestion, diabetes mellitus with evidence of end-organ damage, use of narcotics or prokinetics within 5 days before the procedure, clinical hyper-/hypothyroidism, and symptoms suggestive of acute bowel obstruction were excluded. INTERVENTION Gum chewing for at least 20 minutes every 2 hours starting at the time of capsule ingestion. MAIN OUTCOME MEASUREMENTS Small-bowel transit time, gastric transit time, and completion rate were measured. RESULTS Chewing gum did not have any significant effect on gastric transit time (rate ratio 1.06; 95% CI, 0.73-1.55; P = .75), small-bowel transit time (rate ratio 0.91; 95% CI, 0.62-1.35; P = .65), or completion rate (91.67% chewing gum vs 88.71% control, P = .58) of CE. LIMITATION Single-center study involving relatively healthy subjects. Procedures were done on an outpatient basis so participants were not monitored for adherence to protocol. CONCLUSIONS Chewing gum does not speed up capsule transit or increase completion rate of CE in patients without risk factors for incomplete studies. ( CLINICAL TRIAL REGISTRATION NUMBER NCT01241825.).

Src contributes to IL6-induced vascular endothelial growth factor-C expression in lymphatic endothelial cells

Formation of lymphatic capillaries by lymphatic endothelial cells (LECs) occurs both in normal tissues as well as in pathological processes including tumor metastasis. Interleukin-6 (IL-6), a potent pro-inflammatory cytokine, has been shown to be highly elevated in various cancers. IL-6 has also been shown to increase tumor lymphangiogenesis through vascular endothelial growth factor-C (VEGF-C) induction in tumor cells. Although lymphangiogenesis is associated with lymph node metastasis and also resistance to conventional therapy in various cancers, the precise mechanisms of lymphangiogenesis in LECs remain unclear. This study aimed to investigate the signaling cascade involved in IL-6-induced VEGF-C expression in murine LECs (SV-LEC). The VEGF-C mRNA and protein levels were increased in SV-LECs exposed to IL-6. IL-6 time-dependently induced Src phosphorylation and downstream phosphorylation of ERK1/2 and p38MAPK. In contrast, PP2, an inhibitor of Src signaling, abrogated IL-6′s effects on ERK1/2 and p38MAPK phosphorylation. IL-6 exposure also led to increase in VEGF-C promoter-luciferase activity as well as C/EBPβ- and κB-luciferase activities. VEGF-C promoter-, C/EBPβ- and κB-luciferase activities were all suppressed by Src, ERK1/2 or p38MAPK signaling blockades despite presence of IL-6. Finally, C/EBPβ and p65 binding to the VEGF-C promoter region were increased after IL-6 exposure in SV-LECs. Taken together, we report a Src-mediated ERK1/2 and p38MAPK activation resulting in C/EBPβ and p65 binding to the promoter region of VEGF-C, leading to VEGF-C expression in IL-6-exposed SV-LECs.

MAPK phosphatase-1 contributes to trichostatin A inhibition of cyclooxygenase-2 expression in human umbilical vascular endothelial cells exposed to lipopolysaccharide

BACKGROUND Histone deacetylase (HDAC) inhibitors have emerged as a new class of antitumor agents because they were demonstrated to induce cell cycle arrest, promote cell apoptosis, and inhibit metastasis. Recently, HDAC inhibitors were also shown to exhibit pronounced anti-inflammatory properties. However, the underlying mechanism contributing to the suppression of inflammatory responses by HDAC inhibitors remains to be fully defined. In the present study, we explored the actions of trichostatin A (TSA), a potent HDAC inhibitor, on lipopolysaccharide (LPS)-induced cyclooxygenase (COX)-2 expression in human umbilical vascular endothelial cells (HUVECs). METHODS HUVECs were exposed to LPS in the absence or presence of TSA. COX-2 expression and signaling molecules (JNK, p38MAPK and c-jun) activated by LPS were assessed. RESULTS The LPS-induced cox-2 messenger RNA and protein were markedly suppressed by TSA. TSA inhibited JNK and p38MAPK phosphorylation in cells exposed to LPS. Treatment of cells with a JNK signaling inhibitor (JNK inhibitor II) or a p38MAPK inhibitor (p38MAPK inhibitor III) markedly inhibited LPS-induced COX-2 expression. TSA suppression of JNK and p38MAPK phosphorylation and subsequent COX-2 expression were restored by selective inhibition of MKP-1 using MKP-1 siRNA. In addition, TSA caused an increase in MKP-1 phosphatase activity in HUVECs. In conclusion, TSA may cause MKP-1 activation to dephosphorylate JNK and p38MAPK, leading to the downregulation of COX-2 in HUVECs stimulated by LPS, a proinflammatory stimulus. GENERAL SIGNIFICANCE MKP-1 contributes to TSAs protective actions in HUVECs exposed to LPS. The present study also supports the therapeutic value of TSA in treating inflammatory vascular diseases.