Veerapol Kukongviriyapan

Exome sequencing of liver fluke-associated cholangiocarcinoma

Opisthorchis viverrini–related cholangiocarcinoma (CCA), a fatal bile duct cancer, is a major public health concern in areas endemic for this parasite. We report here whole-exome sequencing of eight O. viverrini–related tumors and matched normal tissue. We identified and validated 206 somatic mutations in 187 genes using Sanger sequencing and selected 15 genes for mutation prevalence screening in an additional 46 individuals with CCA (cases). In addition to the known cancer-related genes TP53 (mutated in 44.4% of cases), KRAS (16.7%) and SMAD4 (16.7%), we identified somatic mutations in 10 newly implicated genes in 14.8–3.7% of cases. These included inactivating mutations in MLL3 (in 14.8% of cases), ROBO2 (9.3%), RNF43 (9.3%) and PEG3 (5.6%), and activating mutations in the GNAS oncogene (9.3%). These genes have functions that can be broadly grouped into three biological classes: (i) deactivation of histone modifiers, (ii) activation of G protein signaling and (iii) loss of genome stability. This study provides insight into the mutational landscape contributing to O. viverrini–related CCA.

Mulberry leaf extract restores arterial pressure in streptozotocin-induced chronic diabetic rats

Free radical-induced vascular dysfunction plays a key role in the pathogenesis of vascular disease found in chronic diabetic patients. Morus alba (MA) leaf extract is promoted for good health especially in diabetic patients. Interestingly, antidiabetic and antioxidant activities of MA have been reported in experimental animals. Thus, the hypothesis of this study was that the long-term treatment with MA could improve vascular reactivity of chronic diabetic rats. To test this hypothesis, we examined the effect of long-term treatment with MA on the vascular responses to vasoactive agents in streptozotocin-induced chronic diabetic rats. The diabetic rats were either orally administered with distilled water, MA (0.25, 0.5 and 1 g/kg per day) or subcutaneously injected with insulin (4 U/kg per day) for 8 weeks. After each treatment, the fasting blood glucose, blood pressure, vascular responses to vasoactive agents and tissue malondialdehyde were examined. Morus alba at the doses of 0.5 and 1 g/kg, which significantly reduced blood glucose level, also significantly decreased the high blood pressure in diabetic rats. Vascular responses of the chronic diabetic rats to vasodilators, acetylcholine (3-30 nmol/kg) and sodium nitroprusside (1-10 nmol/kg) were significantly suppressed by 26% to 44% and 45% to 77% respectively, whereas those to vasoconstrictor, phenylephrine (0.01-0.1 micromol/kg) were significantly increased by 23% to 38% as compared to normal rats. Interestingly, the administration of 0.5 and 1 g/kg MA or 4 U/kg insulin significantly restored the vascular reactivities of diabetic rats. Moreover, 8 weeks of diabetes resulted in the elevation of malondialdehyde content in tissues (liver, kidney, heart, and aorta), and MA treatment significantly lessened this increase. These results provide the first evidence for the efficacy of MA in restoring the vascular reactivity of diabetic rats, the mechanism of which may associate with the alleviation of oxidative stress.

Quercetin and EGCG exhibit chemopreventive effects in cholangiocarcinoma cells via suppression of JAK/STAT signaling pathway.

Quercetin and epigallocatechin‐3‐gallate (EGCG) are dietary phytochemicals with antiinflammatory and antitumor effects. In the present study, we examined the effects of these two compounds on Janus‐like kinase (JAK)/signal transduction and transcription (STAT) pathway of cholangiocarcinoma (CCA) cells, because CCA is one of the aggressive cancers with very poor prognosis and JAK/STAT pathway is critically important in inflammation and carcinogenesis. The results showed that the JAK/STAT pathway activation by proinflammatory cytokine interleukin‐6 and interferon‐γ in CCA cells was suppressed by pretreatment with quercetin and EGCG, evidently by a decrease of the elevated phosphorylated‐STAT1 and STAT3 proteins in a dose‐dependent manner. The cytokine‐mediated up‐regulation of inducible nitric oxide synthase (iNOS) and intercellular adhesion molecule‐1 (ICAM‐1) via JAK/STAT cascade was abolished by both quercetin and EGCG pretreatment. Moreover, these flavonoids also could inhibit growth and cytokine‐induced migration of CCA cells. Pretreatment with specific JAK inhibitors, AG490 and piceatannol, abolished cytokine‐induced iNOS and ICAM‐1 expression. These results demonstrate beneficial effects of quercetin and EGCG in the suppression of JAK/STAT cascade of CCA cells. Quercetin and EGCG would be potentially useful as cancer chemopreventive agents against CCA. Copyright

Crucial role of heme oxygenase-1 on the sensitivity of cholangiocarcinoma cells to chemotherapeutic agents.

Cancer cells acquire drug resistance via various mechanisms including enhanced cellular cytoprotective and antioxidant activities. Heme oxygenase-1 (HO-1) is a key enzyme exerting potent cytoprotection, cell proliferation and drug resistance. We aimed to investigate roles of HO-1 in human cholangiocarcinoma (CCA) cells for cytoprotection against chemotherapeutic agents. KKU-100 and KKU-M214 CCA cell lines with high and low HO-1 expression levels, respectively, were used to evaluate the sensitivity to chemotherapeutic agents, gemcitabine (Gem) and doxorubicin. Inhibition of HO-1 by zinc protoporphyrin IX (ZnPP) sensitized both cell types to the cytotoxicity of chemotherapeutic agents. HO-1 gene silencing by siRNA validated the cytoprotective effect of HO-1 on CCA cells against Gem. Induction of HO-1 protein expression by stannous chloride enhanced the cytoprotection and suppression of apoptosis caused by anticancer agents. The sensitizing effect of ZnPP was associated with increased ROS formation and loss of mitochondrial transmembrane potential, while Gem alone did not show any effects. A ROS scavenger, Tempol, abolished the sensitizing effect of ZnPP on Gem. Combination of ZnPP and Gem enhanced the release of cytochrome c and increased p21 levels. The results show that HO-1 played a critical role in cytoprotection in CCA cells against chemotherapeutic agents. Targeted inhibition of HO-1 may be a strategy to overcome drug resistance in chemotherapy of bile duct cancer.

Tetrahydrocurcumin alleviates hypertension, aortic stiffening and oxidative stress in rats with nitric oxide deficiency.

Tetrahydrocurcumin (THC), a major metabolite of curcumin, possesses strong antioxidant and cardioprotective properties. However, the activities of THC in hypertension and its associated complications remain unknown. The aim of this study was to investigate the effect of THC on hemodynamic status, aortic elasticity and oxidative stress in rats with N-nitro-L-arginine methyl ester (L-NAME)-induced hypertension. Hypertension was induced in male Sprague-Dawley rats by administration of L-NAME (50 mg kg−1 body weight) in drinking water for 5 weeks. THC at a dose of 50 or 100 mg kg−1 per day was administered daily during the fourth and fifth weeks when the hypertensive state had been established. The effects of THC on hemodynamics, aortic elasticity, endothelial nitric oxide synthase (eNOS) protein expression and oxidative stress markers were assessed. Marked increases in blood pressure, peripheral vascular resistance, aortic stiffness and oxidative stress were found in rats after L-NAME administration. THC significantly reversed these deleterious effects by reducing aortic wall thickness and stiffness. These effects were associated with increased aortic eNOS expression, elevated plasma nitrate/nitrite, decreased oxidative stress with reduced superoxide production and enhanced blood glutathione. Our results provide the first evidence that THC attenuates the detrimental effect of L-NAME by improving the hemodynamic status and aortic elasticity concomitant with reduction of oxidative stress. The present study suggests that THC might be used as a dietary supplement to protect against cardiovascular alterations under nitric oxide-deficient conditions.

Asiatic Acid Alleviates Hemodynamic and Metabolic Alterations via Restoring eNOS/iNOS Expression, Oxidative Stress, and Inflammation in Diet-Induced Metabolic Syndrome Rats

Asiatic acid is a triterpenoid isolated from Centella asiatica. The present study aimed to investigate whether asiatic acid could lessen the metabolic, cardiovascular complications in rats with metabolic syndrome (MS) induced by a high-carbohydrate, high-fat (HCHF) diet. Male Sprague-Dawley rats were fed with HCHF diet with 15% fructose in drinking water for 12 weeks to induce MS. MS rats were treated with asiatic acid (10 or 20 mg/kg/day) or vehicle for a further three weeks. MS rats had an impairment of oral glucose tolerance, increases in fasting blood glucose, serum insulin, total cholesterol, triglycerides, mean arterial blood pressure, heart rate, and hindlimb vascular resistance; these were related to the augmentation of vascular superoxide anion production, plasma malondialdehyde and tumor necrosis factor-alpha (TNF-α) levels (p < 0.05). Plasma nitrate and nitrite (NOx) were markedly high with upregulation of inducible nitric oxide synthase (iNOS) expression, but dowregulation of endothelial nitric oxide synthase (eNOS) expression (p < 0.05). Asiatic acid significantly improved insulin sensitivity, lipid profiles, hemodynamic parameters, oxidative stress markers, plasma TNF-α, NOx, and recovered abnormality of eNOS/iNOS expressions in MS rats (p < 0.05). In conclusion, asiatic acid improved metabolic, hemodynamic abnormalities in MS rats that could be associated with its antioxidant, anti-inflammatory effects and recovering regulation of eNOS/iNOS expression.

Mulberry leaf extract stimulates glucose uptake and GLUT4 translocation in rat adipocytes.

Mulberry (Morus alba L.) leaf tea is promoted for its health benefits and the control of diabetes in Asian nations. The blood glucose lowering activity of mulberry leaf extract (MA) has been proven; however, the molecular basis underlying this effect remains unclear. The aim of the present work is to elucidate its mechanism of the antihyperglycemic action, by examining the effect of MA on glucose uptake and the translocation of glucose transporter 4 protein (GLUT4) to the plasma membrane of adipocytes isolated from diabetic rats. The incubation of adipocytes with 5-45 μg/ml MA resulted in 31-54% increase of glucose uptake in a dose-dependent manner. This glucose uptake enhancing effect was inhibited by the phosphoinositol 3-kinase (PI3-K) inhibitor, wortmannin (100 nM). The GLUT4 protein on the plasma membrane fraction of adipocytes was markedly increased after treatment with 15 μg/ml MA extract. Interestingly, gallic acid, one of the phenolic compounds found in MA extract, increased glucose uptake and enhanced the translocation of GLUT4 at concentrations comparable to the amount of gallic acid in the effective concentration ranges of MA. Thus, it is likely that gallic acid contributes, at least in part, to its antihyperglycemic activity. The present results suggest that the antihyperglycemic action of MA is mediated by increasing glucose uptake via the activation of PI3-K signaling pathway and translocation of GLUT4 to the plasma membrane. These findings are the first molecular evidence supporting the mulberry tea as herbal medicine for diabetic patients.

Curcumin improves vascular function and alleviates oxidative stress in non-lethal lipopolysaccharide-induced endotoxaemia in mice

Oxidative stress is implicated in various pathological conditions, including septic shock, and other diseases associated with local or systemic inflammation. Curcumin, a major component from turmeric (Curcuma longa), possesses diverse anti-inflammatory, anti-tumour and antioxidant properties. The aim of this study was to investigate the effect of curcumin on modulation of vascular dysfunction and oxidative stress induced by lipopolysaccharide (LPS) in mice. Male ICR mice were treated with curcumin (50 or 100 mg/kg), administered intragastrically, either before or after intraperitoneal injection of LPS (10 mg/kg). Fifteen hours after LPS administration, arterial blood pressure was measured and vascular response to vasoactive agents were assessed. Aortic tissues and blood samples were taken for assays of antioxidant and oxidative stress markers. LPS caused marked hypotension, tachycardia and vascular hyporeactivity. The mean arterial pressures in responses to phenylephrine, acetylcholine, and sodium nitroprusside of LPS-treated mice were significantly decreased when compared with the untreated controls. Curcumin modulated heart rate and restored arterial blood pressure in a dose-dependent manner in both protectively- and therapeutically-treated regimens. Furthermore, the vascular responsiveness of LPS-treated mice was improved by curcumin. Interestingly, the improvements of haemodynamics and vascular response during endotoxaemia were related to alleviation of oxidative stress by reducing aortic-derived superoxide production, suppression of lipid peroxidation and protein oxidation, and decrease in urinary nitric oxide metabolites with preservation of the ratio of glutathione/glutathione disulfide. This study provides the first evidence for the potential role of curcumin in prevention and treatment of vascular dysfunction in mice with endotoxaemia elicited by LPS.

Curcumin improves endothelial dysfunction and vascular remodeling in 2K-1C hypertensive rats by raising nitric oxide availability and reducing oxidative stress.

Oxidative stress plays a role in maintaining high arterial blood pressure and contributes to the vascular changes that lead to hypertension. Consumption of polyphenol-rich foods has demonstrated their beneficial role in the prevention and treatment of hypertension. Curcumin (CUR), a phenolic compound present in the rhizomes of turmeric, possesses cardiovascular protective, anti-inflammatory and antioxidant properties. The present study was designed to investigate the protective effect of CUR on 2kidney-1clip (2K-1C)-induced hypertension, endothelial dysfunction, vascular remodeling and oxidative stress in male Sprague-Dawley rats. Sham operated or 2K-1C rats were treated with CUR at a dose of 50 or 100 mg/kg/day (or vehicle). After 6 weeks of treatment, CUR ameliorated hemodynamic performance in 2K-1C hypertensive rats (P< 0.05), by reducing blood pressure, increasing hindlimb blood flow and decreasing hindlimb vascular resistance. Hemodynamic restoration was associated with a reduction in plasma angiotensin converting enzyme level. Endothelium-dependent vasorelaxation, in response to acetylcholine, of aortic rings isolated from 2K-1C hypertensive rats-treated with CUR was significantly increased (P< 0.05). CUR also attenuated hypertension-induced oxidative stress and vascular structural modifications. These effects were associated with elevated plasma nitrate/nitrite, upregulated eNOS expression, downregulated p47phox NADPH oxidase and decreased superoxide production in the vascular tissues. The overall findings of this study suggest the mechanisms responsible for the antihypertensive action of CUR in 2K-1C hypertension-induced endothelial dysfunction and vascular remodeling involve the improvement NO bioavailability and a reduction in oxidative stress.

Curcumin Protects against Cadmium-Induced Vascular Dysfunction, Hypertension and Tissue Cadmium Accumulation in Mice

Curcumin from turmeric is commonly used worldwide as a spice and has been demonstrated to possess various biological activities. This study investigated the protective effect of curcumin on a mouse model of cadmium (Cd)—induced hypertension, vascular dysfunction and oxidative stress. Male ICR mice were exposed to Cd (100 mg/L) in drinking water for eight weeks. Curcumin (50 or 100 mg/kg) was intragastrically administered in mice every other day concurrently with Cd. Cd induced hypertension and impaired vascular responses to phenylephrine, acetylcholine and sodium nitroprusside. Curcumin reduced the toxic effects of Cd and protected vascular dysfunction by increasing vascular responsiveness and normalizing the blood pressure levels. The vascular protective effect of curcumin in Cd exposed mice is associated with up-regulation of endothelial nitric oxide synthase (eNOS) protein, restoration of glutathione redox ratio and alleviation of oxidative stress as indicated by decreasing superoxide production in the aortic tissues and reducing plasma malondialdehyde, plasma protein carbonyls, and urinary nitrate/nitrite levels. Curcumin also decreased Cd accumulation in the blood and various organs of Cd-intoxicated mice. These findings suggest that curcumin, due to its antioxidant and chelating properties, is a promising protective agent against hypertension and vascular dysfunction induced by Cd.