Diagnostic Efficacy and Possible Underlying Mechanisms of Noninvasive Clinical Markers in Hepatocellular Carcinoma.

Background and Aims: In this study, we aimed to evaluate the diagnostic values of alpha-fetoprotein (AFP), soluble AXL (sAXL), des-γ-carboxy prothrombin (DCP), the aspartate aminotransferase-to-platelet ratio index (APRI), and the gamma-glutamyl transpeptidase-to-platelet ratio (GPR) in hepatocellular carcinoma (HCC) and the possible underlying mechanisms of the correlations between them. Methods: We collected serum samples from 190, 128, and 75 patients with HCC, cirrhosis, and chronic viral hepatitis, and from 82 healthy subjects. Serum levels of AFP, sAXL, and DCP were determined, and APRI and GPR values were calculated. Receiver operating characteristic (ROC) curves were used to analyze the diagnostic value of single and combined biomarkers. Results: We detected significant differences between the HCC group and other groups regarding serum AFP, sAXL, DCP, and APRI levels. GPR significantly differed between the HCC group and other groups, except for the liver cirrhosis group. AFP, sAXL, DCP, APRI, and GPR had positive correlations with each other, and AFP showed a higher area under the curve (AUC) and Youden index values, while APRI and DCP showed the highest sensitivity and specificity. Also, when AFP was combined with sAXL, DCP, APRI, and GRP, the highest AUC (0.911) and a higher net reclassification improvement value were obtained compared with those obtained for the individual biomarkers. Conclusions: AFP, sAXL, DCP, APRI, and GPR are independent risk factors for HCC, and the diagnostic performance of AFP combined with sAXL, DCP, APRI, and GPR for HCC diagnosis was superior to that of the individual biomarkers.

A functional polymorphism on chromosome 15q25 associated with survival of early stage non-small-cell lung cancer.

INTRODUCTION: The 15q25 region has been associated with lung-cancer risk and might also be associated with the prognosis of lung cancer. This study was conducted to determine the impact of a functional polymorphism in the CHRNA3 gene on chromosome 15q25 in the survival of patients with early-stage non-small-cell lung cancer (NSCLC). METHODS: Five hundred and eighty-three consecutive patients with surgically resected NSCLC were enrolled. The rs6495309C > T polymorphism in the promoter of the CHRNA3 gene was investigated. The association between genotype and overall survival (OS) and disease-free survival (DFS) was analyzed. RESULTS: Patients with the rs6495309 CT or TT genotype had a significantly better OS and DFS than the rs6495309 CC genotype (adjusted hazard ratio for OS = 0.56, 95% confidence interval = 0.41-0.75, p = 0.0001; and adjusted hazard ratio for DFS = 0.61, 95% confidence interval = 0.48-0.79, p = 0.0001). An association between the rs6495309C > T polymorphism and survival outcome was demonstrated in smokers and never-smokers, and in squamous-cell carcinomas and adenocarcinomas. CONCLUSION: The CHRNA3 rs6495309C > T polymorphism may affect survival in patients with early-stage NSCLC. Analysis of the rs6495309C > T polymorphism can help identify patients at high risk of a poor disease outcome.

Heat shock augments angiotensin II-induced vascular contraction through increased production of reactive oxygen species.

A temporal increase in temperature triggers a series of stress responses and alters vascular smooth muscle (VSM) contraction induced by agonist stimulation. Here we examined the role of reactive oxygen species (ROS) in heat shock-dependent augmentation of angiotensin II (AngII)-induced VSM contraction. Endothelium-denuded rat aortic rings were treated with heat shock for 45 min at 42 degrees C and then subjected to assays for the production of force, ROS, and the expression of ROS-related enzymes. AngII-induced contraction was enhanced in heat shock-treated aorta. AngII-induced production of hydrogen peroxide and superoxide were elevated in response to the heat shock treatment. Pre-treatment with superoxide dismutases (SOD) mimetic and inhibitors for glutathione peroxidase and NADPH oxidase but not for xanthine oxidase eliminated an increase in the AngII-induced contraction in the heat shock-treated aorta. Heat shock increased the expression of p47phox, a cytosolic subunit of NADPH oxidase, but not Cu-Zn-SOD and Mn-SOD. In addition, heat shock increased contraction that was evoked by hydrogen peroxide and pyrogallol. These results suggest that heat shock causes an elevation of ROS as well as a sensitization of ROS signal resulting in an augmentation of VSM contraction in response to agonist.

Promoter hypomethylation upregulates Na+-K+-2Cl- cotransporter 1 in spontaneously hypertensive rats.

The Na(+)-K(+)-2Cl(-) cotransporter 1 (NKCC1) is one of several transporters that have been implicated for development of hypertension since NKCC1 activity is elevated in hypertensive aorta and vascular contractions are inhibited by bumetanide, an inhibitor of NKCC1. We hypothesized that promoter hypomethylation upregulates the NKCC1 in spontaneously hypertensive rats (SHR). Thoracic aortae and mesenteric arteries were excised, cut into rings, mounted in organ baths and subjected to vascular contraction. The expression levels of nkcc1 mRNA and protein in aortae and heart tissues were measured by real-time PCR and Western blot, respectively. The methylation status of nkcc1 promoter region was analyzed by combined bisulfite restriction assay (COBRA) and bisulfite sequencing. Phenylephrine-induced vascular contraction in a dose-dependent manner, which was inhibited by bumetanide. The inhibition of dose-response curves by bumetanide was much greater in SHR than in Wistar Kyoto (WKY) normotensive rats. The expression levels of nkcc1 mRNA and of NKCC1 protein in aortae and heart tissues were higher in SHR than in WKY. Nkcc1 gene promoter was hypomethylated in aortae and heart than those of WKY. These results suggest that promoter hypomethylation upregulates the NKCC1 expression in aortae and heart of SHR.

Calcium sensitization induced by sodium fluoride in permeabilized rat mesenteric arteries.

It was hypothesized that NaF induces calcium sensitization in Ca(2+)-controlled solution in permeabilized rat mesenteric arteries. Rat mesenteric arteries were permeabilized with beta-escin and subjected to tension measurement. NaF potentiated the concentration-response curves to Ca(2+) (decreased EC(50) and increased E(max)). Cumulative addition of NaF (4.0, 8.0 and 16 mM) also increased vascular tension in Ca(2+)-controlled solution at pCa 7.0 or pCa 6.5, but not at pCa 8.0. NaF-induced vasocontraction and GTPgammaS-induced vasocontraction were not additive. NaF-induced vasocontraction at pCa 7.0 was inhibited by pretreatment with Rho kinase inhibitors H1152 or Y27632 but not with a MLCK inhibitor ML-7 or a PKC inhibitor Ro31-8220. NaF induces calcium sensitization in a Ca(2+)-dependent manner in beta-escin-permeabilized rat mesenteric arteries. These results suggest that NaF is an activator of the Rho kinase signaling pathway during vascular contraction.

Fluoride induces vascular contraction through activation of RhoA/Rho kinase pathway in isolated rat aortas.

We hypothesized that fluoride induces vascular contraction through activation of the RhoA/Rho kinase pathway in isolated rat aortas. Rat aortic rings were mounted in organ baths and contracted with sodium fluoride (NaF). We measured the amount of GTP-RhoA as well as vascular tension. We also determined the level of phosphorylation of the myosin light chain (MLC(20)), myosin phosphatase targeting subunit 1 (MYPT1) and PKC-potentiated inhibitory protein for heterotrimeric MLCP of 17kDa (CPI17). In both physiological salt solution and Ca(2+)-free solution, NaF increased vascular tension and MLC(20) phosphorylation in dose-dependent manners. NaF increased not only phosphorylation level of MYPT1(Thr855) and CPI17(Thr38), but also the amount of GTP-RhoA. Both H1152 and Y27632, inhibitors of Rho kinase, but not Ro31-8220, an inhibitor of PKC, attenuated NaF-induced contraction and phosphorylation level of MLC(20), MYPT1(Thr855) and CPI17(Thr38). In conclusion, fluoride induces vascular contraction through activation of the RhoA/Rho kinase pathway.

Flavone Attenuates Vascular Contractions by Inhibiting RhoA/Rho Kinase Pathway.

Our previous study demonstrated that flavone inhibits vascular contractions by decreasing the phosphorylation levelof the myosin phosphatase target subunit (MYPT1). In the present study, we hypothesized that flavone attenuates vascular contractions through the inhibition of the RhoA/Rho kinase pathway. Rat aortic rings were denuded of endothelium, mounted in organ baths, and contracted with either 30 nM U46619 (a thromboxane A2 analogue) or 8.0 mM NaF 30 min after pretreatment with either flavone (100 or 300 microM) or vehicle. We determined the phosphorylation level of the myosin light chain (MLC(20)), the myosin phophatase targeting subunit 1 (MYPT1) and the protein kinase C-potentiated inhibitory protein for heterotrimeric myosin light chain phophatase of 17-kDa (CPI17) by means of Western blot analysis. Flavone inhibited, not only vascular contractions induced by these contractors, but also the levels of MLC(20) phosphorylation. Furthermore, flavone inhibited the activation of RhoA which had been induced by either U46619 or NaF. Incubation with flavone attenuated U46619-or NaF-induced phosphorylation of MYPT1(Thr855) and CPI17(Thr38), the downstream effectors of Rho-kinase. In regards to the Ca(2+)-free solution, flavone inhibited the phosphorylation of MYPT1(Thr855) and CPI17(Thr38), as well as vascular contractions induced by U46619. These results indicate that flavone attenuates vascular contractions, at least in part, through the inhibition of the RhoA/Rho-kinase pathway.

17beta-estradiol attenuates vascular contraction through inhibition of RhoA/Rho kinase pathway.

We hypothesized that 17beta-estradiol attenuates vascular contraction through inhibition of RhoA/Rho kinase pathway. Rat aortic rings were contracted with cumulative addition of U46619, NaF, KCl or PDBu 30 min after pretreatment with 17beta-estradiol (10, 30, and 100 microM) or vehicle. We measured the amount of GTP RhoA and the level of phosphorylation of the myosin light chain (MLC(20)), myosin phosphatase targeting subunit 1 (MYPT1) and PKC-potentiated inhibitory protein for heterotrimeric MLCP of 17 kDa (CPI17). Pretreatment with 17beta-estradiol dose-dependently inhibited the concentration-response curves in response to U46619, NaF or KCl, but not to PDBu. 17beta-Estradiol decreased not only the level of phosphorylation of MYPT1(Thr855) and CPI17(Thr38) as well as MLC(20), but also the activity of RhoA induced by U46619 or NaF. However, 17beta-estradiol did not affect the level of phosphorylation of CPI17 induced by PDBu. 17beta-Estradiol attenuates vascular contraction through inhibition of RhoA/Rho kinase pathway.

Vasorelaxation by Samhwangsasim-tang, an herb medicine, is associated with decreased phosphorylation of the myosin phosphatase target subunit.

Samhwangsasim-tang (SST) is a widely used herbal medicine with vasodilatory actions in oriental countries. We hypothesized that SST modulates vascular contractility by decreasing phosphorylation of the myosin phosphatase target subunit. Rat aortic ring preparations were mounted in organ baths and subjected to contractions or relaxations. Phosphorylation of 20kDa myosin light chains (MLC(20)) and MYPT1, a target subunit of myosin phosphate 1, were examined with immunoblots. SST relaxed aortic ring preparations precontracted with phenylephrine whether endothelium was intact or denuded. Treatment of aortic rings with N(ω)-nitro-l-arginine methyl ester (l-NAME), an inhibitor of endothelial nitric oxide synthase or methylene blue, an inhibitor of guanylyl cyclase, did not affect the relaxing action of SST. Furthermore, SST inhibited vascular contractions induced by NaF or phenylephrine, but not by phorbol dibutyrate. SST also decreased vascular tension precontracted by 8.0mmol/L NaF or 1.0µmol/L phenylephrine, but not by 1.0µmol/L phorbol dibutyrate. In vascular strips, SST decreased the phosphorylation level of both MLC(20) and MYPT1 induced by 8.0mmol/L NaF. In conclusion, SST inhibited vascular contraction by decreasing phosphorylation of the myosin phosphatase target subunit.