KEAP1/NRF2 signaling pathway mutations in cervical cancer.

OBJECTIVE: The aim of the present study was to explore the potential involvement of mutations in the KEAP1/NRF2 signaling pathway in Chinese samples with cervical cancer. PATIENTS AND METHODS: 236 Chinese patients with various types of cervical cancer were recruited, and the coding exons and the corresponding intron-exon boundaries of the KEAP1 and NRF2 genes were analyzed for the potential mutations in the KEAP1/NRF2 signaling pathway. RESULTS: A novel KEAP1 missense somatic mutation (c.1408C>T, p.R470C) and 5 NRF2 missense somatic mutations (c.72G>C, p.W24C; c.85G>T, p.D29Y; c.101G>A, p.R34Q; c.230A>C, p.D77A and c.242G>A p.G81D) were identified in 187 patients with cervical squamous cell carcinoma, respectively; no mutations were detected in other subtypes. All these mutations were heterozygous and predicted to be pathogenic by PolyPhen-2, MutationTaster programs, and evolutionary conservation analysis. Among these mutations, the KEAP1 (p.R470C) and 3 NRF2 mutations (p.D29Y, p.D77A, and p.G81D) were detected in cervical cancer for the first time. Also, no mutations were identified in our 21 adenosquamous carcinomas or 25 adenocarcinomas. CONCLUSIONS: We identified 6 potential diseases causing mutations in the KEAP1/NRF2 signaling pathway in 187 (3.2%) Chinese cases with cervical squamous cell carcinoma, implicating KEAP1/NRF2 signaling pathway might play an active role in the pathogenesis of this subtype of cervical cancer. Furthermore, among these detected mutations, the KEAP1 and 3 NRF2 mutations were reported in cervical cancer for the first time.

[Study on Urinary Metabolic Profile in Rats with Deep Venous Thrombosis Based on Pattern Recognition].

OBJECTIVES: To study the urinary metabolic profile in rats with deep venous thrombosis （DVT） based on metabolomics and to screen out small molecular biomarkers for the diagnosis and forensic identification of DVT. METHODS: Inferior vena cava of rats was ligated to construct DVT models. The rats were randomly divided into three groups： DVT, sham, and control groups, 10 in each group. The urine of DVT and sham rats was collected during 24 hours in the metabolic cage at 48 hours after operating, meanwhile, 24 hours urine was collected in control group. The metabolic profile was analyzed by nuclear magnetic resonance. SIMCA-P 14.1 software was used for pattern recognition. The variable importance in projection （VIP） value from orthogonal PLS-DA （OPLS-DA） model combined with Mann-Whitney U test were used to search the different metabolites in the urine. RESULTS: The metabolic profiles of urine from DVT, sham, and control groups had significant differences. The DVT, sham, and control groups could be distinguished by the partial least squares method-discriminant analysis （PLS-DA） model. Compared with the urine of the rats in control groups, the levels of leucine, glutamine, creatine, creatinine and sucrose in the urine of DVT rats were up-regulated, and the levels of 3-hydroxybutyrate, lactate, acetone, α-oxoglutarate, citrate and hippurate were down-regulated. CONCLUSIONS: The different metabolites in the urine of DVT rats are expected to become its candidate biomarkers. The results can provide a research basis for the diagnosis, treatment and forensic identification of DVT.

[Effects of RhoA on the adherens junction of murine ameloblasts].

OBJECTIVE: To investigate the regulation mechanism of RhoA signaling pathway during the enamel formation by using the EGFP-RhoADominant Negative (EGFP-RhoADN) transgenic mice model, from the aspect of adherens junctions, and to provide a theory basis for mechanism of enamel development defects. METHODS: The enamel thickness of mandibular first molars of EGFP-RhoADN transgenic mice and wild type (WT) mice were observed by scanning electronic microscopy at 20 kV, and the enamel thickness of the distal face of the central cusp was measured at 10 locations via analysis by ImageJ (Rasband, 1997-2009). The enamel organs from mandibular first molars from postnatal-4-day (P4) EGFP-RhoADN mice and wild type mice were isolated, and the total RNA and protein were extracted from the epithelium of the enamel organs. The expression level of the adherens junctions components in ameloblasts layer of the postnatal-4-day EGFP-RhoADN transgenic mice and wild type mice mandibular first molars were detected by real-time PCR and Western blot assay. RESULTS: The EGFP-RhoADN transgenic mice had decreased enamel thickness in their bilateral mandibular first molars versus those of control group (n=20), and enamel thickness was (84.60±0.20) µm vs. (106.24±0.24) µm, P<0.05. The protein expressions of E-cadherin, α-E-catenin and pan-cadherin in ameloblasts layer of postnatal-4-day EGFP-RhoADN transgenic mice molars were down-regulated, and the protein level of ß-catenin in ameloblasts layer of P4 EGFP-RhoADN transgenic mice molars was up-regulated. The mRNA level of E-cadherin in ameloblasts layer of P4 EGFP-RhoADN transgenic mice molars was down-regulated versus that of WT mice, and the gene expression of E-cadherin was 0.93±0.01 vs. 1.00±0.02, P<0.05. The mRNA level of ß-catenin in ameloblasts layer of P4 EGFP-RhoADN transgenic mice molars was up-regulated versus that of WT mice, and the gene expression of ß-catenin was 1.23±0.03 vs. 1.00±0.05, P<0.05. CONCLUSION: In the mandibular first molars of EGFP-RhoADN transgenic mice, the enamel formation was disrupted and the adherens junctions of EGFP-RhoADN transgenic mice ameloblasts were implicated during amelogenesis. RhoA signaling pathway may play a critical role in enamel development by altering the adherens junctions in ameloblasts.

Down-regulation of lncRNA MIR31HG correlated with aggressive clinicopathological features and unfavorable prognosis in esophageal squamous cell carcinoma.

OBJECTIVE: Long non-coding RNA MIR31HG (MIR31HG) has been shown to affect numerous tumorigenesis. However, the function of MIR31HG in esophageal squamous cell carcinoma (ESCC) remains unclear. The aim of this study was to investigate whether the levels of MIR31HG could be served as a prognostic factor in patients with ESCC. PATIENTS AND METHODS: MIR31HG expression was detected in 185 samples of surgically resected ESCC and matched normal tumor-adjacent tissues by qRT-PCR. The association between MIR31HG expression levels in tissue and characteristics was examined. Overall survival (OS) curves were conducted to compare MIR31HG level and clinical characteristics. Cox regression analysis was conducted to determine the prognostic value of MIR31HG. RESULTS: The levels of MIR31HG were decreased in the ESCC tissues from patients with ESCC compared with the control (p < 0.01). In malignant cases, lower expression MIR31HG levels were significantly associated with poor differentiation (p < 0.001), advanced lymph node metastasis (p = 0.006), positive distant metastasis (p = 0.005) and TNM stage (p = 0.004). Kaplan-Meier analysis indicated that patients presenting with reduced MIR31HG expression exhibited poorer OS (p = 0.0002). Univariate and multivariate analysis suggested that MIR31HG expression was an independent prognostic marker for survival in patients with ESCC. CONCLUSIONS: We observed that down-regulated MIR31HG in ESCC patients was associated with malignant clinical characteristics. MIR31HG might be considered as a potential prognostic indicator and a potential target for therapeutic targets in ESCC.

[Clinical application of titanium plate reconstruction of mandibular defect: 10 years of follow up].

OBJECTIVE: To summarize the postoperative complications of reconstruction of mandible defect with titanium reconstruction plate. METHODS: A total of 111 cases of the mandibular defect caused by various reasons and repaired by titanium reconstruction plate in the Department Oral and Maxillofacial Surgery of the affiliated Hospital of Qingdao University from 2003 to 2012 were collected and followed up. The complications were analyzed. RESULTS: Thirty-seven percent of 111 cases showed long term complications. The titanium fracture was the main complication(16%[18/111]), followed by stress-shielding (9%[10/111]), infection(8%[9/111]), and titanium plate exposure(4%[4/111]). Titanium plate fracture occurred within 8 months and 3 years after surgery. The simple titanium plate reconstruction had the highest rate of plate fracture(30%[15/50]). Stress-shielding in non-vascularized bone graft was more significant than that in vascularized bone graft(P<0.05). When replaced by mini-titanium plate, the stress-shielding effect disappeared gradually. When the retention of mandibular margin height was less than 1 cm with the use of reconstruction plate, the postoperative complications were prone to occur. CONCLUSIONS: Bone graft is the best way to reconstruct mandibular defect, and simple reconstruction plate repair is applied only as a transitional means for high degree of malignancy, obvious recurrence tendency tumor or special reasons such as age etc, which are not suitable for bone graft. The reconstruction plate fixation is not recommended for bone graft, especially non-vascularized bone graft. The retention of mandibular margin with reconstruction plate fixation is open to discussion.

Clinical significance of lymphatic vessel invasion in stage I non-small cell lung cancer patients.

The aim of this retrospective study was to evaluate the prognostic influence of lymphatic vessel invasion (LVI) in stage I non-small cell lung cancer (NSCLC) patients. From January 2004 to December 2007, LVI was detected in 57 patients with T1N0M0 NSCLC; therefore, 114 patients with the same pathology, T stage, and surgery method, but without LVI, were selected as the control group to compare survival. The overall survival and relapse-free survival rates were estimated using the Kaplan-Meier method, log-rank test, and Cox proportional hazards analysis. The average follow-up length was 59.94 ± 23.1 months. The 5-year overall survival rates of the LVI-negative and the LVI-positive groups were 90.54 and 70.1%, respectively (P = 0.002). A multivariate analysis revealed LVI to be an independent predictive factor (hazard ratio = 4.562; P = 0.004). The 5-year overall survival rates for patients who received postoperative adjunctive therapy and those who did not in the LVI-positive group were 88.2 and 61.5%, respectively, with a P value less than 0.05 in both univariate and multivariate analyses. LVI is a poor prognostic factor in stage I NSCLC patients; postoperative adjunctive therapy is needed to improve the prognosis of NSCLC patients with LVI.

Photoluminescence properties of highly dispersed ZnO quantum dots in polyvinylpyrrolidone nanotubes prepared by a single capillary electrospinning.

Highly dispersed ZnO quantum dots (QDs) in polyvinylpyrrolidone (PVP) nanotubes have been prepared by a single capillary electrospinning. The structure and optical properties characterizations were performed by x-ray diffraction, scanning and transmission electron microscopy, absorption, photoluminescence, and resonant Raman spectra. In the composites, PVP molecules passivate the surface defects of ZnO QDs and prevent the aggregations of ZnO QDs. As a result, the composites exhibit narrower band edge emissions and less laser thermal effects. Blueshifted band gap, enlarged exciton energy, and less exciton-longitudinal optical (LO) phonon interaction due to the quantum confinement effect have also been observed.

Correlation between epithelial cell permeability of cephalexin and expression of intestinal oligopeptide transporter.

The proton-coupled oligopeptide transporter (PEPT1) has been shown to mediate mucosal cell transport of di- and tripeptide, and some peptidomimetic drugs. In this study, we determined the correlation between PEPT1 protein expression and the permeability of cephalexin, a substrate of PEPT1, in human PEPT1 (hPEPT1)-overexpressed Caco-2 cells (Caco-2/hPEPT1 cells) and rat jejunum. Caco-2/hPEPT1 cells with various levels of hPEPT1 expression were established by an adenoviral transfection system. The effective intestinal permeability (P(eff)) in rat jejunum was evaluated using a single pass in situ perfusion method. The level of PEPT1 in Caco-2/hPEPT1 cells and rat intestinal mucosal samples was quantitated by densitometry after immunoblotting and enhanced chemiluminescence detection. In Caco-2/hPEPT1 cells, an excellent correlation was observed between cephalexin uptake and hPEPT1 expression (R2 = 0.96, P < 0.005). This demonstrates that cephalexin uptake is directly proportional to hPEPT1 expression. In the rat perfusion study, the mean P(eff) +/- S.D. (n = 15) of cephalexin was 3.89 +/- 1.63 x 10(-5) cm/s. A very significant correlation between PEPT1 expression and cephalexin permeability with an R2 = 0.63 (P < 0.001) was observed. This indicates that the variation in PEPT1 expression is one of the major factors accounting for variable intestinal cephalexin absorption. To our knowledge, this is the most direct evidence that variation of PEPT1 expression is correlated with absorption permeability variation of peptide-like compounds in vitro and in vivo.

Function and expression of multidrug resistance-associated protein family in human colon adenocarcinoma cells (Caco-2).

Several organic anions are actively extruded from intestinal epithelial cells into the lumen and vascular sides. To examine the role of the multidrug resistance-associated protein (MRP) family in the intestinal efflux of organic anions, the function and expression of these proteins were investigated with Caco-2, a human adenocarcinoma cell line that retains many of the characteristics of normal enterocytes. [(3)H]2,4-Dinitrophenyl-S-glutathione (DNP-SG) and [(3)H]17beta-estradiol 17-beta-D-glucuronide (E(2)17betaG), typical substrates for MRP1 and cMOAT (canalicular multispecific organic anion transporter)/MRP2, were taken up into brush-border membrane vesicles (BBMVs) from Caco-2 in an ATP-dependent manner, with K(m) values of 16.9 +/- 7.2 and 9.4 +/- 1.2 microM, respectively. The uptake of [(3)H]DNP-SG into BBMVs was osmotically sensitive and stimulated to some extent by other nucleotide triphosphates (GTP, CTP, and UTP) but not by ADP or AMP. An ATPase inhibitor, vanadate, inhibited the ATP-dependent uptake of [(3)H]DNP-SG to some extent. Reverse-transcriptase polymerase chain reaction resulted in the amplification of MRP1, MRP3, and MRP5. Northern blot analysis indicated extensive expression of cMOAT/MRP2 and MRP3 and only minimal expression of MRP1 and MRP5. Although cMOAT/MRP2 was continuously expressed throughout the culture period, MRP3 was not expressed immediately after the confluent state was reached. Collectively, the presence of ATP-dependent transport systems for DNP-SG and E(2)17betaG was demonstrated in Caco-2 cells. Because cMOAT/MRP2 and MRP3 may be expressed on brush-border and basolateral membranes in epithelial cells, respectively, the transport activity associated with BBMVs may result from the function of cMOAT/MRP2.

Possible involvement of P-glycoprotein in biliary excretion of CPT-11 in rats.

In our previous work, we found that the biliary excretion of the carboxylate form of irinotecan, CPT-11, on rat bile canalicular membrane consists of two components, the low-affinity one being canalicular multispecific organic anion transporter (cMOAT). In the present study, we have investigated the high-affinity component by studying the uptake in canalicular membrane vesicles. The ATP-dependent uptake of the carboxylate form of CPT-11 was inhibited significantly by several substrates and/or modulators of P-glycoprotein, including PSC-833, verapamil, and cyclosporin A, at a substrate concentration of 5 microM, at which the high-affinity component is involved predominantly in CPT-11 transport. When the concentration of the carboxylate form of CPT-11 was 250 microM, at which the low-affinity component (cMOAT) is involved predominantly in its transport, the inhibitory effect of the above compounds was reduced greatly. Similarly, there was also much lower inhibition of the ATP-dependent uptake of S-(2,4-dinitrophenyl)-glutathione, a substrate of cMOAT, by the above compounds. Taurocholic acid, a substrate of canalicular bile acid transporter, failed to inhibit the uptake of CPT-11 at the substrate concentration of both 5 and 250 microM. These results suggest that P-glycoprotein may act as the high-affinity component in the biliary excretion of the carboxylate form of CPT-11 in rats.

Active efflux of CPT-11 and its metabolites in human KB-derived cell lines.

To investigate the possible involvement of P-glycoprotein (P-gp), multidrug resistance-associated protein (MRP), and/or other glutathione S-conjugate export pump (GS-X pump) family members on the active efflux of irinotecan [(7-ethyl-10-[4-(1-piperidino)-1-pipertidino)-1-piperidino]carb onylox y camptothecin (CPT-11)] and its metabolites, as well as their contribution to the acquisition of resistance, we studied the uptake of CPT-11, its active metabolite SN-38, and glucuronide conjugate (SN38-Glu) using membrane vesicles from human epidermoid KB-3-1-derived cell lines. These lines included KB-C2, C-A500, and KCP-4, which overexpress P-gp, MRP, and the unidentified GS-X pump, respectively. The carboxylate form of SN-38 exhibited significant ATP-dependent transport, with a Michaelis constant of 17 microM, into membrane vesicles from C-A500 but not from other cell lines. Among these KB-derived cells, significant ATP-dependent uptake of the carboxylate form of CPT-11 was only observed in KB-C2 vesicles. In addition, the uptake of the lactone and carboxylate forms of SN38-Glu into membrane vesicles from C-A500 and KB-C2, but not KCP-4, was ATP dependent, although the transport activity in C-A500 was much higher than that in KB-C2. The 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay revealed that the resistance of KB-C2 to CPT-11 and SN-38, compared with that of KB-3-1, was 6.3- and 6.8-fold, respectively; the corresponding figures for C-A500 were 12- and 27-fold, respectively, whereas those for KCP-4 were 2.3- and 20-fold, respectively. These results suggest that MRP and P-gp are involved in the active efflux of SN-38 and CPT-11, respectively, from human KB-derived cells. In addition, a difference in substrate specificity among GS-X pump members was demonstrated.

Biliary excretion mechanism of CPT-11 and its metabolites in humans: involvement of primary active transporters.

After administration of CTP-11, a camptothecin derivative exhibiting a wide spectrum of antitumor activity, dose-limiting gastrointestinal toxicity with great interpatient variability is observed. Because the biliary excretion is a major elimination pathway for CPT-11 and its metabolites [an active metabolite, 7-ethyl-10-hydroxy-camptothecin (SN-38), and its glucuronide, SN38-Glu], several hypotheses for the toxicity involve biliary excretion. Here, we investigated whether primary active transport is involved in the biliary excretion of anionic forms of CPT-11 and its metabolites in humans using bile canalicular membrane vesicles (cMVs). Uptake of the carboxylate form of CPT-11 and the carboxylate and lactone forms of SN38-Glu by cMVs prepared from five human liver samples was ATP dependent. The concentration dependence of the ATP-dependent uptake of the carboxylate form of CPT-11 and SN38-Glu suggests the involvement of at least two saturable transport components, both with lower affinity and higher capacity than in rats. The ATP-dependent uptake of the carboxylate form of SN-38 showed a single saturable component but was detectable only in one human cMV sample. Both carboxylate and lactone forms of SN38-Glu uptake also showed a large intersample variability, although the variability was less than that observed for the carboxylate form of SN-38. On the other hand, the carboxylate form of CPT-11 exhibited much less variability. The carboxylate forms of SN38-Glu and SN-38 almost completely inhibited the ATP-dependent uptake of leukotriene C4, a well-known substrate of canalicular multispecific organic anion transporter, whereas the inhibition by the carboxylate form of CPT-11 was not as marked. Thus, multiple primary active transport systems are responsible for the biliary excretion of CPT-11 and its metabolites, and the major transport system for CPT-11 differs from that for the other two compounds. A greater degree of inter-cMV variability in the uptake of SN-38 and SN38-Glu may imply that interindividual variability in biliary excretion of these metabolites might contribute to interpatient variability in the toxicity caused by CPT-11.

Multiplicity of biliary excretion mechanisms for irinotecan, CPT-11, and its metabolites in rats.

We have reported previously that a canalicular multispecific organic anion transporter (cMOAT) is responsible for the biliary excretion of carboxylate forms of irinotecan, 7-ethyl-10-[4-(1-piperidino)-1 piperidino] carbonyloxy camptothecin (CPT-11), its active metabolite SN-38, and glucuronide conjugate (SN38-Glu) and the lactone form of SN38-Glu in rats. In this paper, the multiplicity of biliary excretion mechanisms for these four anionic compounds was investigated using isolated liver bile canalicular membrane vesicles (CMVs) obtained from Sprague Dawley rats. For the carboxylate form of CPT-11 and the lactone and carboxylate forms of SN38-Glu, ATP-dependent uptake consisted of both high- and low-affinity components in CMVs. Mutual inhibition studies with S-(2,4-dinitrophenyl)glutathione, a representative substrate for cMOAT, and the uptake study using CMVs from Eisai hyperbilirubinemic rats revealed that cMOAT is responsible for the biliary excretion of the low-affinity component of the carboxylate form of CPT-11 and the high-affinity component of both the lactone and carboxylate forms of SN38-Glu, whereas the high-affinity component for CPT-11 and the low-affinity component for SN38-Glu, which are expressed in Eisai hyperbilirubinemic rats, are governed by a transporter different from cMOAT. The carboxylate form of SN-38 was found to be transported by cMOAT alone. We conclude that multiple transporters, including cMOAT, are responsible for the biliary excretion of CPT-11 and its metabolites (anionic forms), and the contribution of each transporter differs greatly, depending on the substrates.

Multispecific organic anion transporter is responsible for the biliary excretion of the camptothecin derivative irinotecan and its metabolites in rats.

Irinotecan, 7-ethyl-10-[4-(1-piperidino)-1-piperidino]-carbonyloxycamptothecin (CPT-11), is a potent anticancer drug that is increasingly used in chemotherapy. A frequent limiting side effect involves gastrointestinal toxicity (diarrhea), which is thought to be related to the biliary excretion of CPT-11 and its metabolites. Accordingly, the biliary excretion mechanisms for both the lactone and carboxylate forms of CPT-11 and its metabolites, SN-38 and its glucuronide (SN38-Glu), were investigated using Sprague-Dawley (SD) rats and Eisai hyperbilirubinemic rats (EHBR), with the latter being mutant rats with a genetic deficiency of the canalicular multispecific organic anion transporter. After i.v. administration of CPT-11, the biliary excretion clearance, defined as the biliary excretion rate normalized to the hepatic concentration, of both the lactone and carboxylate forms of SN38-Glu was much lower in EHBR. The biliary excretion clearance for the carboxylate form of both CPT-11 and SN-38 was also substantially smaller in EHBR and showed marked saturation with increasing dose only in SD rats. On the other hand, the biliary excretion clearance for the lactone forms of CPT-11 and SN-38 showed only a minimal difference in EHBR, compared with SD rats. These results suggest that, for the carboxylate form of CPT-11 and SN-38 and the carboxylate and lactone forms of SN38-Glu, there exists a specific transport system at the bile canalicular membrane that is deficient in EHBR. To confirm this hypothesis, the uptake of these substrates by isolated hepatic canalicular membrane vesicles (CMV) was examined. ATP-dependence was clearly observed for the uptake of these four compounds by CMV prepared from SD rats but not by CMV from EHBR. In addition, the compounds inhibited the ATP-dependent uptake of S-(2,4-dinitrophenyl) glutathione by CMV from SD rats, in a concentration-dependent manner. These results suggest that the biliary excretion of the carboxylate forms of CPT-11 and SN-38 and the carboxylate and lactone forms of SN38-Glu is mediated by the multispecific organic anion transporter, which is deficient in EHBR.

[Pharmacokinetics and disposition of vinpocetine in rats].

A reverse-phase HPLC method for determination of vinpocetine in biological samples was developed. The method was simple, highly specific and accurate. After i.v. administration of vinpocetine in rats, the plasma concentration-time curves of vinpocetine was found to be fitted to a two-compartment open model. Dosing 5 and 10 mg.kg-1 vinpocetine in rats, the elimination of the drug from plasma accorded with linear kinetics and the elimination half-lives were shown to be 1.76 +/- 0.27 h. The volumes of distribution were 7.30 +/- 0.49 L.kg-1 (5 mg.kg-1) and 6.07 +/- 0.67 L.kg-1 (10 mg.kg-1) respectively. The drug levels were high in the lung, spleen, liver and kidney, moderate in brain, fat and testis and low in heart, muscle and blood. Our results demonstrate that vinpocetine was eliminated in a rapid rate and distributed widely in the body. The bioavailability of vinpocetine after ig administration was 54.54% in rats. A small amount of unchanged vinpocetine was detected in urine, feces and bile after i.v. and ig administration.