Tumor-derived extracellular vesicles confer 5-fluorouracil resistance in esophageal cancer via long noncoding RNA AC116025.2 delivery.

5-Fluorouracil (5-FU) resistance is one of the main causes for treatment failure in esophageal cancer (EC). Here, we intended to elucidate the mechanism of tumor-derived extracellular vesicles (TEVs)-encapsulated long noncoding RNAs (lncRNAs) AC116025.2 in 5-FU resistance in EC. EVs were isolated from the serum samples of EC patients and HEEC, TE-1, and TE-1/5-FU cells, followed by RT-qPCR detection of AC116025.2 expression in EVs. The relationship among AC116025.2, microRNA (miR)-4496, and SEMA5A was evaluated. Next, EC cells were cocultured with EVs, followed by lentivirus transduction and plasmid transfection for studying the role of TEVs-AC116025.2 in EC cells in relation to miR-4496 and SEMA5A. Tumor formation in nude mice was applied for in vivo confirmation. Elevated AC116025.2 expression was seen in the EVs from the serum of 5-FU insensitive patients and from 5-FU-resistant EC cells. Mechanistically, AC116025.2 bound to miR-4496 that inversely targeted SEMA5A in EC cells. EVs-oe-AC116025.2 augmented EC cell viability, colony formation, and 5-FU resistance, but diminished their apoptosis through miR-4496-mediated SEMA5A. Furthermore, EVs-oe-AC116025.2 augmented tumor formation and 5-FU resistance of EC cells in vivo. Conclusively, our data offered evidence of the promoting mechanism of TEVs in the 5-FU resistance of EC by delivering AC116025.2.

DNA methylation mediates the genetic variants on ticagrelor major metabolite elimination and platelet function recovery after ticagrelor discontinuation.

Aim: To investigate the influence of DNA methylation on ticagrelor major metabolite M8 elimination and platelet function recovery after ticagrelor discontinuation. Materials & methods: Among healthy Chinese subjects, a causal inference test was conducted to identify CpG sites located on absorption, distribution, metabolism and excretion genes that mediate genetic variants on M8 elimination. Colocalization analysis was used to identify the CpG sites that shared causal variants with platelet function recovery. Results: cg05300248 (CHST9), cg05640674 (SLC22A5) and cg00846580 (DHRS7) mediated genetic variants on the M8 elimination. cg06338150 (NOTCH1) and cg17456097 (RPS6KA1) were demonstrated to have strong evidence of colocalization with platelet function recovery. Conclusion: The results provide new biological insights into the impact of DNA methylation on M8 elimination and platelet function recovery after ticagrelor discontinuation. Clinical trial registration: clinicaltrials.gov, identifier: NCT03092076.

Tryptophan Metabolites as Biomarkers for Esophageal Cancer Susceptibility, Metastasis, and Prognosis.

Background: Perturbation of tryptophan (TRP) metabolism contributes to the immune escape of cancer; however, the explored TRP metabolites are limited, and their efficacy in clarifying the susceptibility and progression of esophageal cancer (EC) remains ambiguous. Our study sought to evaluate the effects of the TRP metabolic profile on the clinical outcomes of EC using a Chinese population cohort; and to develop a risk prediction model targeting TRP metabolism. Method: A total of 456 healthy individuals as control subjects and 393 patients with EC who were followed up for one year as case subjects were enrolled. Quantification of the plasma concentrations of TRP and its metabolites was performed using HPLC-MS/MS. The logistic regression model was applied to evaluate the effects of the clinical characteristics and plasma metabolites of the subjects on susceptibility and tumor metastasis events, whereas Cox regression analysis was performed to assess the overall survival (OS) of the patients. Results: Levels of creatinine and liver enzymes were substantially correlated with multiple metabolites/metabolite ratios in TRP metabolism, suggesting that hepatic and renal function would exert effects on TRP metabolism. Age- and sex-matched case-control subjects were selected using propensity score matching. Plasma exposure to 5-HT was found to be elevated 3.94-fold in case subjects (N = 166) compared to control subjects (N = 203), achieving an AUC of 0.811 for predicting susceptibility event. Subsequent correlation analysis indicated that a higher plasma exposure to 5-HIAA significantly increased the risk of lymph node metastasis (OR: 2.16, p = 0.0114). Furthermore, it was figured out that OS was significantly shorter for patients with elevated XA/KYN ratio (HR: 1.99, p = 0.0016), in which medium and high levels of XA/KYN versus low level had a significantly lower OS (HR: 0.48, p = 0.0080 and HR: 0.42, p = 0.0031, respectively). Conclusion: This study provides a pivotal basis for targeting endogenous TRP metabolism as a potential therapeutic intervention.

Estimation of Mycophenolic Acid Exposure in Heart Transplant Recipients by Population Pharmacokinetic and Limited Sampling Strategies.

Purpose: The aim of this study is i) to establish a strategy to estimate the area under the curve of the dosing interval (AUC0-12h) of mycophenolic acid (MPA) in the heart transplant recipients and ii) to find the covariates that significantly affect the pharmacokinetics of MPA exposure. Methods: This single-center, prospective, open-label, observational study was conducted in 91 adult heart transplant recipients orally taking mycophenolate mofetil dispersible tablets. Samples collected intensively and sparsely were analyzed by the enzyme-multiplied immunoassay technique, and all the data were used in PPK modeling. Potential covariates were tested stepwise. The goodness-of-fit plots, the normalized prediction distribution error, and prediction-corrected visual predictive check were used for model evaluation. Optimal sampling times by ED-optimal strategy and multilinear regression (MLR) were analyzed based on the simulated data by the final PPK model. Moreover, using intensive data from 14 patients, the accuracy of AUC0-12h estimation was evaluated by Passing-Bablok regression analysis and Bland-Alman plots for both the PPK model and MLR equation. Results: A two-compartment model with first-order absorption and elimination with a lag time was chosen as the structure model. Co-medication of proton pump inhibitors (PPIs), estimated glomerular filtration rate (eGFR), and albumin (ALB) were found to significantly affect bioavailability (F), clearance of central compartment (CL/F), and the distribution volume of the central compartment (V2/F), respectively. Co-medication of PPIs decreased F by 27.6%. When eGFR decreased by 30 ml/min/1.73 m2, CL/F decreased by 23.7%. However, the impact of ALB on V2/F was limited to MPA exposure. The final model showed an adequate fitness of the data. The optimal sampling design was pre-dose and 1 and 4 h post-dose for pharmacokinetic estimation. The best-fit linear equation was finally established as follows: AUC0-12h = 3.539 × C0 + 0.288 × C0.5 + 1.349 × C1 + 6.773 × C4.5. Conclusion: A PPK model was established with three covariates in heart transplant patients. Co-medication of PPIs and eGFR had a remarkable impact on AUC0-12h of MPA. A linear equation was also concluded with four time points as an alternative way to estimate AUC0-12h for MPA.

Association of Malnutrition, Left Ventricular Ejection Fraction Category, and Mortality in Patients Undergoing Coronary Angiography: A Cohort With 45,826 Patients.

Background: The regulatory effect of the left ventricular ejection fraction (LVEF) categories on the association of malnutrition and all-cause mortality in patients undergoing coronary angiography (CAG) have not been adequately addressed. Methods: Forty-five thousand eight hundred and twenty-six patients consecutively enrolled in the Cardiorenal ImprovemeNt (CIN) study (ClinicalTrials.gov NCT04407936) from January 2008 to July 2018 who underwent coronary angiography (CAG). The Controlling Nutritional Status (CONUT) score was applied to 45,826 CAG patients. The hazard ratios of mortality across combined LVEF and/or malnutrition categories were estimated by Cox regression models. Variables adjusted for in the Cox regression models included: age, gender, hypertension (HT), DM, PCI, coronary artery disease (CAD), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), triglyceride (TRIG), chronic kidney disease (CKD), statins, atrial fibrillation (AF), anemia, and stroke. Population attributable risk (PAR) was estimated for eight groups stratified by nutritional status and LVEF categories. Results: In our study, 42,181(92%) of patients were LVEF ≥ 40%, of whom, 41.55 and 9.34% were in mild and moderate or severe malnutrition status, respectively, while 46.53 and 22.28% in mild and moderate or severe malnutritional status among patients with LVEF < 40%. During a median follow-up time of 4.5 years (percentile 2.8-7.1), 5,350 (11.7%) patients died. After fully adjustment, there is no difference of mortality on malnutrition in LVEF < 40% group (mild, moderate and severe vs. normal, HR (95%CI): [1.00 (0.83-0.98)], [1.20 (0.95-1.51)], [1.41 (0.87-2.29)], respectively, p for trend =0.068), but malnutrition was related to markedly increased risk of mortality in LVEF ≥ 40% group (mild, moderate, and severe vs. normal, HR (95%CI): [1.21 (1.12-1.31)], [1.56 (1.40-1.74)], and [2.20(1.67-2.90)], respectively, p for trend < 0.001, and p for interaction < 0.001). Patients with LVEF ≥ 40% had a higher malnutrition-associated risk of mortality and a higher PAR than those with LVEF < 40%. Conclusions: Malnutrition is common in CAG patients and it has a greater effect on all-cause mortality and a higher PAR in patients with LVEF ≥ 40% than LVEF < 40%.

A 4-aminonaphthalimide-based fluorescent traceable prodrug with excellent photoinduced cytotoxicity.

A blue light activated anti-cancer prodrug, NST, was designed based on a photoactive 4-aminonaphthalimide derivative and an anticancer drug, 10-hydroxycamptothecin. NST was hard to be taken up by living cells and showed negligible dark cytotoxicity. The irradiation caused photocleavage of NST and resulted in high cytotoxicity.

Comprehensive Metabolomics Identified the Prominent Role of Glycerophospholipid Metabolism in Coronary Artery Disease Progression.

Background: Coronary stenosis severity determines ischemic symptoms and adverse outcomes. The metabolomic analysis of human fluids can provide an insight into the pathogenesis of complex disease. Thus, this study aims to investigate the metabolomic and lipidomic biomarkers of coronary artery disease (CAD) severity and to develop diagnostic models for distinguishing individuals at an increased risk of atherosclerotic burden and plaque instability. Methods: Widely targeted metabolomic and lipidomic analyses of plasma in 1,435 CAD patients from three independent centers were performed. These patients were classified as stable coronary artery disease (SCAD), unstable angina (UA), and myocardial infarction (MI). Associations between CAD stages and metabolic conditions were assessed by multivariable-adjusted logistic regression. Furthermore, the least absolute shrinkage and selection operator logistic-based classifiers were used to identify biomarkers and to develop prediagnostic models for discriminating the diverse CAD stages. Results: On the basis of weighted correlation network analysis, 10 co-clustering metabolite modules significantly (p < 0.05) changed at different CAD stages and showed apparent correlation with CAD severity indicators. Moreover, cross-comparisons within CAD patients characterized that a total of 72 and 88 metabolites/lipid species significantly associated with UA (vs. SCAD) and MI (vs. UA), respectively. The disturbed pathways included glycerophospholipid metabolism, and cysteine and methionine metabolism. Furthermore, models incorporating metabolic and lipidomic profiles with traditional risk factors were constructed. The combined model that incorporated 11 metabolites/lipid species and four traditional risk factors represented better discrimination of UA and MI (C-statistic = 0.823, 95% CI, 0.783-0.863) compared with the model involving risk factors alone (C-statistic = 0.758, 95% CI, 0.712-0.810). The combined model was successfully used in discriminating UA and MI patients (p < 0.001) in a three-center validation cohort. Conclusion: Differences in metabolic profiles of diverse CAD subtypes provided a new approach for the risk stratification of unstable plaque and the pathogenesis decipherment of CAD progression.

Adult mesenchymal stem cell ageing interplays with depressed mitochondrial Ndufs6.

Mesenchymal stem cell (MSC)-based therapy has emerged as a novel strategy to treat many degenerative diseases. Accumulating evidence shows that the function of MSCs declines with age, thus limiting their regenerative capacity. Nonetheless, the underlying mechanisms that control MSC ageing are not well understood. We show that compared with bone marrow-MSCs (BM-MSCs) isolated from young and aged samples, NADH dehydrogenase (ubiquinone) iron-sulfur protein 6 (Ndufs6) is depressed in aged MSCs. Similar to that of Ndufs6 knockout (Ndufs6-/-) mice, MSCs exhibited a reduced self-renewal and differentiation capacity with a tendency to senescence in the presence of an increased p53/p21 level. Downregulation of Ndufs6 by siRNA also accelerated progression of wild-type BM-MSCs to an aged state. In contrast, replenishment of Ndufs6 in Ndufs6-/--BM-MSCs significantly rejuvenated senescent cells and restored their proliferative ability. Compared with BM-MSCs, Ndufs6-/--BM-MSCs displayed increased intracellular and mitochondrial reactive oxygen species (ROS), and decreased mitochondrial membrane potential. Treatment of Ndufs6-/--BM-MSCs with mitochondrial ROS inhibitor Mito-TEMPO notably reversed the cellular senescence and reduced the increased p53/p21 level. We provide direct evidence that impairment of mitochondrial Ndufs6 is a putative accelerator of adult stem cell ageing that is associated with excessive ROS accumulation and upregulation of p53/p21. It also indicates that manipulation of mitochondrial function is critical and can effectively protect adult stem cells against senescence.

The resistance of esophageal cancer cells to paclitaxel can be reduced by the knockdown of long noncoding RNA DDX11-AS1 through TAF1/TOP2A inhibition.

Esophageal cancer (EC) is one of the most common malignancies in the world. The currently used chemotherapeutic drug for the treatment of EC is paclitaxel (PTX), the efficacy of which is affected by the development of drug resistance. The present study aims to define the role of the long noncoding RNA (lncRNA) DDX11-AS1 in the progression of EC with the involvement of PTX-resistant EC cells. First, EC and adjacent normal tissue samples were collected from 82 patients with EC, after which the expression levels of DDX11-AS1, TOP2A and TAF1 were determined. The results showed that DDX11-AS1, TOP2A and TAF1 were highly expressed in EC tissues, and there was a positive correlation between the expression levels of DDX11-AS1 and TOP2A. A PTX-resistant EC cell line was constructed. Next, we evaluated the effects of DDX11-AS1 and TOP2A on the resistance of EC cells to PTX, and the regulatory relationships between DDX11-AS1, TOP2A and TAF1 were investigated. DDX11-AS1 could promote TOP2A transcription via TAF1, and the knockdown of TOP2A or DDX11-AS1 could increase the sensitivity of EC cells to PTX. The effect of DDX11-AS1 on the growth of PTX-inhibited tumors was confirmed using a tumor formation assay in nude mice. It was verified that knocking down DDX11-AS1 reduced the expression level of TOP2A and inhibited tumor growth. In conclusion, our findings suggest that DDX11-AS1 knockdown results in reduced resistance of EC cells to PTX by inhibiting TOP2A transcription via TAF1. Therefore, DDX11-AS1 knockdown could be a promising therapeutic strategy for EC.

MicroRNA-188 aggravates contrast-induced apoptosis by targeting SRSF7 in novel isotonic contrast-induced acute kidney injury rat models and renal tubular epithelial cells.

BACKGROUND: Contrast media (CM) is widely used in cardiac catheterization; however, it may cause contrast-induced acute kidney injury (CI-AKI) which severely increases mortality. MicroRNA (miRNA) has been found to participate in the process of acute kidney injury (AKI), and this discovery has great potential for diagnosis and treatment. However, the role of miRNA in CI-AKI is still unclear. This study aimed to investigate the regulatory effect miRNAs exert in CI-AKI. METHODS: We established a novel, representative, isotonic CI-AKI model by using CM iodixanol, a CM which is commonly used in clinic. Next-generation sequencing and reverse transcription polymerase chain reaction (RT-qPCR) were performed to determine the expression of miRNA-188 in CI-AKI. Western blot analysis of the apoptosis regulator protein and TUNEL assay were ordered to evaluate apoptosis. Bioinformatics and double luciferin reporter gene assay were performed to predict and to confirm the interaction between microRNA-188 and SRSF7. RESULTS: The novel isotonic CI-AKI rat model we established exhibited typical characteristics of CI-AKI in serum creatinine, cystatin C, HE staining, and under electron microscope observation. Sequencing and RT-qPCR demonstrated that miRNA-188 was significantly up-regulated both in CI-AKI rat and HK-2 cell models while overexpression of miRNA-188 significantly aggravated apoptosis in CI-AKI cell models. SRSF7 was identified as a direct target gene of miRNA-188, and dual luciferase reporter assay determined the direct interaction between SRSF7 and miRNA-188. In addition, SRSF7 silencing reduced the cell viability rate of the CI-AKI cell model. CONCLUSIONS: The present study's findings indicate that miRNA-188 aggravated contrast-induced apoptosis by regulating SRSF7, which may serve as a potential drug target for CI-AKI intervention.

A genome-wide association study on lipoprotein (a) levels and coronary artery disease severity in a Chinese population.

Lipoprotein (a) [Lp(a)] is a genetically determined risk factor of coronary artery disease (CAD). Previous genome-wide association studies (GWASs), which were mostly carried out in Caucasians, have identified many Lp(a)-associated SNPs. Here, we performed a GWAS on Lp(a) levels and further explored the relationships between Lp(a)-associated SNPs and CAD severity in 1,403 Han Chinese subjects. We observed that elevated Lp(a) levels were significantly associated with the increased synergy between percutaneous coronary intervention with TAXUS and cardiac surgery (SYNTAX) score and the counts of heavily calcified lesions and long-range lesions (LRLs; P < 0.05), which are defined as lesions spanning >20 mm. Moreover, we identified four independent SNPs, namely, rs7770628, rs73596816, and rs6926458 in LPA, and rs144217738 in SLC22A2, that were significantly associated with Lp(a) levels. We also found that rs7770628 was associated with high SYNTAX scores [odds ratio (OR) (95% CI): 1.37 (1.05-1.80), P = 0.0213, false discovery rate (FDR) = 0.0852], and that rs7770628 and rs73596816 were associated with high risk of harboring LRLs [OR (95% CI): 1.53 (1.17-2.01), P = 0.0018, FDR = 0.0072 and 1.72 (1.19-2.49), P = 0.0040, FDR = 0.0080, respectively]. Our study was a large-scale GWAS to identify Lp(a)-associated variants in the Han Chinese population. Our findings highlight the importance and potential of Lp(a) intervention and expand our understanding of CAD prevention and treatment.

Effects of SLCO1B1 and GATM gene variants on rosuvastatin-induced myopathy are unrelated to high plasma exposure of rosuvastatin and its metabolites.

Myotoxicity is a significant factor contributing to the poor adherence and reduced effectiveness in the treatment of statins. Genetic variations and high drug plasma exposure are considered as critique causes for statin-induced myopathy (SIM). This study aims to explore the sequential influences of rosuvastatin (RST) pharmacokinetic and myopathy-related single-nucleotide polymorphisms (SNPs) on the plasma exposure to RST and its metabolites: rosuvastatin lactone (RSTL) and N-desmethyl rosuvastatin (DM-RST), and further on RST-induced myopathy. A total of 758 Chinese patients with coronary artery disease were enrolled and followed up SIM incidents for 2 years. The plasma concentrations of RST and its metabolites were determined through a validated ultra-performance liquid chromatography mass spectrometry method. Nine SNPs in six genes were genotyped by using the Sequenom MassArray iPlex platform. Results revealed that ABCG2 rs2231142 variations were highly associated with the plasma concentrations of RST, RSTL, and DM-RST (Padj < 0.01, FDR < 0.05). CYP2C9 rs1057910 significantly affected the DM-RST concentration (Padj < 0.01, FDR < 0.05). SLCO1B1 rs4149056 variant allele was significantly associated with high SIM risk (OR: 1.741, 95% CI: 1.180-2.568, P = 0.0052, FDR = 0.0468). Glycine amidinotransferase (GATM) rs9806699 was marginally associated with SIM incidents (OR: 0.617, 95% CI: 0.406-0.939, P = 0.0240, FDR = 0.0960). The plasma concentrations of RST and its metabolites were not significantly different between the SIM (n = 51) and control groups (n = 707) (all P > 0.05). In conclusion, SLCO1B1 and GATM genetic variants are potential biomarkers for predicting RST-induced myopathy, and their effects on SIM are unrelated to the high plasma exposure of RST and its metabolites.

Gender Differences in the Hepatotoxicity and Toxicokinetics of Emodin: The Potential Mechanisms Mediated by UGT2B7 and MRP2.

Emodin is a main anthraquinone compound which exists in Chinese traditional medicines including Polygonum multiflorum and Rhubarb. It is documented to have obvious liver and kidney toxicity. This study aims to (a) estimate gender differences of the hepatotoxicity and toxicokinetics in rats after oral administration of emodin (60 and 150 mg/kg/d) for a consecutive 28 days and (b) clarify relative mechanisms caused by glucuronidation and disposition. Hepatotoxicity was significantly higher in female rats than that in male rats, as evidenced by histopathological and biochemical tests. Similarly, the toxicokinetic profiles of emodin have time and gender differences, which could cause time and gender differences in hepatotoxicity. The metabolic and transcriptomics data of 55 human liver and 36 human kidney samples demonstrated that UDP-glucuronosyltransferase 2B7 (UGT2B7) was the predominant enzyme for emodin glucuronidation. A genome-wide association study (GWAS) identified that rs11726899 located within ∼50 kb of the transcript of UGT2B could significantly affect emodin metabolism. Knockdown of UGT2B7 in HepG2 cells significantly decreased emodin glucuronidation and increased cytotoxicity of emodin. The gene expression and protein levels of UGT2B7 were decreased, but those of the multidrug-resistant-protein 2 (MRP2) were increased in HepG2 cells after being treated with 50 µM emodin for 48 h. Long-term use of emodin could decrease the intrinsic clearance (CLint, decreased by 18.5%-35.4%) values of zidovidue (UGT2B7 substrate) glucuronide in both male and female liver microsomes from rats administrated with emodin for 28 days, thus causing the accumulation of emodin. However, higher self-induced MRP2 expression and lower hepatotoxicity were observed in emodin-treated male rats compared to that in female rats. Therefore, gender differences in the hepatotoxicity and toxicokinetics of emodin are potentially mediated by the coupling of UGT2B7 and MRP2 in vivo.

SLCO1B1 521T > C polymorphism associated with rosuvastatin-induced myotoxicity in Chinese coronary artery disease patients: a nested case-control study.

PURPOSE: This nested case-control study aimed to evaluate the association of candidate genetic variants with statin-induced myotoxicity in Chinese patients with coronary artery disease (CAD). METHODS: One hundred forty-eight Chinese patients experiencing statin-induced myotoxicity were included in our study, and 255 patients without muscular side effects served as controls. Five SNPs in CYP3A5, SLCO1B1, and APOE were genotyped. The effect of genetic variants on statin-induced myotoxicity was assessed. RESULTS: Patients who carried at least one SLCO1B1 521C allele had a higher risk for myotoxicity (OR = 1.69, 95%CI = 1.07-2.67, P = 0.024). Significant association was found between SLCO1B1 521C mutant allele mutation and risk of myotoxicity in individuals that received rosuvastatin (OR = 3.67, 95%CI = 1.42-9.47, P = 0.007). However, non-significant association was observed between 521C mutant allele and risk of myotoxicity (P > 0.5) in patients that received atorvastatin and simvastatin. The other four single nucleotide polymorphisms (SNPs), namely rs776746, rs2306283, rs7412, and rs429358, showed no significant association with any statin induced myotoxicity (P > 0.5). CONCLUSIONS: SLCO1B1 (rs4149056, 521T > C) is associated with statin-induced myotoxicity in Chinese patients with coronary artery disease. In addition, SLCO1B1 521C mutant allele increased the risk of rosuvastatin-associated myotoxicity.

Relation of Transcriptional Factors to the Expression and Activity of Cytochrome P450 and UDP-Glucuronosyltransferases 1A in Human Liver: Co-Expression Network Analysis.

Cytochrome P450 (CYPs) and UDP-glucuronosyltransferases (UGTs) play important roles in the metabolism of exogenous and endogenous compounds. The gene transcription of CYPs and UGTs can be enhanced or reduced by transcription factors (TFs). This study aims to explore novel TFs involved in the regulatory network of human hepatic UGTs/CYPs. Correlations between the transcription levels of 683 key TFs and CYPs/UGTs in three different human liver expression profiles (n = 640) were calculated first. Supervised weighted correlation network analysis (sWGCNA) was employed to define hub genes among the selected TFs. The relationship among 17 defined TFs, CYPs/UGTs expression, and activity were evaluated in 30 liver samples from Chinese patients. The positive controls (e.g., PPARA, NR1I2, NR1I3) and hub TFs (NFIA, NR3C2, and AR) in the GreysWGCNA Module were significantly and positively associated with CYPs/UGTs expression. And the cancer- or inflammation-related TFs (TEAD4, NFKB2, and NFKB1) were negatively associated with mRNA expression of CYP2C9/CYP2E1/UGT1A9. Furthermore, the effect of NR1I2, NR1I3, AR, TEAD4, and NFKB2 on CYP450/UGT1A gene transcription translated into moderate influences on enzyme activities. To our knowledge, this is the first study to integrate Gene Expression Omnibus (GEO) datasets and supervised weighted correlation network analysis (sWGCNA) for defining TFs potentially related to CYPs/UGTs. We detected several novel TFs involved in the regulatory network of hepatic CYPs and UGTs in humans. Further validation and investigation may reveal their exact mechanism of CYPs/UGTs regulation.

Simultaneous Determination of Ticagrelor and Its Metabolites in Human Plasma and Urine Using Liquid Chromatography-Tandem Mass Spectrometry.

We have developed and validated a rapid, selective and sensitive method using high-performance liquid chromatography-tandem mass spectrometry (MS) for the quantification of ticagrelor and all of its as-yet-identified metabolites in human plasma and urine. For the analysis of ticagrelor, its metabolites and the internal standard (IS) plasma samples were processed by liquid-liquid extraction using ethyl acetate and urine was processed by protein precipitation. Separations were performed on an Ultimate XB-C18 column (2.1 mm × 150 mm, 3 µm), using aqueous ammonium acetate (0.025 mM)/acetonitrile (35 : 65, v:v) as the mobile phase. Ticagrelor and all 11 metabolites were eluted within 4.5 min. Quantification was performed using electrospray ionization, operating in negative ion mode. The ticagrelor and metabolite M8 (AR-C124910XX) responses were optimized at the m/z 521.2 → 361.2 and m/z 477.2 → 361.1 transitions, respectively. The assay was validated over the linear range of 0.5-2,000 ng/mL for ticagrelor and M8. The intra- and inter-assay precisions were ≤14.6% for ticagrelor and ≤14.7% for M8, respectively. The matrix effects of plasma and urine were in the range of 98.3-110.7% for ticagrelor and 102.1-112.3% for M8. The relative quantification of other metabolites was performed by assessing the ratio of metabolite to IS peaks. The newly developed method was successfully used in a pharmacokinetic study characterizing ticagrelor metabolism in human volunteers.

Smad3 inactivation and MiR-29b upregulation mediate the effect of carvedilol on attenuating the acute myocardium infarction-induced myocardial fibrosis in rat.

Carvedilol, a nonselective ß-adrenoreceptor antagonist, protects against myocardial injury induced by acute myocardium infarction (AMI). The mechanisms underlying the anti-fibrotic effects of carvedilol are unknown. Recent studies have revealed the critical role of microRNAs (miRNAs) in a variety of cardiovascular diseases. This study investigated whether miR-29b is involved in the cardioprotective effect of carvedilol against AMI-induced myocardial fibrosis. Male SD rats were randomized into several groups: the sham surgery control, left anterior descending (LAD) surgery-AMI model, AMI plus low-dose carvedilol treatment (1 mg/kg per day, CAR-L), AMI plus medium-dose carvedilol treatment (5 mg/kg per day, CAR-M) and AMI plus high-dose carvedilol treatment (10 mg/kg per day, CAR-H). Cardiac remodeling and impaired heart function were observed 4 weeks after LAD surgery treatment; the observed cardiac remodeling, decreased ejection fraction, and fractional shortening were rescued in the CAR-M and CAR-H groups. The upregulated expression of Col1a1, Col3a1, and α-SMA mRNA was significantly reduced in the CAR-M and CAR-H groups. Moreover, the downregulated miR-29b was elevated in the CAR-M and CAR-H groups. The in vitro study showed that Col1a1, Col3a1, and α-SMA were downregulated and miR-29b was upregulated by carvedilol in a dose-dependent manner in rat cardiac fibroblasts. Inhibition of ROS-induced Smad3 activation by carvedilol resulted in downregulation of Col1a1, Col3a1, and α-SMA and upregulation of miR-29b derived from the miR-29b-2 precursor. Enforced expression of miR-29b significantly suppressed Col1a1, Col3a1, and α-SMA expression. Taken together, we found that smad3 inactivation and miR-29b upregulation contributed to the cardioprotective activity of carvedilol against AMI-induced myocardial fibrosis.

Pharmacokinetics, excretion, and distribution of combretastatin A4 phosphate in rats.

In order to characterize the pharmacokinetics, excretion, and distribution of combretastatin A4 phosphate (CA4P) and its active metabolite, combretastatin A4 (CA4), in rats, a reliable gradient HPLC-based method has been developed and validated. The pharmacokinetic profiles of CA4P and CA4 in rats after CA4P intravenous injection at doses of 0.7, 1 and 4 mg x kg(-1) were best described by a two-compartment model. The terminal half-lives of CA4P or CA4 were similar at different CA4P dose levels, 5-9 min for CA4P and 39-60 min for CA4, while t1/2alpha, and Vd of CA4P or CA4 were very different. CA4 was largely distributed to the heart, intestine, lung, spleen and liver during 15 to 40 min after intravenous injection of CA4P. CA4P was predominantly excreted into urine (10.72%) and feces (9.703%) and to a lesser extent into bile (0.897%), whereas a greater portion of CA4 were excreted into feces (6.235%) and to a lesser extent into urine (0.782%) and bile (0.496%) during 0-28 h after intravenous injection of 1 mg x kg(-1) to rats. This is the first study to characterize the distribution of the active CA4P metabolite, CA4, in rat.

Phosphoinositide-3-kinase, catalytic, alpha polypeptide RNA interference inhibits growth of colon cancer cell SW948.

AIM: To investigate the gene knock-down effect by the phosphoinositide-3-kinase, catalytic, alpha polypeptide (PIK3CA)-targeted double-stranded RNA (dsRNA) and its effect on cell proliferation and cycle distribution in SW948. METHODS: Two PIK3CA-targeted dsRNAs were constructed and transfected into SW948 cells. Transfections were performed using lipofectamine™ 2000. The transfection effectiveness was calculated basing on the rate of fluorescence cell of SW948 at 6 h after transfection. Total messenger RNA was extracted from these cells using the RNeasy kit, and semiquantitative reverse transcription polymerase chain reaction was performed to detect the down-regulation of PIK3CA, AKT1, MYC, and CCND1 gene expression. Cells were harvested, proteins were resolved, and western blot was employed to detect the expression levels of PIK3CA, AKT1, MYC, and CCND1 gene. Cell proliferation was assessed by 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di-phenytetrazoliumromide assay and the inhibition rate was calculated. Soft agar colony formation assay was performed basing on colonies greater than 60 µm in diameter at ×100 magnification. The effect on cell cycle distribution and apoptosis was assessed by flow cytometry. All experiments were performed in triplicate. RESULTS: Green fluorescence was observed in SW948 cell transfected with plasmid Pgenesil-1, and the transfection effectiveness was about 65%. Forty-eight hours post-transfection, mRNA expression of PIK3CA in SW948 cells was 0.51 ± 0.04 vs 0.49 ± 0.03 vs 0.92 ± 0.01 vs 0.93 ± 0.03 (P = 0.001 ) in Pgenesil-CA1, Pgenesil-CA2, negative and blank group respectively. mRNA expression of AKT1 was 0.50 ± 0.03 vs 0.48 ± 0.01 vs 0.93 ± 0.04 vs 0.92 ± 0.02 (P = 0.000) in Pgenesil-CA1, Pgenesil-CA2, negative and blank group respectively. mRNA expression of MYC was 0.49 ± 0.01 vs 0.50 ± 0.04 vs 0.90 ± 0.02 vs 0.91 ± 0.03 (P = 0.001) in the four groups respectively. mRNA expression of CCND1 was 0.45 ± 0.02 vs 0.51 ± 0.01 vs 0.96 ± 0.03 vs 0.98 ± 0.01 (P = 0.001) in the four groups respectively. The protein level of PIK3CA was 0.53 ± 0.01 vs 0.54 ± 0.02 vs 0.92 ± 0.03 vs 0.91 ± 0.02 (P = 0.001) in Pgenesil-CA1, Pgenesil-CA2, negative and blank group respectively. The protein level of AKT1 in the four groups was 0.49 ± 0.02 vs 0.55 ± 0.03 vs 0.94 ± 0.03 vs 0.95 ± 0.04, P = 0.000). The protein level of MYC in the four groups was 0.51 ± 0.03 vs 0.52 ± 0.04 vs 0.92 ± 0.02 vs 0.95 ± 0.01 (P = 0.000). The protein level of CCND1 in the four groups was 0.54 ± 0.04 vs 0.56 ± 0.03 vs 0.93 ± 0.01 vs 0.93 ± 0.03 (P = 0.000). Both Pgenesil-CA1 and Pgenesil-CA2 plasmids significantly suppressed the growth of SW948 cells when compared with the negative or blank group at 48 h after transfection (29% vs 25% vs 17% vs 14%, P = 0.001), 60 h after transfection (38% vs 34% vs 19% vs 16%, P = 0.001), and 72 h after transfection (53% vs 48% vs 20% vs 17%, P = 0.000). Numbers of colonies in negative, blank, CA1, and CA2 groups were 42 ± 4, 45 ± 5, 8 ± 2, and 10 ± 3, respectively (P = 0.000). There were more than 4.5 times colonies in the blank and negative control groups as there were in the CA1 and CA2 groups. In addition, the colonies in blank and negative control groups were also larger than those in the CA1 and CA2 groups. The percentage of cells in the CA1 and CA2 groups was significantly higher in G0/G1 phase, but lower in S and G2/M phase when compared with the negative and control groups. Moreover, cell apoptosis rates in the CA1 and CA2 groups were 5.11 ± 0.32 and 4.73 ± 0.32, which were significantly higher than those in negative (0.95 ± 0.11, P = 0.000) and blank groups (0.86 ± 0.13, P = 0.001). No significant difference was found between CA1 and CA2 groups in cell cycle distribution and apoptosis. CONCLUSION: PIK3CA-targeted short hairpin RNAs can block the phosphoinositide 3-kinase-Akt signaling pathway and inhibit cell growth, increase apoptosis, and induce cell cycle arrest in the PIK3CA-mutant colon cancer SW948 cells.

High levels of glucose induce "metabolic memory" in cardiomyocyte via epigenetic histone H3 lysine 9 methylation.

Diabetic patients continue to develop inflammation and cardiovascular complication even after achieving glycemic control, suggesting a "metabolic memory". Metabolic memory is a major challenge in the treatment of diabetic complication, and the mechanisms underlying metabolic memory are not clear. Recent studies suggest a link between chromatin histone methylation and metabolic memory. In this study, we tested whether histone 3 lysine-9 tri-methylation (H3K9me3), a key epigenetic chromatin marker, was involved in high glucose (HG)-induced inflammation and metabolic memory. Incubating cardiomyocyte cells in HG resulted in increased levels of inflammatory cytokine IL-6 mRNA when compared with myocytes incubated in normal culture media, whereas mannitol (osmotic control) has no effect. Chromatin immunoprecipitation (ChIP) assays showed that H3K9me3 levels were significantly decreased at the promoters of IL-6. Immunoblotting demonstrated that protein levels of the H3K9me3 methyltransferase, Suv39h1, were also reduced after HG treatment. HG-induced apoptosis, mitochondrial dysfunction and cytochrome-c release were reversible. However, the effects of HG on the expression of IL-6 and the levels of H3K9me3 were irreversible after the removal of HG from the culture. These results suggest that HG-induced sustained inflammatory phenotype and epigenetic histone modification, rather than HG-induced mitochondrial dysfunction and apoptosis, are main mechanisms responsible for metabolic memory. In conclusion, our data demonstrate that HG increases expression of inflammatory cytokine and decreases the levels of histone-3 methylation at the cytokine promoter, and suggest that modulating histone 3 methylation and inflammatory cytokine expression may be a useful strategy to prevent metabolic memory and cardiomyopathy in diabetic patients.