PCSK9 inhibitor recaticimab for hypercholesterolemia on stable statin dose: a randomized, double-blind, placebo-controlled phase 1b/2 study.

BACKGROUND: Recaticimab (SHR-1209, a humanized monoclonal antibody against PCSK9) showed robust LDL-C reduction in healthy volunteers. This study aimed to further assess the efficacy and safety of recaticimab in patients with hypercholesterolemia. METHODS: In this randomized, double-blind, placebo-controlled phase 1b/2 trial, patients receiving stable dose of atorvastatin with an LDL-C level of 2.6 mmol/L or higher were randomized in a ratio of 5:1 to subcutaneous injections of recaticimab or placebo at different doses and schedules. Patients were recruited in the order of 75 mg every 4 weeks (75Q4W), 150Q8W, 300Q12W, 150Q4W, 300Q8W, and 450Q12W. The primary endpoint was percentage change in LDL-C from the baseline to end of treatment (i.e., at week 16 for Q4W and Q8W schedule and at week 24 for Q12W schedule). RESULTS: A total of 91 patients were enrolled and received recaticimab and 19 received placebo. The dose of background atorvastatin in all 110 patients was 10 or 20 mg/day. The main baseline LDL-C ranged from 3.360 to 3.759 mmol/L. The least-squares mean percentage reductions in LDL-C from baseline to end of treatment relative to placebo for recaticimab groups at different doses and schedules ranged from -48.37 to -59.51%. No serious treatment-emergent adverse events (TEAEs) occurred. The most common TEAEs included upper respiratory tract infection, increased alanine aminotransferase, increased blood glucose, and increased gamma-glutamyltransferase. CONCLUSION: Recaticimab as add-on to moderate-intensity statin therapy significantly and substantially reduced the LDL-C level with an infrequent administration schedule (even given once every 12 weeks), compared with placebo. TRIAL REGISTRATION: ClinicalTrials.gov , number NCT03944109.

Triiodothyronine maintains cardiac transverse-tubule structure and function.

Subclinical hypothyroidism and low T3 syndrome are commonly associated with an increased risk of cardiovascular disease (CVD) and mortality. We examined effects of T3 on T-tubule (TT) structures, Ca2+ mobilization and contractility, and clustering of dyadic proteins. Thyroid hormone (TH) deficiency was induced in adult female rats by propyl-thiouracil (PTU; 0.025%) treatment for 8 weeks. Rats were then randomized to continued PTU or triiodo-L-thyronine (T3; 10 µg/kg/d) treatment for 2 weeks (PTU + T3). After in vivo echocardiographic and hemodynamic recordings, cardiomyocytes (CM) were isolated to record Ca2+ transients and contractility. TT organization was assessed by confocal microscopy, and STORM images were captured to measure ryanodine receptor (RyR2) cluster number and size, and L-type Ca2+ channel (LTCC, Cav1.2) co-localization. Expressed genes including two integral TT proteins, junctophilin-2 (Jph-2) and bridging integrator-1 (BIN1), were analyzed in left ventricular (LV) tissues and cultured CM using qPCR and RNA sequencing. The T3 dosage used normalized serum T3, and reversed adverse effects of TH deficiency on in vivo measures of cardiac function. Recordings of isolated CM indicated that T3 increased rates of Ca2+ release and re-uptake, resulting in increased velocities of sarcomere shortening and re-lengthening. TT periodicity was significantly decreased, with reduced transverse tubules but increased longitudinal tubules in TH-deficient CMs and LV tissue, and these structures were normalized by T3 treatment. Analysis of STORM data of PTU myocytes showed decreased RyR2 cluster numbers and RyR localizations within each cluster without significant changes in Cav1.2 localizations within RyR clusters. T3 treatment normalized RyR2 cluster size and number. qPCR and RNAseq analyses of LV and cultured CM showed that Jph2 expression was T3-responsive, and its increase with treatment may explain improved TT organization and RyR-LTCC coupling.

BNP as a New Biomarker of Cardiac Thyroid Hormone Function.

BACKGROUND: Cardiac re-expression of fetal genes in patients with heart failure (HF) suggests the presence of low cardiac tissue thyroid hormone (TH) function. However, serum concentrations of T3 and T4 are often normal or subclinically low, necessitating an alternative serum biomarker for low cardiac TH function to guide treatment of these patients. The clinical literature suggests that serum Brain Natriuretic Peptide (BNP) levels are inversely associated with serum triiodo-L-thyronine (T3) levels. The objective of this study was to investigate BNP as a potential serum biomarker for TH function in the heart. METHODS: Two animal models of thyroid hormone deficiency: (1) 8-weeks of propyl thiouracil-induced hypothyroidism (Hypo) in adult female rats were subsequently treated with oral T3 (10 µg/kg/d) for 3, 6, or 14 days; (2) HF induced by coronary artery ligation (myocardial infarction, MI) in adult female rats was treated daily with low dose oral T3 (5 µg/kg/d) for 8 or 16 wks. RESULTS: Six days of T3 treatment of Hypo rats normalized most cardiac functional parameters. Serum levels of BNP increased 5-fold in Hypo rats, while T3 treatment normalized BNP by day 14, showing a significant inverse relationship between serum BNP and free or total T3 concentrations. Myocardial BNP mRNA was increased 2.5-fold in Hypo rats and its expression was decreased to normal values by 14 days of T3 treatment. Measurements of hemodynamic function showed significant dysfunction in MI rats after 16 weeks, with serum BNP increased by 4.5-fold and serum free and total T3 decreased significantly. Treatment with T3 decreased serum BNP while increasing total T3 indicating an inverse correlation between these two biologic factors (r 2 = 0.676, p < 0.001). Myocardial BNP mRNA was increased 5-fold in MI rats which was significantly decreased by T3 over 8 to 16 week treatment periods. CONCLUSIONS: Results from the two models of TH dysfunction confirmed an inverse relationship between tissue and serum T3 and BNP, such that the reduction in serum BNP could potentially be utilized to monitor efficacy and dosing of T3 treatment. Thus, serum BNP may serve as a reliable biomarker for cardiac TH function.

Adverse transverse-tubule remodeling in a rat model of heart failure is attenuated with low-dose triiodothyronine treatment.

Pre-clinical animal studies have shown that triiodothyronine (T3) replacement therapy improves cardiac contractile function after myocardial infarction (MI). We hypothesized that T3 treatment could prevent adverse post-infarction cardiomyocyte remodeling by maintaining transverse-tubule (TT) structures, thus improving calcium dynamics and contractility. METHODS: Myocardial infarction (MI) or sham surgeries were performed on female Sprague-Dawley rats (aged 12 wks), followed by treatment with T3 (5µg/kg/d) or vehicle in drinking water for 16 wks (n = 10-11/group). After in vivo echocardiographic and hemodynamic analyses, left ventricular myocytes were isolated by collagenase digestion and simultaneous calcium and contractile transients in single cardiomyocytes were recorded using IonOptix imaging. Live cardiomyocytes were stained with AlexaFluor-488 conjugated wheat germ agglutinin (WGA-488) or di-8-ANEPPS, and multiple z-stack images per cell were captured by confocal microscopy for analysis of TT organization. RTqPCR and immunoblot approaches determined expression of TT proteins. RESULTS: Echocardiography and in vivo hemodynamic measurements showed significant improvements in systolic and diastolic function in T3- vs vehicle-treated MI rats. Isolated cardiomyocyte analysis showed significant dysfunction in measurements of myocyte relengthening in MI hearts, and improvements with T3 treatment: max relengthening velocity (Vmax, um/s), 2.984 ± 1.410 vs 1.593 ± 0.325, p < 0.05 and time to Vmax (sec), 0.233 ± 0.037 vs 0.314 ± 0.019, p < 0.001; MI + T3 vs MI + Veh, respectively. Time to peak contraction was shortened by T3 treatment (0.161 ± 0.021 vs 0.197 ± 0.011 s., p < 0.01; MI + T3 vs MI + Veh, respectively). Analysis of TT periodicity of WGA- or ANEPPS-stained cardiomyocytes indicated significant TT disorganization in MI myocytes and improvement with T3 treatment (transverse-oriented tubules (TE%): 9.07 ± 0.39 sham, 6.94 ± 0.67 MI + Veh and 8.99 ± 0.38 MI + T3; sham vs MI + Veh, p < 0.001; MI + Veh vs MI + T3, p < 0.01). Quantitative RT-PCR showed that reduced expression of BIN1 (Bridging integrator-1), Jph2 (junctophilin-2), RyR2 (ryanodine receptor) and Cav1.2 (L-type calcium channel) in the failing myocardium were increased by T3 and immunoblot analysis further supporting a potential T3 effect on the TT-associated proteins, BIN1 and Jph2. In conclusion, low dose T3 treatment initiated immediately after myocardial infarction attenuated adverse TT remodeling, improved calcium dynamics and contractility, thus supporting the potential therapeutic utility of T3 treatment in heart failure.

Efficacy and safety of levothyroxine (L-T4) replacement on the exercise capability in chronic systolic heart failure patients with subclinical hypothyroidism: Study protocol for a multi-center, open label, randomized, parallel group trial (ThyroHeart-CHF).

BACKGROUND: Subclinical hypothyroidism is a common condition in patients with heart failure and is defined as elevated serum thyroid hormone (TSH) with normal circulating free thyroxine (FT4). Evidence on the effect of thyroid hormone treatment is lacking. We designed a randomized controlled trial to compare the efficacy and safety of thyroid hormone supplementation in patients with chronic heart failure complicated with subclinical hypothyroidism. METHODS/DESIGN: Eligible participants were identified from the cardiology units of five study centers based on the following criteria: 18 years or older, systolic heart failure with NewYork Heart Association (NYHA) class II-III, left ventricular ejection fraction ≤ 40%, and subclinical hypothyroidism (TSH > 4.78µIU/ml, < 10 µIU/ml + FT4 level within reference range). Eligible patients will be randomly assigned in a 1:1 manner to receive thyroxine replacement therapy plus standard chronic heart failure (CHF) treatment or only standard CHF therapy. Levothyroxine will be administered at an initial dose of 12.5 µg once daily and will be titrated until TSH is within the normal range. The primary endpoints include the difference in distance of the six-minute walk test between 24 weeks and baseline. The secondary endpoints include differences in plasma NT-proBNP levels and serum lipid profiles, changes in the NYHA classification, cardiovascular death, re-hospitalization, differences in echocardiographic and cardiac magnetic resonance imaging measures, and Minnesota Living With Heart Failure Questionnaire (MLHFQ) results between 24 weeks and baseline. DISCUSSION: ThyroHeart-CHF is designed as a prospective, multi-center, randomized, controlled clinical trial to study the efficacy and safety of thyroid hormone supplementation in patients with chronic heart failure complicated with subclinical hypothyroidism. The study findings will have significant implications for discovering the new therapeutic targets and methods of heart failure. TRAIL REGISTRATION: ClinicalTrials.gov, NCT03096613 . Registered on 30 March 2017.

Epicardial adipose tissue volume is associated with non-alcoholic fatty liver disease and cardiovascular risk factors in the general population.

BACKGROUND: Epicardial adipose tissue (EAT) is considered an important source of bioactive molecules that can influence coronary arteries directly and is related to the concurrent presence of both obstructive coronary stenosis and myocardial ischemia independently. Non-alcoholic fatty liver disease (NAFLD) has become an emergent health problem worldwide. AIM: This cross-sectional study aimed to address the relationship between the volume of EAT and NAFLD and other cardiovascular risk factors in the general population. MATERIALS AND METHODS: In this study, we selected a total of 2,238 participants aged at least 40 years from the Jidong community in Tangshan, China. The 64-slice CT was used to survey the volume of EAT and liver ultrasonography was used for the diagnosis of NAFLD. The study cohorts were compared according to EAT volume. RESULTS: Cardiovascular risk factors, such as coronary artery calcium score, carotid intima-media thickness, NAFLD, and ideal cardiovascular health metrics were also found to be related to EAT. In multivariate logistic regression analysis, NAFLD groups showed significant association with higher EAT volume, after correcting for main cardiovascular disease risk factors (OR [95% CI], 1.407 [1.117, 1.773]). CONCLUSION: Our findings in a general community population provide evidence that EAT is strongly associated with NAFLD and other cardiovascular risk factors.

Association between nonalcoholic fatty liver disease and subclinical atherosclerosis: a cross-sectional study on population over 40 years old.

BACKGROUND: Nonalcoholic fatty liver disease (NAFLD) refers to fatty infiltration of liver in the absence of excessive alcohol abuse. However, the problem that whether NAFLD is correlated with subclinical atherosclerosis assessed by carotid intima-media thickness (CIMT) and brachial-ankle pulse wave velocity (ba-PWV) remains a source of controversy. This can be attributed to the differences in diagnosis methods, population ethnicity, sampling size and bias. This study aimed to further investigate the association of NAFLD with subclinical atherosclerosis. METHODS: A cross-sectional study was carried out in the current study on population aged over 40 years derived from Kailuan community-based prospective study among Chinese adults from June 2010 to June 2011. NAFLD was evaluated through ultrasonography and histories of alcohol consumption. Clinical parameters and medical histories of patients were collected in the manner of interview performed by trained investigators using the standardized questionnaires. The biochemical parameters were analyzed at the central laboratory. CIMT and ba-PWV of each patient were measured. Multivariate logistic regression was used to analyze the associations of NAFLD with subclinical atherosclerosis assessed by CIMT or ba-PWV. RESULTS: A total of 4112 participants aged over 40 years were enrolled from Kailuan cohort, including 2229 men and 1883 women. The overall prevalence of NAFLD was 38.2% in the total population. Statistically significant differences were found in CIMT (P < 0.0001) and ba-PWV (P = 0.0007) according to the presence of NAFLD. It is notably that the multivariate logistic regression revealed NAFLD was independently associated with elevated CIMT after adjusting the conventional cardiovascular and metabolic risk factors (OR = 1.663, 95% CI = 1.391-1.989, P < 0.0001). In addition, NAFLD was also found to be positively associated with elevated ba-PWV after adjusting age, gender, BMI, current smoking and regular exercising (OR = 1.319, 95% CI = 1.072-1.624, P = 0.0089). CONCLUSIONS: Our findings suggest that NAFLD is remarkably correlated with subclinical atherosclerosis, which should be strongly advised to engage in the preventive strategies for cardiovascular diseases (CVDs).

Development and validation of Women Acute Myocardial Infarction in-Hospital Mortality Score (WAMI Score).

BACKGROUND: A variety of risk models have been developed to predict acute myocardial infarction (AMI) in-hospital mortality risk. As a distinct, higher-risk population, women with AMI have different risk profiles from their men counterparts. Published researches have indicated that the interaction between variables in these models for in-hospital mortality and gender are significant. Due to the interaction and gender differences, the predicting value of these risk models for women could be controversial. METHODS: Databases from the China Patient-centered Evaluative Assessment of Cardiac Events (China PEACE) Retrospective AMI Study were utilized for model derivation (n=16,100, women were 4896) and databases from the China PEACE Prospective AMI Study for model validation (n=6207, women were 2090). A multivariable backward stepwise logistic regression was used to examine correlates of in-hospital mortality, and the variables were subsequently weighted and integrated into a scoring system. RESULTS: We constructed a novel risk-predicting tool to estimate the baseline risk of in-hospital mortality among women with AMI. The risk score includes 8 variables [age, systolic blood pressure, heart rate, initial glomerular filtration rate (GFR), serum glucose, Killip class, cardiac arrest, ventricular tachycardia/ventricular fibrillation (VT/VF)]. The prognostic discriminatory capacity of the Women Acute Myocardial Infarction in-Hospital Mortality (WAMI) risk score was excellent (c statistic 0.84, 95% confidence interval [CI]: 0.83 to 0.86, p<0.001). External validation of the model showed better prognostic capacity (c statistic 0.87, 95% CI: 0.84 to 0.90, p<0.001) than the GRACE risk score (0.77, 95% CI 0.72-0.82, p<0.001) and TIMI risk score (0.72, 95% CI 0.68-0.77, p<0.001). CONCLUSIONS: The WAMI Score is a simple robust tool for predicting the in-hospital mortality risk of women with AMI. TRIAL REGISTRATION: "China PEACE-Retrospective AMI Study", NCT01624883, retrospectively registered: April 2012. Date of enrolment of the first participant to the trial: June 17, 2012. "China PEACE-Prospective AMI Study", NCT01624909, prospectively registered: December 2012. Date of enrolment of the first participant to the trial: June 17, 2012.

Prognostic Value of Free Triiodothyronine Level in Patients With Hypertrophic Obstructive Cardiomyopathy.

Context: Thyroid hormone acts as a fundamental regulator in cardiovascular homeostasis in pathophysiological conditions. Objective: This study aims to determine whether thyroid hormone could be an independent predictor of adverse events in patients with hypertrophic obstructive cardiomyopathy (HOCM). Design, Patients, and Outcome Measures: The original cohort consisted of 965 consecutive patients with HOCM who were admitted to Fuwai Hospital from October 2009 to December 2014, and 756 patients completed thyroid function evaluations. Patients were divided into three groups according to free triiodothyronine (FT3) levels: tertile 1 (<2.81 pg/mL, n = 247), tertile 2 (2.81 to 3.11 pg/mL, n = 250), tertile 3 (3.12 to 4.09 pg/mL, n = 259). Results: In correlation analysis, FT3 showed significantly positive correlation with left ventricular ejection fraction (r = 0.109, P = 0.003). After a median follow-up of 44 months, a total of 45 (6.0%) endpoints (all-cause mortality or cardiac transplantation) occurred with rates of 13.4%, 3.6%, and 1.2% in tertiles 1, 2, and 3, respectively. Univariate Cox analysis established FT3 as a predictor of endpoint [hazard ratio (HR), 0.111; 95% confidence interval (CI), 0.065, 0.189; P < 0.001]. After adjustment for traditional risk factors, the prognostic value of FT3 level was still significant (HR, 0.216; 95% CI, 0.083, 0.559; P = 0.002). Compared with patients in tertile 3, those in tertile 1 were at a much higher risk of endpoint (HR, 4.918; 95% CI, 1.076, 22.485; P = 0.040). Conclusions: FT3 correlated with cardiac function and could serve as an independent predictor of all-cause mortality and cardiac transplantation in patients with HOCM. These results suggest that monitoring thyroid function in HOCM patients is necessary.

Hepatocyte DUSP14 maintains metabolic homeostasis and suppresses inflammation in the liver.

Nonalcoholic fatty liver disease (NAFLD) is a prevalent and complex disease that confers a high risk of severe liver disorders. Despite such public and clinical health importance, very few effective therapies are currently available for NAFLD. We report a protective function and the underlying mechanism of dual-specificity phosphatase 14 (DUSP14) in NAFLD and related metabolic disorders. Insulin resistance, hepatic lipid accumulation, and concomitant inflammatory responses, key pathological processes involved in NAFLD development, were significantly ameliorated by hepatocyte-specific DUSP14 overexpression (DUSP14-HTG) in high-fat diet (HFD)-induced or genetically obese mouse models. By contrast, specific DUSP14 deficiency in hepatocytes (DUSP14-HKO) aggravated these pathological alterations. We provided mechanistic evidence that DUSP14 directly binds to and dephosphorylates transforming growth factor ß-activated kinase 1 (TAK1), resulting in the reduced activation of TAK1 and its downstream signaling molecules c-Jun N-terminal kinase 1 (JNK), p38, and nuclear factor kappa B NF-κB. This effect was further evidenced by the finding that inhibiting TAK1 activity effectively attenuated the deterioration of glucolipid metabolic phenotype in DUSP14-HKO mice challenged by HFD administration. Furthermore, we identified that both the binding domain and the phosphatase activity of DUSP14 are required for its protective role against hepatic steatosis, because interruption of the DUSP14-TAK1 interaction abolished the mitigative effects of DUSP14. CONCLUSION: Hepatocyte DUSP14 is required for maintaining hepatic metabolic homeostasis and for suppressing inflammation, a novel function that relies on constraining TAK1 hyperactivation. (Hepatology 2018;67:1320-1338).

Interleukin-6 identified as an important factor in hypoxia- and aldehyde dehydrogenase-based gefitinib adaptive resistance in non-small cell lung cancer cells.

Gefitinib resistance and relapse of the disease were the greatest challenges facing clinical therapy of non-small-cell lung cancer (NSCLC). Of note, regarding the hypoxia condition in solid tumor tissues in vivo, roles of hypoxia in gefitinib adaptive resistance and its association with lung cancer stem cells (LCSCs) have not been fully elucidated. In the present study, the role of hypoxia in gefitinib adaptive resistance and its association with aldehyde dehydrogenase (ALDH)-based LCSC gefitinib resistance were comparatively studied using RNA-sequencing (RNA-seq) technology. Co-treatment of PC9 cells with gefitinib and hypoxia (1% O2) significantly enhanced adaptive resistance compared with gefitinib or hypoxia treatment alone. An ALDEFLUOR assay demonstrated that there was a significant increase of ALDH expression level in hypoxia and gefitinib co-treated PC9 cells, in addition to a higher ratio of G0/G1 quiescent cell enrichment and acquisition of epithelial-mesenchymal transition. RNA-seq analysis revealed that interleukin-6 (IL-6) is an important common factor in hypoxia and ALDH-based gefitinib resistance, supported by inflammation-associated tumor necrosis factor, nuclear factor-κB and Janus kinase-signal transducer and activator of transcription signaling pathway enrichment. Furthermore, exposure of PC9 and HCC827 cells to IL-6 increased gefitinib adaptive resistance. Consequently, IL-6 expression level was a poor prognostic marker for patients with NSCLC and adenocarcinoma. Thus, targeting IL-6 combined with tyrosine kinase inhibitor treatment may be promising in NSCLC clinical therapy in the future.

USP18 protects against hepatic steatosis and insulin resistance through its deubiquitinating activity.

Nonalcoholic fatty liver disease (NAFLD) is characterized by hepatic steatosis, impaired insulin sensitivity, and chronic low-grade inflammation. However, the pathogenic mechanism of NAFLD is poorly understood, which hinders the exploration of possible treatments. Here, we report that ubiquitin-specific protease 18 (USP18), a member of the deubiquitinating enzyme family, plays regulatory roles in NAFLD progression. Expression of USP18 was down-regulated in the livers of nonalcoholic steatohepatitis patients and high-fat diet (HFD)-induced or genetically obese mice. When challenged with HFD, hepatocyte-specific USP18 transgenic mice exhibited improved lipid metabolism and insulin sensitivity, whereas mice knocked out of USP18 expression showed adverse trends regarding hepatic steatosis and glucose metabolic disorders. Furthermore, the concomitant inflammatory response was suppressed in USP18-hepatocyte-specific transgenic mice and promoted in USP18-hepatocyte-specific knockout mice treated with HFD. Mechanistically, hepatocyte USP18 ameliorates hepatic steatosis by interacting with and deubiquitinating transforming growth factorß-activated kinase 1 (TAK1), which inhibits TAK1 activation and subsequently suppresses the downstream c-Jun N-terminal kinase and nuclear factor kappa B signaling pathways. This is further validated by alleviated steatotic phenotypes and highly activated insulin signaling in HFD-fed USP18-hepatocyte-specific knockout mice administered a TAK1 inhibitor. The therapeutic effect of USP18 on NAFLD relies on its deubiquitinating activity because HFD-fed mice injected with active-site mutant USP18 failed to inhibit hepatic steatosis. CONCLUSION: USP18 associates with and deubiquitinates TAK1 to protect against hepatic steatosis, insulin resistance, and the inflammatory response. (Hepatology 2017;66:1866-1884).

Role of α7-nicotinic acetylcholine receptor in nicotine-induced invasion and epithelial-to-mesenchymal transition in human non-small cell lung cancer cells.

Nicotine via nicotinic acetylcholine receptors (nAChRs) stimulates non-small cell lung cancer (NSCLC) cell invasion and epithelial to mesenchymal transition (EMT) which underpin the cancer metastasis. However, the receptor subtype-dependent effects of nAChRs on NSCLC cell invasion and EMT, and the signaling pathway underlying the effects remain not fully defined. We identified that nicotine induced NSCLC cell invasion, migration, and EMT; the effects were suppressed by pharmacological intervention using α7-nAChR selective antagonists or by genetic intervention using α7-nAChR knockdown via RNA inference. Meanwhile, nicotine induced activation of MEK/ERK signaling in NSCLC cells; α7-nAChR antagonism or MEK/ERK signaling pathway inhibition suppressed NSCLC cell invasion and EMT marker expression. These results indicate that nicotine induces NSCLC cell invasion, migration, and EMT; the effects are mediated by α7-nAChRs and involve MEK/ERK signaling pathway. Delineating the effect of nicotine on the NSCLC cell invasion and EMT at receptor subtype level would improve the understanding of cancer biology and offer potentials for the exploitation of selective ligands for the control of the cancer metastasis.

Autocrine glutamatergic transmission for the regulation of embryonal carcinoma stem cells.

Glutamate behaves as the principal excitatory neurotransmitter in the vertebrate central nervous system and recently demonstrates intercellular signaling activities in periphery cancer cells. How the glutamatergic transmission is organized and operated in cancer stem cells remains undefined. We have identified a glutamatergic transmission circuit in embryonal carcinoma stem cells. The circuit is organized and operated in an autocrine mechanism and suppresses the cell proliferation and motility. Biological analyses determined a repertoire of glutamatergic transmission components, glutaminase, vesicular glutamate transporter, glutamate NMDA receptor, and cell membrane excitatory amino-acid transporter, for glutamate biosynthesis, package for secretion, reaction, and reuptake in mouse and human embryonal carcinoma stem cells. The glutamatergic components were also identified in mouse transplanted teratocarcinoma and in human primary teratocarcinoma tissues. Released glutamate acting as the signal was directly quantified by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Genetic and pharmacological abolishment of the endogenously released glutamate-induced tonic activation of the NMDA receptors increased the cell proliferation and motility. The finding suggests that embryonal carcinoma stem cells can be actively regulated by establishing a glutamatergic autocrine/paracrine niche via releasing and responding to the transmitter.

Concomitant BET and MAPK blockade for effective treatment of ovarian cancer.

Ovarian cancer is the most lethal gynecologic malignancy, and it is imperative to develop new treatments to ameliorate patient survival. Using an anti-cancer drug library containing 180 small molecule inhibitors, we performed a high-content image-based screen and found that BET and MEK inhibitors are among the candidates which were able to effectively inhibit ovarian cancer cell growth. However, BET inhibition alone was largely cytostatic, possibly due to feedback activation of the MAPK pathway. Consequently, the combination of MEK and BET inhibitors suppressed both cell proliferation and survival, and was more efficacious than single agent. Mechanistically, BET and MEK inhibitors exerted synergistic effects on apoptosis regulators including BIM and BAD. Our findings support concomitant BET and MAPK blockade as an effective therapeutic strategy in ovarian cancer.

A Phase II Study of Concurrent Chemoradiotherapy With Paclitaxel and Cisplatin for Inoperable Esophageal Squamous Cell Carcinoma.

OBJECTIVES: A phase II study was performed to investigate the efficacy and the safety of a 3-week schedule of paclitaxel (PTX) plus cisplatin (DDP) combined with concurrent radiotherapy for esophageal squamous cell cancer. PATIENTS AND METHODS: Patients with newly diagnosed esophageal squamous cell cancer who had histologic proof of local-regional carcinoma of the esophagus, a Karnofsky performance status of 80 or greater, and normal liver, renal, and bone marrow functions were enrolled in the phase II trial. Chemotherapy consisted of DDP (25 mg/m/d) for 3 days plus PTX (175 mg/m) given for 3 hours, every 3 weeks for 4 cycles. The total dose of concurrent radiation with 68.4 Gy/44 Fx (late course-accelerated radiotherapy) or 61.2 Gy/34 Fx (conventional radiotherapy) was given at the first day of chemotherapy. RESULTS: Between July 2008 and November 2011, 76 patients were enrolled in this trial. The median age was 58 years (range, 37 to 74 y). The stages were stage II (21 patients), stage III (27 patients), and stage IV (28 patients). A total of 89.5% (68/76) and 63.2% (48/76) patients completed ≥2 cycles and all 4 cycles of chemotherapy, respectively. With the median follow-up of 36 months, the overall median survival time was 28.5 months and the progression-free survival time was 14.7 months. One- and 3-year survival rates were 75% and 41%, respectively. Neutropenia grade 3 and 4 occurred in 30.3% and 31.6% of the patients, respectively. CONCLUSIONS: Radiotherapy concurrent with a 3-week schedule of PTX and DDP resulted in an encouraging overall survival rate, but a relatively higher hematological toxicity.

Adrenergic DNA damage of embryonic pluripotent cells via ß2 receptor signalling.

Embryonic pluripotent cells are sensitive to genotoxicity though they need more stringent genome integrity to avoid compromising multiple cell lineages and subsequent generations. However it remains unknown whether the cells are susceptible to adrenergic stress which can induce somatic cell genome lesion. We have revealed that adrenergic stress mediators cause DNA damage of the cells through the ß2 adrenergic receptor/adenylate cyclase/cAMP/PKA signalling pathway involving an induction of intracellular reactive oxygen species (ROS) accumulation. The adrenergic stress agonists adrenaline, noradrenaline, and isoprenaline caused DNA damage and apoptosis of embryonic stem (ES) cells and embryonal carcinoma stem cells. The effects were mimicked by ß2 receptor-coupled signalling molecules and abrogated by selective blockade of ß2 receptors and inhibition of the receptor signalling pathway. RNA interference targeting ß2 receptors of ES cells conferred the cells the ability to resist the DNA damage and apoptosis. In addition, adrenergic stimulation caused a consistent accumulation of ROS in the cells and the effect was abrogated by ß2 receptor blockade; quenching of ROS reversed the induced DNA damage. This finding will improve the understanding of the stem cell regulatory physiology/pathophysiology in an adrenergic receptor subtype signalling mechanism.

ß2-Adrenoreceptor-Mediated Proliferation Inhibition of Embryonic Pluripotent Stem Cells.

Adrenoreceptors (ARs) are widely expressed and play essential roles throughout the body. Different subtype adrenoceptors elicit distinct effects on cell proliferation, but knowledge remains scarce about the subtype-specific effects of ß2-ARs on the proliferation of embryonic pluripotent stem (PS) cells that represent different characteristics of proliferation and cell cycle regulation with the somatic cells. Herein, we identified a ß2-AR/AC/cAMP/PKA signaling pathway in embryonic PS cells and found that the pathway stimulation inhibited proliferation and cell cycle progression involving modulating the stem cell growth and cycle regulatory machinery. Embryonic stem (ES) cells and embryonal carcinoma stem (ECS) cells expressed functional ß-ARs coupled to AC/cAMP/PKA signaling. Agonistic activation of ß-ARs led to embryonic PS cell cycle arrest and proliferation inhibition. Pharmacological and genetic analyzes using receptor subtype blocking and RNA interference approaches revealed that this effect selectively depended on ß2-AR signaling involving the regulation of AKT, ERK, Rb, and Cyclin E molecules. Better understanding of the effects of ß2-ARs on embryonic PS cell proliferation and cycle progression may provide new insights into stem cell biology and afford the opportunity for exploiting more selective ligands targeting the receptor subtype for the modulation of stem cells.

Simultaneous determination of the repertoire of classical neurotransmitters released from embryonal carcinoma stem cells using online microdialysis coupled with hydrophilic interaction chromatography-tandem mass spectrometry.

Dynamic, continuous, and simultaneous multi-analysis of transmitters is important for the delineation of the complex interactions between the neuronal and intercellular communications. But the analysis of the whole repertoire of classical transmitters of diverse structure is challenging due to their different physico-chemical properties and to their high polarity feature which leads to poor retention in traditional reversed-phase columns during LC-MS analysis. Here, an online microdialysis coupled with hydrophilic interaction chromatography-tandem mass spectrometry (online MD-HILIC-MS/MS) detection method was developed for the simultaneous measurement of the repertoire of classical transmitters (acetylcholine, serotonin, dopamine, norepinephrine, glutamate, GABA, and glycine). Stable isotope labeled internal standards and authentic matrix have been applied to guarantee reliable results. The method was successfully employed to reveal the characteristics of transmitter release from embryonal carcinoma stem cells. The method features simple procedure (no sample preparation), high recovery (≥ 73%), high accuracy (89.36%≤RE≤116.89%), good reproducibility (2.18%≤ RSD ≤14.56%), and sensitive limits of detection (2 pg for acetylcholine, serotonin, and glutamate, 10 pg for dopamine, norepinephrine, GABA, and glycine). It can be flexibly applied to determine the contents of the classical transmitters in other biological matrix samples with minor changes.

Characterization of combinatorial histone modifications on lineage-affiliated genes during hematopoietic stem cell myeloid commitment.

Hematopoietic stem cells (HSCs) are multipotent stem cells capable of self-renewal and multilineage differentiation. Mechanisms regulating the maintenance of HSCs' multipotency and differentiation are still unclear. In this study, we observed the role of combinatorial histone modification pattern in the maintenance of HSCs' pluripotency and differentiation. HSCs (CD34(+)CD38(low)) were collected from human umbilical cord blood and induced to differentiate to committed cells in vitro. The histone modifications on lineage-specific transcription factors/genes in multipotent HSCs and differentiated progenies, including megakaryocytes, granulocytes, and erythrocytes, were analyzed by chromatin immunoprecipitation-quantitative polymerase chain reaction. Our results showed that a certain level of acH4 and acH3 together with high level of H3K4me2, low level of H3K4me3, and a certain level of H3K9me3 and H3K27me3 were present in lineage-specific genes in CD34(+)CD38(low) HSCs. As CD34(+)CD38(low) cells differentiated into granulocytes, erythroid cells, and megakaryocytes, the modification levels of acH3, acH4, and H3K4me2 on lineage-specific genes remained the same or elevated, while H3K4me3 level was increased greatly. At the same time, H3K9me3 and H3K27me3 modification levels became lower. In non-lineage-specific genes, the acH3 and acH4 levels were decreased, and H3K4me3 level remained at low level, while H3K9me3 and H3K27me3 levels were increased drastically. Our data suggest that combinatorial histone modification patterns have implicated function in maintaining the multipotency of HSCs and keeping the accuracy of gene expression program during HSC differentiation.