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.

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.

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.

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.

Non-neuronal release of gamma-aminobutyric Acid by embryonic pluripotent stem cells.

γ-Aminobutyric acid (GABA), the principle inhibitory transmitter in the mature central nervous system, is also involved in activities outside the nervous system. Recent studies have shown that functional GABA receptors are expressed in embryonic stem (ES) cells and these receptors control ES cell proliferation. However, it is not clear whether ES cells have their own GABAergic transmission output machinery that can fulfill GABA release or whether the cells merely process the GABA receptors by receiving and responding to the diffused GABA released elsewhere. To get further insight into this unresolved problem, we detected the repertoire of components for GABA synthesis, storage, reaction, and termination in ES and embryonal carcinoma stem cells by biological assays, and then directly quantified released GABA in the intercellular milieu from these pluripotent stem (PS) cells by an analytical chemical assay based on high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS). We found that embryonic PS cells processed a GABAergic circuit machinery and spontaneously released GABA, which suggests the potential that embryonic PS cells could autonomously establish a GABA niche via release of the transmitter.

[Growing of human embryonic stem cells on feeders derived from themselves].

This study was carried out to determine whether mesenchymal stem cells (MSCs) derived from teratoma of human embryonic stem cells (hESCs) function as feeder cells to support hESCs growth. Approximately 5x10(6) hESCs were injected into the hind limb muscle of each SCID-beige mouse to form teratoma. After 8 weeks, the MSCs were isolated from the teratoma and cultured in Mesencult medium. Purified MSCs were then used as the feeder cells for hESCs culture. High purity MSCs derived from teratoma were isolated. The cells were morphologically similar to bone marrow MSCs (bMSCs). The teratoma-derived MSCs were negative for CD34 and CD45 but positive for CD29, CD49b, CD105, CD73, and CD90, which resembled those expressed by bMSCs. After passaged on MSCs feeder cells more than 10 passages, hESCs maintained hESC characteristics in morphology. Reverse PCR showed the expression of Oct4 and Nanog. SSEA-1 was negative and SSEA-4, TRA-1-60, and TRA-1-81 were positive. Alkaline phosphatase staining showed positive results.The karyotype remained normal. Moreover, the hECSs cultured on teratoma-derived MSCs formed teratoma in vivo and embryoid body in vitro confirmed their pluripotency. Accordingly, MSCs derived from hESCs by in vivo differentiation can be used as the feeder cells for hESCs culture.