NRF2-GPX4/SOD2 axis imparts resistance to EGFR-tyrosine kinase inhibitors in non-small-cell lung cancer cells.

Epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) have achieved satisfactory clinical effects in the therapy of non-small cell lung cancer (NSCLC), but acquired resistance limits their clinical application. NRF2 has been shown to enhance the resistance to apoptosis induced by radiotherapy and some chemotherapy. In this study, we investigated the role of NRF2 in resistance to EGFR-TKIs. We showed that NRF2 protein levels were markedly increased in a panel of EGFR-TKI-resistant NSCLC cell lines due to slow degradation of NRF2 protein. NRF2 knockdown overcame the resistance to EGFR-TKIs in HCC827ER and HCC827GR cells. Furthermore, we demonstrated that NRF2 imparted EGFR-TKIs resistance in HCC827 cells via upregulation of GPX4 and SOD2, and suppression of GPX4 and SOD2 reversed resistance to EGFR-TKIs. Thus, we conclude that targeting NRF2-GPX4/SOD2 pathway is a potential strategy for overcoming resistance to EGFR-TKIs.

UPLC-MS-based metabolomics reveals metabolic dysregulation in ALDH1A1-overexpressed lung adenocarcinoma cells.

INTRODUCTION: Specific oncogenotypes can produce distinct metabolic changes in cancer. Recently it is considered that metabolic reprograming contributes heavily to drug resistance. Aldehyde dehydrogenase 1A1 (ALDH1A1), is overexpressed in drug resistant lung adenocarcinomas and may be the cause of acquired drug resistance. However, how ALDH1A1 affects metabolic profiling in lung adenocarcinoma cells remains elusive. OBJECTIVE: We sought to investigate metabolic alterations induced by ALDH1A1 in lung adenocarcinoma in order to better understand the reprogramming and metabolic mechanism of resistance induced by ALDH1A1. METHODS: Metabolic alterations in lung adenocarcinoma HCC827-ALDH1A1 cells were analyzed by ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS). HCC827-ALDH1A1 metabolic signatures were extracted by univariate and multivariate statistical analysis. Furthermore, metabolite enrichment analysis and pathway analysis were performed using MetaboAnalyst 4.0 software. RESULTS: Twenty-two metabolites were positively identified using authentic standards, including uridine monophosphate (UMP), uridine diphosphate (UDP), adenosine diphosphate (ADP), malic acid, malonyl-coenzyme A, nicotinamide adenine dinucleotide (NAD), coenzyme A and so on. Furthermore, metabolic pathway analysis revealed several dysregulated pathways in HCC827-ALDH1A1 cells, including nucleotide metabolism, urea cycle, tricarboxylic acid (TCA) cycle, and glycerol phospholipid metabolism etc. CONCLUSION: Lung cancer is the most frequent cause of cancer-related deaths worldwide. Nearly all patients eventually undergo disease progression due to acquired resistance. Mechanisms of biological acquired resistance need to be identified. Our study identified altered metabolites in HCC827-ALDH1A1 cells, enhancing our knowledge of lung adenocarcinoma metabolic alterations induced by ALDH1A1, creating a novel therapeutic pathway. These metabolic signatures of ALDH1A1 overexpression may shed light on molecular mechanisms in drug-resistant tumors, and on candidate drug targets. Furthermore, new molecular targets may provide the foundation for potential anticancer strategies for lung cancer therapy.

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.

Cancer cell-autonomous cGAS-STING response confers drug resistance.

The cGAS-STING pathway has long been recognized as playing a crucial role in immune surveillance and tumor suppression. Here, we show that when the pathway is activated in a cancer-cell-autonomous response manner, it confers drug resistance. Targeted or conventional chemotherapy drugs promoted cytosolic DNA accumulation in cancer cells, activating the cGAS-STING pathway and downstream TBK1-IRF3/NF-κB signaling. This cancer cell-intrinsic response enabled the cells to counteract drug stress, allowing treatment resistance to be acquired and maintained. Blockade of stimulator of interferon genes (STING) signaling delayed and overcame resistance in models in vitro and in vivo. This finding uncovers an alternative face of cGAS-STING signaling other than the well-reported modulation of microenvironmental immune cells. It also implies a caution for the combination of STING agonist with targeted or conventional chemotherapy drug treatment, a strategy prevailing in current clinical trials.

Metabolic and Nonmetabolic Functions of PSAT1 Coordinate Signaling Cascades to Confer EGFR Inhibitor Resistance and Drive Progression in Lung Adenocarcinoma.

Emerging evidence demonstrates that the dysregulated metabolic enzymes can accelerate tumorigenesis and progression via both metabolic and nonmetabolic functions. Further elucidation of the role of metabolic enzymes in EGFR inhibitor resistance and metastasis, two of the leading causes of death in lung adenocarcinoma, could help improve patient outcomes. Here, we found that aberrant upregulation of phosphoserine aminotransferase 1 (PSAT1) confers erlotinib resistance and tumor metastasis in lung adenocarcinoma. Depletion of PSAT1 restored sensitivity to erlotinib and synergistically augmented the tumoricidal effect. Mechanistically, inhibition of PSAT1 activated the ROS-dependent JNK/c-Jun pathway to induce cell apoptosis. In addition, PSAT1 interacted with IQGAP1, subsequently activating STAT3-mediated cell migration independent of its metabolic activity. Clinical analyses showed that PSAT1 expression positively correlated with the progression of human lung adenocarcinoma. Collectively, these findings reveal the multifunctionality of PSAT1 in promoting tumor malignancy through its metabolic and nonmetabolic activities. SIGNIFICANCE: Metabolic and nonmetabolic functions of PSAT1 confer EGFR inhibitor resistance and promote metastasis in lung adenocarcinoma, suggesting therapeutic targeting of PSAT1 may attenuate the malignant features of lung cancer.

Huperzine A lowers intraocular pressure via the M3 mAChR and provides retinal neuroprotection via the M1 mAChR: a promising agent for the treatment of glaucoma.

BACKGROUND: Glaucoma is a neurodegenerative disease that shares similar pathological mechanisms with Alzheimer's disease (AD). Drug treatments for glaucoma increasingly rely upon both lowering of intraocular pressure (IOP) and optic nerve protection, as lowering of IOP alone has been unsatisfactory. Huperzine A (HupA) is an acetylcholinesterase inhibitor (AChEI) used for AD. This study investigated the potential of HupA as a treatment for glaucoma. METHODS: The ability of HupA to lower IOP via causing pupil constriction was assessed using New Zealand rabbits. The retinal neuroprotective effects of HupA were assessed in vivo using rat retinas subjected to ischemia-reperfusion (I/R) and in vitro using primary retinal neurons (PRNs) suffering from oxygen-glucose deprivation (OGD). RESULTS: HupA caused pupil constriction in a dose-time dependent manner which was reversed by the nonselective muscarinic acetylcholine receptor (mAChR) antagonist atropine and the selective M3 mAChR antagonist 4-DAMP. However, HupA had no effect on isolated iris muscle tension and calcium flow indicating an indirect M3 mAChR mediated effect. HupA exerted a neuroprotective effect against I/R and OGD to attenuate the retinal pathological lesion, improve retinal neuronal cell viability, reverse oxidative stress injury by increasing GSH levels and SOD activity, and decreasing MDA content and reduce the retinal neuronal apoptosis by decreasing Bax/Bcl-2 ratio and caspase-3 expression with no effect on the calcium flow tests. The effects were abolished by atropine and the selective M1 mAChR antagonist pirenzepine in OGD-induced PRNs suggesting an indirect M1 mAChR-mediated effect via inhibiting AChE activity to increase endogenous ACh level. Furthermore, HupA increased phosphorylated AKT level and decreased the levels of phosphorylated JNK, P38 MAPK and ERK via M1 mAChR antagonists indicating an involvement of activating the M1 mAChR and the downstream AKT/MAPK signaling pathway in the protective effects of HupA. CONCLUSIONS: HupA could significantly decrease IOP via activating M3 mAChR indirectly and produce retinal neuroprotective effect through M1 mAChR/AKT/MAPK by increasing endogenous ACh level. These investigations demonstrated that HupA was an effective drug in glaucoma treatment and the clinical application of HupA and other AChEIs for glaucoma patients should be further investigated.

Targeting AKR1B1 inhibits glutathione de novo synthesis to overcome acquired resistance to EGFR-targeted therapy in lung cancer.

Acquired resistance represents a bottleneck to molecularly targeted therapies such as epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) treatment in lung cancer. A deeper understanding of resistance mechanisms can provide insights into this phenomenon and help to develop additional therapeutic strategies to overcome or delay resistance. Here, we identified a pharmacologically targetable metabolic mechanism that drives resistance to EGFR TKIs in lung cancer cell lines and patient-derived xenograft mice. We demonstrated that aldo-keto reductase family 1 member B1 (AKR1B1) interacts with and activates signal transducer and activator of transcription 3 (STAT3) to up-regulate the cystine transporter solute carrier family 7 member 11 (SLC7A11). This leads to enhanced cystine uptake and flux to glutathione de novo synthesis, reactive oxygen species (ROS) scavenging, protection from cell death, and EGFR TKI drug resistance in lung cancer cell lines and xenograft mouse models. Suppression of AKR1B1 with selective inhibitors, including the clinically approved antidiabetic drug epalrestat, restored the sensitivity of resistant cell lines to EGFR TKIs and delayed resistance in lung cancer patient-derived xenograft mice. Our findings suggest a metabolic mechanism for resistance to a molecularly targeted therapy and provide a potential therapeutic target for overcoming resistance to EGFR TKIs, including the third-generation inhibitor osimertinib.

Erratum: Overcoming erlotinib resistance in EGFR mutation-positive lung adenocarcinomas through repression of phosphoglycerate dehydrogenase: Erratum.

[This corrects the article DOI: 10.7150/thno.23177.].

Development and validation of a rapid, robust and sensitive UPLC-QQQ-MS/MS method for simultaneous quantification of GSH metabolism in lung cancer cells.

Changes in cellular metabolism accompany tumor therapeutic resistance. Metabolite concentrations specifically reflect the cellular state. Glutathione (GSH) metabolism maintains the redox homeostasis while also confers therapeutic resistance to cancer cells. However, analytical methods for studying GSH metabolism rely on high-resolution-based untargeted metabolomics. Since the aim of untargeted metabolomics studies is covering as much metabolites as possible, these methods lack sensitivity for simultaneous analysis of intracellular GSH-related metabolites with different polarities and structures. In this study, based on cultured lung cancer cells, we described a rapid, robust and sensitive ultra-performance liquid chromatography-triple quadrupole tandem mass spectrographic method (UPLC-QQQ-MS/MS) to simultaneously quantify a repertoire of GSH-related metabolites, including GSH, GSSG, glycine, cysteine, glutamine, glutamate, cystine, γ-glutamyl-cysteine and cysteinyl-glycine. This method avoided the use of derivatization and/or ion-pairing reagents and was validated according to United States Food and Drug Administration (US FDA) criteria. The lower limit of quantification was determined to be 0.5-100 ng/mL with lower limits of detection at 0.14-10.07 ng/mL. The intra- and inter-day precision values for all the analytes were <15% CV, and the accuracy ranged from 85.4% to 114% at three levels of quality control. This method combined simple preparation with rapid analytical procedure (8 min), allowed for high-throughput analysis of GSH metabolism in basic and therapeutic treatment conditions within cultured cells. Our data showed a significant alteration of GSH metabolism in two independent resistant cells compared to sensitive cells. This method monitored the impact of molecularly targeted drugs on GSH metabolism within lung cancer cells and therefore helped identifying potential metabolic vulnerability for the therapeutic resistance in lung cancer.

Dysregulated Urinary Arginine Metabolism in Older Adults With Amnestic Mild Cognitive Impairment.

Background: Urine samples, which capture an individual's metabolic profile, are ideal for the exploration of non-invasive biomarkers to confirm the amnestic mild cognitive impairment (aMCI) status of patients vs. unimpaired ones. Objective: We aimed to detect differentially metabolized amino acids, which are important objectives in metabolomics, garnering particular attention in biomedical pathogenesis from the urine of aMCI patients, which may give clinicians the possibility to intervene with early treatments that curb Alzheimer's disease (AD). Methods: The study included 208 subjects, 98 of whom were aMCI patients, and 110 who were control subjects without dementia. Urine samples were taken from each participant and supernatant was obtained for analysis. The concentrations of amino acids were measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Results: Urinary arginine levels in aMCI patients are obviously lower than in normal controls (q < 0.2 and p < 0.05). Meanwhile, aMCI patients had significant reduced urinary global arginine bioavailability ratio (GABR), and GABR in urine displayed a positive correlation with the score of CMMSE. Conclusion: Urinary dysregulated arginine metabolism that may serve as a helpful clinical diagnostic biomarker for aMCI in older adults.

A Novel Allosteric Inhibitor of Phosphoglycerate Mutase 1 Suppresses Growth and Metastasis of Non-Small-Cell Lung Cancer.

Phosphoglycerate mutase 1 (PGAM1) plays a pivotal role in cancer metabolism and tumor progression via its metabolic activity and interaction with other proteins like α-smooth muscle actin (ACTA2). Allosteric regulation is considered to be an innovative strategy to discover a highly selective and potent inhibitor targeting PGAM1. Here, we identified a novel PGAM1 allosteric inhibitor, HKB99, via structure-based optimization. HKB99 acted to allosterically block conformational change of PGAM1 during catalytic process and PGAM1-ACTA2 interaction. HKB99 suppressed tumor growth and metastasis and overcame erlotinib resistance in non-small-cell lung cancer (NSCLC). Mechanistically, HKB99 enhanced the oxidative stress and altered multiple signaling pathways including the activation of JNK/c-Jun and suppression of AKT and ERK. Collectively, the study highlights the potential of PGAM1 as a therapeutic target in NSCLC and reveals a distinct mechanism by which HKB99 inhibits both metabolic activity and nonmetabolic function of PGAM1 by allosteric regulation.

Overcoming erlotinib resistance in EGFR mutation-positive lung adenocarcinomas through repression of phosphoglycerate dehydrogenase.

How to improve the efficacy and reverse the resistance to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs), such as erlotinib, remains a major challenge in the targeted therapy of lung adenocarcinoma with EGFR-activating mutation. Phosphoglycerate dehydrogenase (PHGDH) is the key enzyme of de novo serine biosynthesis over-expressed in various types of cancer including lung cancer. Elevated PHGDH expression is correlated with a worse overall survival in clinical lung adenocarcinoma patients. Here we investigated the role of PHGDH in lung adenocarcinoma with the acquisition of resistance to erlotinib. Methods: The necessary genes required for the acquired erlotinib resistance in lung adenocarcinoma cells were screened out by RNA-Seq analysis. Then the protein and mRNA levels of PHGDH were confirmed by immunoblotting and qRT-PCR in the erlotinib resistant cells. The effects of PHGDH inhibition or overexpression on erlotinib resistance were examined using cell culture and tumor xenograft mouse models respectively. To explore mechanism, the ROS level and DNA damage marker, γH2AX, were tested by DCFH-DA staining and immunofluorescence after PHGDH inhibition. Results: We found that PHGDH level was significantly increased in the lung adenocarcinoma PC9ER4 and HCC827ER9 cells that acquired resistance to erlotinib. Perturbation of PHGDH by siPHGDH transfection or NCT-503, a small molecular PHGDH inhibitor, synergistically augmented the tumoricidal effect and restored sensitivity to erlotinib in cell lines and xenografts. Over-expression of PHGDH caused xenografts resistant to erlotinib. Furthermore, multiple DNA damage repair pathways related genes were changed by PHGDH depletion specifically in erlotinib resistant cells. ROS stress and DNA damage marker γH2AX were enhanced by siPHGDH and NCT-503, which was reversed by NAC. Conclusion: Our study indicated that PHGDH inhibition has potential therapeutic value in lung adenocarcinoma with the acquired resistance to EGFR-TKIs.

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.

L-Satropane Prevents Retinal Neuron Damage by Attenuating Cell Apoptosis and Aß Production via Activation of M1 Muscarinic Acetylcholine Receptor.

Muscarinic acetylcholine receptor (mAChR) agonists have been used to treat glaucoma due to their intraocular pressure-lowering effects. Recently, it has been reported that retinal mAChRs activation can also stimulate neuroprotective pathways. PURPOSE: In our study, we evaluated the potential neuroprotective effect of L-satropane, a novel mAChR agonist, on retinal neuronal injury induced by cobalt chloride (CoCl2) and ischemia/reperfusion (I/R). METHODS: CoCl2-induced hypoxia injury in cultured cell models and I/R-induced retinal neuronal damage in rats in vivo were used to evaluate the abilities of L-satropane. In detail, we measured the occurrence of retinal pathological changes including molecular markers of neuronal apoptosis and Aß expression. RESULTS: Pretreatment with L-satropane protects against CoCl2-induced neurotoxicity in PC12 and primary retinal neuron (PRN) cells in a dose-dependent manner by increasing retinal neuron survival. CoCl2 or I/R-induced cell apoptosis by upregulating Bax expression and downregulating Bcl-2 expression, which resulted in an increased Bax/Bcl-2 ratio, and upregulating caspase-3 expression/activity was significantly reversed by L-satropane treatment. In addition, L-satropane significantly inhibited the upregulation of Aß production in both retinal neurons and tissue. We also found that I/R-induced histopathological retinal changes including cell loss in the retinal ganglion cell layer (GCL) and increased TUNEL positive retinal ganglion cells in GCL and thinning of the inner plexiform layer (IPL) and inner nuclear layer (INL) were markedly improved by L-satropane. The effects of L-satropane were largely abolished by the nonselective mAChRs antagonist atropine and M1-selective mAChR antagonist pirenzepine. CONCLUSION: These results demonstrated that L-satropane might be effective in preventing retinal neuron damage caused by CoCl2 or I/R. The neuroprotective effects of L-satropane may be attributed to decreasing cell apoptosis and Aß production through activation of M1 mAChR.

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.

DCTPP1 attenuates the sensitivity of human gastric cancer cells to 5-fluorouracil by up-regulating MDR1 expression epigenetically.

Gastric cancer (GC) is among the most malignant cancers with high incidence and poor prognoses worldwide as well as in China. dCTP pyrophosphatase 1 (DCTPP1) is overexpressed in GC with a poor prognosis. Given chemotherapeutic drugs share similar structures with pyrimidine nucleotides, the role of DCTPP1 in affecting the drug sensitivity in GC remains unclear and is worthy of investigation. In the present study, we reported that DCTPP1-knockdown GC cell line BGC-823 exhibited more sensitivity to 5-fluorouracil (5-FU), demonstrated by the retardation of cell proliferation, the increase in cell apoptosis, cell cycle arrest at S phase and more DNA damages. Multidrug resistance 1 (MDR1) expression was unexpectedly down-regulated in DCTPP1-knockdown BGC-823 cells together with more intracellular 5-FU accumulation. This was in large achieved by the elevated methylation in promoter region of MDR1 gene. The intracellular 5-methyl-dCTP level increased in DCTPP1-knockdown BGC-823 cells as well. More significantly, the strong correlation of DCTPP1 and MDR1 expression was detectable in clinical GC samples. Our results thus imply a novel mechanism of chemoresistance mediated by the overexpression of DCTPP1 in GC. It is achieved partially through decreasing the concentration of intracellular 5-methyl-dCTP, which in turn results in promoter hypomethylation and hyper-expression of drug resistant gene MDR1. Our study suggests DCTPP1 as a potential indicative biomarker for the predication of chemoresistance in GC.

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.

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.