bluuebunny/crossref_metadata_2025_split huggingface dataset:
Soft Three Dimensional Stromal Matrix Confines Pancreatic Cancer Cells Through Induction of Autophagy Lysosome Flux
Authors: Han Yuanping , Cheng Jingjing , Wu Pengfei , Yang Huan , Zhang Chunyang , Zeng Yilan , Liu Yong , Luo Mei , Pandol Stephen L 
Published: June 2021

Pancreas is a soft orang. Malignancy of pancreatic ductal adenocarcinoma (PDAC) is intimately associated with pancreatic fibrogenesis and stiffness, generated by chronic pancreatitis, along with early oncogenetic transformation, indicating that soft stroma may confine tumor growth. Little is understood as to how soft three‐dimensional extracellular matrix (3D ECM) in the stroma can restain tumor progression even the cancer cells having acquired oncogenetic transformation. We noticed that embedded in soft 3D ECM, the pancreatic ductal adenocarcinoma cells (KRas/Trp53R172H) can be arrested at G1 phase, showing up‐regulation of p21 and Pten. In the soft 3D ECM, the cancer cells form tight junctions and epithelial ductal phenotypes. Conversely, on 2D stiff interface, the cancer cells are disseminated and exhibited EMT features. Among the 800 up‐regulated genes by the cancer cells in 3D ECM are the lysosomal genes, including cysteine cathepsins, acidic lipase, and v‐ATPase. Indeed, autophagy is highly active by the cells embedded in 3D ECM, featured by elevation of Atg8/LC3‐II, and turnover of p62, a cargo protein for auto‐lysosomal flux and substrate degradation. Lysosomal stress, induced by drugs of lipophilic amines, can inhibit the acidic proteinases, leading to accumulation of HDAC4 and Yap1, suggesting they are degraded through auto‐lysosome. On stiff interface (2D ECM), turnover of the cellular HDAC4 and is mostly operated by proteosomal pathway, wherein the auto‐lysosomal biogenesis is impaired, leading to HDAC4 and Yap1 accumulation. Animal experiments showed that pre‐programing of PDAC cells (Kras/Trp53) in soft 3D ECM could attenuate tumorigenicity. Conversely, existing liver fibrosis and stiffness can promote the tumorigenicity. Clinical studies showed that high levels of auto‐lysosoamal components such as cathepsins H and cathepsin L are related to better survival rate, indicating auto‐lysosomal flux in tumor suppression.Support or Funding InformationNational Science Foundation of China, and National Institutes of Health, USAThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Expression of FOXA1 and GATA3 correlates with invasivity and pathological stage a grade of primary transitional cell carcinoma of the bladder
Authors: Ziaran Stanislav , Novakova Zuzana Varchulova , Bevizova Katarina , Harsanyi Stefan , Bujdak Peter , Galbavy Stefan , Danisovic Lubos 
Published: June 2021

Bladder cancer is the fifth most common malignancy in men and the second most commonly diagnosed urogenital tumor. Approximately 400,000 new patients with this disease occur each year in the world. Incidence rates are 4 times higher in men than in women. Diagnosis is based on cystoskopy, radiological examination and histological examination. Histological evaluation provides definitive diagnosis and also provides information about pathological stage and grade. Recently, methods of molecular biology are used to predict biological behavior an prognosis more precisely. In this study we evaluated 73 patients with primary transitional cell carcinoma (TCC) of urinary bladder (39 patients pTa and 34 pT1 TCC). Total RNA was extracted from bladder tissue samples using GeneJET RNA Purification Kit (Thermo Scientific, USA) according to the manufacturer's recommendations. First‐strand cDNA was synthesized from total RNA with Maxima First Strand cDNA Synthesis Kit for RT‐qPCR (Thermo Scientific, USA). Reaction was performed according to protocol recommended by manufacturer. Obtained cDNA was used as a template for quantitative PCR to determine the expression level of selected genes (TP53, BCL2, FOXA1 and GATA3). The PCR reactions were performed on Eco Real‐Time PCR System (Illumina, USA). Expression of all analyzed genes was normalized to housekeeping gene, glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH). Obtained results showed that expression levels of TP53 and BCL2 showed no significant differences in both groups. Conversely, significantly lower expression of FOXA1 and GATA3 were detected in pT1 TCC when compared to pTa TCC. Moreover, significantly higher expression of FOXA1 and GATA3 was detected in high grade TCC when compared to low grade tumors. Based on our findings, expression of FOXA1 and GATA3 correlates with invasivity and pathological stage a grade of primary bladder TCC, and may be used for further research in prospective studies.Support or Funding InformationVEGA No. 1/0207/16.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Characterization of Microcystin‐Induced Apoptosis in HepG2 Cells
Authors: Krishnan Anjali , Koski Gary , Mou Xiaozhen 
Published: June 2021

Microcystins (MCs) are a class of hepatotoxins produced by freshwater cyanobacteria. Hepatocytes possess the organic anion transporting polypeptide 1B2, which results in the uptake of microcystins followed by inhibition of protein phosphatase 1 and 2A. Previously, programmed cell death, i.e., apoptosis, has been observed in MC‐treated hepatocytes, but, there is still a knowledge gap regarding the pathway adopted for apoptosis observed in hepatic cells. MC‐LR, the most common form of MCs, was administered to HepG2 cells at a 5μM final concentration, and microscopic observation along with Alamar Blue assay was performed to evaluate the induction of cytotoxicity. MC‐LR treated hepatic cells were also stained with caspases specific stains and the presence of caspases was determined based on flow cytometric analysis. Loss of viability and confluency was observed in MC‐LR exposed (24 hrs) HepG2 cells by microscopic analysis and Alamar Blue assay. Caspases 8, 9 and 3 were induced after 24 hrs of MC‐LR treatment. Expression of these caspases indicated the presence of both extrinsic and intrinsic pathway in MC‐LR induced apoptosis, where, extrinsic apoptotic pathway was triggered before the intrinsic apoptotic pathway.Support or Funding InformationKent State UniversityThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
α1D–Adrenoceptor Mediated Components to Nerve‐Evoked Contractions in Mouse Vas Deferens
Authors: Docherty James R 
Published: June 2021

Contractions of the mouse vas deferens are mediated by α1‐adrenoceptors and P2X1‐purinoceptors (Brown et al., 1983). α1D‐Adrenoceptors are involved in the nerve‐evoked contraction to a single stimulus (Bexis et al., 2008). In this study, the role of these receptors in contractions to low and high frequency nerve stimulation have been investigated. Whole vas deferens from C57 mice were set up between electrodes in organ baths under 0.5 g tension in Krebs‐Henseleit solution, and stimulated electrically with 10 pulses at 1Hz or 40 pulses at 10 Hz using a Grass stimulator. The response to 1Hz stimulation consisted of discrete peaks of response corresponding to each stimulus. Threshold concentrations of the α1A‐adrenoceptor antagonist RS100329 (1nM) and the α1D‐adrenoceptor antagonist BMY7378 (100nM) significantly reduced contractions to the first pulse, and the non‐selective antagonist prazosin (10 nM) significantly reduced contractions to the first 4 pulses, at 1 Hz, but the combination of prazosin and the purinoceptor antagonist suramin produced marked inhibition of the response to all 10 pulses. The response to 10 Hz stimulation consisted of an early peak and later plateau of contraction. BMY7378 (100 nM) did not significantly affect the 10 Hz response but prazosin (10 nM) and RS100329 (1 nM) significantly reduced contractions, particularly the peak response, and higher concentrations markedly reduced responses. In the presence of the L‐type calcium channel blocker nifedipine, contractions to 10Hz stimulation were markedly reduced, particularly the later plateau component. In the presence of nifedipine, the peak response to 10 Hz stimulation was significantly reduced by prazosin (1nM) and by BMY7378 (100nM), demonstrating a small α1D‐adrenoceptor mediated component. In summary, contractions to 1 Hz stimulation have early α1A‐ and α1D‐adrenoceptor mediated components, but later responses are predominantly purinergic. Contractions even to 10 Hz stimulation have an early small α1D‐adrenoceptor component to the peak response, but both peak and plateau contractions largely involve α1A‐adrenoceptors and purinoceptors. The α1A‐adrenoceptor and purinoceptor components largely involve calcium entry through L‐type channels, but the α1D‐adrenoceptor mediated component involves T‐type calcium channels (Seto et al., 2010). In conclusion, the α1D‐adrenoceptor mediated component to both low and high frequency stimulation is very short‐lived, occuring in the first second of stimulation at low and high frequencies.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Novel Kinase Inhibitors Reduce Hypoxia‐Induced Apoptosis in Cardiomyocytes
Authors: Moyer Christopher S , Engman Samuel , Butel Bryan , Keomanivang Faithe , Wauson Eric M 
Published: June 2021

Our previous data suggest that bone morphogenic protein 2‐inducible kinase (BMP2K) plays a significant role in the pathophysiology of hypoxia/reoxygenation injury in cardiomyocytes, and that a reduction in BMP2K expression reduces apoptosis in cardiomyocytes. BMP2K and adaptor‐associated kinase 1 (AAK1) are human homologs of the drosophila Numb‐associated protein kinase (NAK). The function of BMP2K is unknown, and there are no identified physiological substrates. BMP2K shares a 74% sequence homology with the kinase domain of AAK1. Novel dual inhibitors of AAK1 and BMP2K, SGC‐AAK1‐1 and LP935509, have recently been developed. Because knockdown of BMP2K in cardiomyocytes protects against hypoxia‐induced cell death, we hypothesized that SGC‐AAK1‐1 and LP935509 would produce the same effect. To test our hypothesis, we pretreated H9c2 cardiomyocyte cells with SGC‐AAK1‐1, LP935509, or an inactive control compound SGC‐AAK1‐1N before subjecting the cells to hypoxic conditions. By analyzing cleaved caspase‐3 and cleaved PARP levels via immunoblotting, we determined that 3 μM of SGC‐AAK1‐1 or LP935509 reduced apoptosis. Since these compounds inhibit both AAK1 and BMP2K with similar potencies, we wanted to determine which kinase was responsible for reduced apoptosis. We observed that transfection of H9c2 cells with BMP2K siRNA, but not AAK1 siRNA, reduced hypoxia‐induced apoptosis. In addition, BMP2K siRNA increased overall cell viability. Phosphoantibody arrays are currently being done to assess the effect of SCG‐AAK1‐1 on cellular kinase signaling. Ongoing experimental objectives are to identify the mechanism by which these kinase inhibitors protect cardiomyocytes in hypoxic conditions, and characterize the substrates of BMP2K.Support or Funding InformationAmerican Heart Association (15SDG25090279) and Iowa Osteopathic Education and Research Funds (E.M.W.)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The effect of Polydatin in dry eye disease in vivo and in vitro
Authors: Park Bongkyun , Lee Tae Gu , Hyun Soo‐Wang , Jo Kyuhyung , Lee Ik Soo , Kim Jin Sook , Kim Chan‐Sik 
Published: June 2021

Polydatin (PD, also named pieceid, (E)‐piceid, (E)‐polydatin, trans‐polydatin, 3,4′, 5‐trihydroxystilbene‐3‐b‐D‐glucoside) is a monocrystalline compound originally isolated from the root and rhizome of Polygonum cuspidatum Sieb. et Zucc. (Polygonaceae), a traditional East Asia medicine. Previous study demonstrated that Polydatin has antioxidant and anti‐inflammatory effect. However, the effect of Polydatin in dry eye diseases (DED) has not been mentioned. We investigated the effect of Polydatin in the models of dry eye in vivo and in vitro. The measurement of tear volume showed that the decreased tear volume in the extraorbital lacrimal gland excised rats was significantly recovered by the Polydatin 0.5% eye drops. In addition, the corneal irregularity and lissamine green staining score revealed that Polydatin eye drops (0.05% and 0.5%) remarkably inhibited the increased corneal irregularity and lissamine green staining scores by the extraorbital lacrimal gland. Tear film breakup time assay showed that the shortened tear film breakup time was reasonably increased in the Polydatin 0.5% eye dropped group. Moreover, we examined the effect of Polydatin in the hyperosmolarity‐induced human conjunctival epithelium. The results from realtime PCR demonstrated that Polydatin significantly inhibited the mRNA expression of TNF‐α, IL‐6 and MMP9 in a concentration dependent manner. Taken together, our results suggest that Polydatin may a potential therapeutic agent to treat dry eye disease through the recovery of damaged corneal layer and inhibition of inflammation in conjunctival epithelium.Support or Funding InformationThis work was supported by Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry and Fisheries(IPET) through (High Value‐added Food Technology Development Program), funded by Ministry of Agriculture, Food and Rural Affairs(MAFRA) (#317033‐03).This work was also supported by Korea Institute of Oriental Medicine (Project No. KSN1812080).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Cannabinoid receptor antagonists and G protein subtypes
Authors: Eldeeb Khalil , Leone‐Kabler Sandra , Howlett Allyn C 
Published: June 2021

The therapeutic importance of inverse agonism for cannabinoid receptors (CB1R) has been proposed. The underlying mechanisms of inverse cannabimimetic effects by the CB1R antagonist/inverse agonist, SR141716 were investigated in both N18TG2 mouse neuroblastoma and human embryonic kidney (HEK)‐293 cells stably expressing CB1Rs (CB1‐HEK). We utilized cell membranes in ligand‐modulated [35S]GTPγS binding and scintillation proximity assay (SPA) (Eldeeb et al., 2016 and 2017). The CB1R agonist, CP55940 stimulated [35S]GTPγS binding (from 1nM: 2.6±5.5% to 1μM: 54±7.3% over basal). Co‐incubation with 1μM of SR141716 inhibited CP55940‐mediated [35S]GTPγS binding to −27±3.2% and −5.6±7%, respectively. In N18TG2 cells treated with the diacylglycerol lipase inhibitor, tetrahydrolipstatin (THL, orlistat), 1μM for 2h, SR141716 antagonizing effects were unchanged. In membranes from cells pretreated with pertussis toxin (PT) (100 ng/ml) for 20 h, SR14171 inhibition of [35S]GTPγS binding was attenuated in PT‐pretreated membranes, indicating that SR141716 effects were Gi/o protein‐dependent. Using SPA, SR14176 alone inhibited [35S]GTPγS binding by Gi/o protein subtypes, primarily Gαi1 and Gαi3 (−46.5±6.6% and −46.7±6.2% with respect to basal). Both previously reported CB1R neutral antagonists, AM4113 and PIMSR, at 1μM were able to reduce maximal CP55940‐stimulated [35S]GTPγS binding for total and individual Gi/o protein subtypes, primarily Gαi1 and Gαi3. Neither AM4113 nor PIMSR (1μM) were able to competitively reverse the SR141716‐mediated inhibition of [35S]GTPγS binding in N18TG2 neuronal cells membranes. In CB1‐HEK cell membranes neither AM4113 nor PIMSR(1μM) behaved as neutral antagonist to the CP55940‐mediated stimulation of [35S]GTPγS binding. The CB1R constitutive activity is mediated by Gi/o protein stimulation, predominantly Gαi1 and Gαi3, and in our experiments, was not associated with endogenously released endocannabinoids. Further research into the molecular mechanism of the competitive antagonist inverse agonist and/or negative allosteric modulator effects of SR141716 and its analogs will be necessary to consider in developing “neutral antagonists” for the CB1 receptor.Support or Funding InformationNIH grants R01‐DA03690, R01‐DA042157, P50‐DA006634, K12‐GM102773This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
C/EBP homologous protein deficiency inhibits statin‐induced myotoxicity
Authors: Woo Chang‐Hoon , Lim Jae Hyang 
Published: June 2021

It has been well established that HMG‐CoA reductase inhibitors (statins) cause adverse side effects in skeletal muscle ranging from mild to fatal myotoxicity upon dose, drug interaction, and exercise. However, the underlying mechanisms by which statins induce myotoxicity have not been fully addressed. Recent reports showed that statins induce endoplasmic reticulum (ER) stress and cell death in immune cells and myoblasts in vitro. Therefore, the goal of study is to investigate the molecular mechanism by which statins induce skeletal muscle cell death and myopathy via the regulation of ER stress. Biochemical data showed that TUDCA, an ER stress inhibitor, inhibited atorvastatin‐ and simvastatin‐induced protein cleavages of PARP‐1 and caspase‐3, respectively. Actually, statin treatment activated marker proteins of unfolded protein responses (UPR) including ATF6, CHOP, and spliced XBP1 and these responses were inhibited by TUDCA. In addition, statin treatment induced mRNA levels of UPR marker genes, suggesting that statins activate ER stress in a transcriptional regulation. The physiological relevance of ER stress in statin‐induced myopathy was demonstrated in a mouse model of myopathy, in which instillation of simvastatin and atorvastatin led to myopathy. Notably, the reduction of muscular endurance in response to statin instillation was significantly improved in TUDCA treating group compared to vehicle control group. Moreover, CHOP deficiency mice showed restoration of statin‐induced reduction of muscular endurance, suggesting that statin induces myopathy via ER stress and in a CHOP‐dependent manner. Taken together, these findings indicate that statins specifically induce myopathy in an ER stress‐dependent manner, suggesting the therapeutic potential of ER stress regulation in preventing adverse effects of statin.Support or Funding InformationThis study was supported by the Medical Research Center Program (2015R1A5A2009124) and the Basic Science Research Program (2018R1A2B6004664) of the National Research Foundation of Korea funded by the Ministry of Science and ICT.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Activation of autophagy protects against myocardial ischemic reperfusion injury by inhibition of NLRP3 inflammasome‐mediated pyroptosis and inflammatory responses in diabetic rats
Authors: Wang Sheng , Zhang Dengwen , Ye Xiaodong , He Yi , Cai Yin , Xia Zhengyuan 
Published: June 2021

BackgroundDiabetic hearts are more vulnerable to ischemia/reperfusion injury (I/RI). The activation of NLRP3 inflammasome can mediate pyroptosis and inflammatory responses, which in turn exacerbate myocardial I/RI. Studies have demonstrated that activation of autophagy can eliminate excess reactive oxygen species (ROS) generation and alleviate myocardial I/RI in diabetes. However, the possible regulatory relationship between autophagy and NLRP3 inflammasome during diabetic myocardial I/RI is still unknown. Thus, the present study aimed to investigate whether the activation of autophagy can alleviate diabetic myocardial I/RI via inhibition of NLRP3 inflammasome‐mediated pyroptosis and inflammatory responses.MethodsA dose of 60 mg/kg streptozotocin was given via tail vein injection to establish type 1 diabetes model in rats. The left anterior descending branch of the coronary artery was ligated for 30 minutes and then allowed for reperfusion for 2 hours to establish rat myocardial I/RI model. RapaLink‐1(1mg/kg, i.p.), the third‐generation mTOR inhibitor, was given 72 hours before inducing myocardial I/RI. The degree of I/RI was assessed by the measurement of plasma CK‐MB, cTnT, LDH, and myocardial infarction size. The expression of NLRP3, ACS, cleaved Caspase‐1, interleulin (IL)‐18, and IL‐1β in the myocardium, and the levels of IL‐18 and IL‐1β in the plasma were tested to assess the activation of inflammasome and inflammatory responses. In addition, LC3 □/□ and p62 were measured to evaluate the level of autophagy.ResultsCompared to non‐diabetic group, levels of cardiac autophagy were inhibited (decreased LC3 □/□ and increased p62) concomitant with activation of NLRP3 inflammasome (increased NLRP3, ASC, cleaved caspase‐1, IL‐18, IL‐1β) in diabetes. Also, more severe myocardial I/RI (elevated CK‐MB, cTnT, LDH, and larger infarction size) was seen in diabetic rats. Administration of RapaLink‐1 activated autophagy, inhibited NLRP3 inflammasome, and alleviated myocardial I/RI in diabetic rats.ConclusionThe activation of NLRP3 inflammasome in the diabetic myocardium may be attributable to the inhibition of autophagy. The activation of autophagy may inhibit NLRP3 inflammasome activation and consequently alleviate myocardial I/RI in diabetes.Support or Funding InformationThe study was supported by Guangdong Natural Science Foundation (2018A030313535, Guangdong, China) and HMRF (05161826) of Hong Kong.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Mesenchymal Stem Cell‐Based Delivery of Gene‐Silencing Products: Toward a Biological Therapy
Authors: Nemeth Erika , Gordon Chris , Devine Dan , Loizos Nick , Brink Peter R. 
Published: June 2021

It has been shown that microRNAs (miRNA) have tumor suppressive effects in an array of human cancers, are highly specific, and are able to be passed through intercellular gap junction channels. However, an effective delivery system for such gene‐silencing products has yet to be optimized for therapeutic applications. This study investigated the regulatory effects of miRNA in human prostate cancer cells (PC3) and the potential of a cell‐based delivery system of miRNA from a donor cell to a recipient cancer cell. Human mesenchymal precursor cells (huMPC; Mesoblast, Ltd.) were selected as donor cells because they enable gap junction‐mediated communication with PC3s and do not elicit an immune response in vivo. Results showed that microRNA‐16 (miR‐16) is a significant regulator of PC3 cell growth in monoculture as miR‐16‐transfected PC3s experienced the onset of a round morphology, decreased proliferation, increased apoptosis, and downregulation of the antiapoptotic protein BCL‐2. Co‐culture of miR‐16‐transfected huMPCs (donor cells) and normal PC3s (recipient cells) resulted in successful delivery of miR‐16. This study presents a preliminary model for therapeutic applications of a mesenchymal stem cell‐based miRNA delivery system in cancer.Support or Funding InformationThis research was supported by the American Physiological Society's Undergraduate Summer Research Fellowship. Human mesenchymal precursor cells (huMPC) were provided by Mesoblast, Ltd.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.