bluuebunny/crossref_metadata_2025_split huggingface dataset:
Mechanisms of a Function‐Selective ERK Inhibitor and Induction of Apoptosis in Melanoma Cells
Authors: Martinez Ramon , Huang Weiliang , Mackowiak Bryan , Shapiro Paul 
Published: June 2021

Various cancer cell types can contribute their proliferative phenotypes to mutations conferring constitutive activity of the extracellular signal‐regulated kinase (ERK1/2) signaling pathway. Recent trends in drug design have suggested that shifting the design of ERK1/2 pathway inhibitors from targeting the catalytic core of the kinase to allosteric inhibition may prove as a promising alternative to therapeutic development. We previously identified a novel thienyl naphthalene sulfonate compound that shows functionally‐selective inhibitory activity against ERK2 by blocking F‐site containing substrates, including Fos family proteins. Proteomic analysis of drug‐treated melanoma cell lysates suggests that treatment with this novel compound results in defects in mitochondrial function as well as upregulation of markers suggestive of activating an oxidative stress response. As well, further mass spectrometry analysis has suggested that the novel compound may generate covalent modifications on cysteine residues of the ERK2 protein. Additional studies with inhibitors of various reactive oxygen species (ROS) have shown that while the generation of ROS may be evident with compound treatment, further cell viability study has indicated that ROS is only partially responsible for the growth inhibition of melanoma cells. In these studies, we provide further insight into the interactions this novel compound makes with the ERK2 protein, and the transcriptomic and proteomic responses that ultimately result in oxidative stress and melanoma cell death.Support or Funding InformationThis work was supported by an NIGMS Institutional Training Grant (T32 GM066706).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Phycocyanin Enhances the Cytotoxic Profile of Cisplatin in Platinum‐Resistant Ovarian Cancer Cells by Targeting Focal Adhesion Kinase
Authors: Levy Arkene , Joseph Shona , Hussain Syed , Saleem Mir , Rathinavelu Appu 
Published: June 2021

Platinum‐based therapy is one of the major treatment approaches in ovarian cancer. Although greater than 80% of advanced stage ovarian cancer patients initially achieve remission with platinum therapy; the majority of this cohort eventually relapse, and develop drug‐resistant disease. Resistance to chemotherapeutic drugs such as cisplatin has been attributed in part to the overexpression of Focal Adhesion Kinase (FAK), which is known to promote cancer cell survival and metastasis. In recent years, there has been heightened interest in targeting focal adhesion kinase in platinum resistant cancer as a new modality in ovarian cancer treatment. In previous studies, we have reported that Phycocyanin (C‐PC), a major accessory pigment in the Limnothrix sp. 37‐2‐1, demonstrates cytotoxicity towards multiple cancer cell lines. Therefore, in the present study we evaluated the ability of C‐PC to sensitize platinum resistant OVCAR3 cells to cisplatin treatment. We also examined the response of FAK activation by phosphorylation, to treatment with cisplatin and CP‐C alone, and in combination (C‐PC +cisplatin). Cell viability was determined by MTT assay, and DNA fragmentation was used to confirm apoptotic cell death. The expression of phosphorylated FAK (pFAK), total FAK, pAKT, as well as the key apoptotic regulators p21, p53, Bax, and Bcl‐2 in response to the treatments were determined through western blotting. The results from our study confirm that C‐PC significantly enhances the cytotoxic profile of cisplatin in OVCAR‐3 cells through the induction of apoptosis as confirmed by the presence of DNA fragmentation patterns in all three treatment groups. The expression of activated pFAK was also significantly reduced in the CPC and CPC + cisplatin treatment groups and this corresponded with increased p53, p21 and Bax expression, while pAkt and Bcl‐2 expression was downregulated. These results confirm a novel effect of CPC on FAK phosphorylation in platinum resistant ovarian cancer, which can be further explored towards the treatment of advanced stage disease through combination strategies.Support or Funding InformationThis project was supported by The Royal Dames of Cancer Research Inc., Ft. Lauderdale, FloridaThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Boswellic Acid Composition of Frankincense Dietary Supplements and Correlation to Cytotoxic Efficacy against Treatment‐Resistant Triple Negative Breast Cancer Cells
Authors: Schmiech Michael , Lang Sophia , Werner Katharina , Schmidt Christoph Q , Syrovets Tatiana , Simmet Thomas 
Published: June 2021

Frankincense is an oleogum resin from Boswellia trees of the Burseraceae family. The oleogum resin contains 15–20 % triterpenic acids, including therapeutically active boswellic acids [1]. Extracts of oleogum resins obtained from Boswellia serrata have been traditionally used in Indian folk medicine (Ayurveda) for the treatment of various chronic inflammatory diseases such as rheumatoid arthritis. Furthermore, boswellic acids are known to possess anticancer efficacy against various human cancer types, e.g. gliomas, prostate cancer, or leukemia [2–4]. The aim of the present study was to analyze the contents of boswellic acids in dietary supplements from various Boswellia species grown in different areas and to correlate the concentrations of distinct boswellic acids to the extracts' cytotoxic efficacy against chemoresistant triple‐negative human breast cancer cells. Accordingly, a new method to quantify a large variety of pentacyclic triterpenic acids was developed. Dietary supplements from various Boswellia species Boswellia serrata, Boswellia sacra, Boswellia carterii, and others were extracted with methanol and analyzed by high performance reversed‐phase liquid chromatography coupled with tandem mass spectrometric detection (HPLC‐MS/MS). Besides, the effects of the extracts against the treatment‐resistant, triple negative human breast cancer cell line MDA‐MB‐231 were investigated in vitro by using a cell viability assay. The extracts exhibited potent cytotoxic activity against the human breast cancer cell line. Correlations between the boswellic acid contents and the cytotoxic efficacy were determined by multiple linear regression and principal component regression. This approach allowed not only the correlation of complex phytochemical mixtures to desired biomedical effects, but it likewise provided evidence for key compounds governing the therapeutic efficacy. Hence, the study provides new insights into differential chemical composition patterns of frankincense dietary supplements from Boswellia species grown in different regions. Besides, the study identified distinct components correlating positively with the desired cytotoxic activity. Thus, the identified key compounds deserve further studies either as chemically pure substances or as defined chemical mixtures permitting development of the most active ingredients as potential anticancer drug candidates.Support or Funding InformationThis work was supported by the Academic Center for Complementary and Integrative Medicine (AZKIM), State Ministry of Baden‐Württemberg for Sciences, Research and Arts.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Constituents of Artemisia annua Dietary Supplements Induce ROS Elevation, ERK Activation, and Apoptosis in Treatment‐Resistant Triple Negative Human Breast Cancer Cells
Authors: Lang Sophia , Schmiech Michael , Hafner Susanne , Paetz Christian , Schmidt Christoph Q , Syrovets Tatiana , Simmet Thomas 
Published: June 2021

In recent years, Artemisia annua L. and especially artemisinin and its semisynthetic derivatives became subject of intense investigations for their potential anticancer activity. Yet, available evidence suggests that artemisinin might not be the most active ingredient of Artemisia annua. For this reason, we have analyzed different Artemisia annua extracts, which did not contain any detectable artemisinin marketed as dietary supplements for their activity against the triple negative human breast cancer cell line MDA‐MB‐231. A bioguided fractionation by semi‐preparative HPLC was conducted and the structures of individual compounds were identified by mass spectrometry and 1H‐ and 13C‐NMR spectroscopy. Extracts as well as the isolated compounds 6,7‐dimethoxy‐coumarin, chrysosplenol D, casticin, arteannuic acid and arteannuin B were further analyzed in vitro and in vivo. The extracts and the individual compounds inhibited the viability of several cancer cell lines. Interestingly, the viability of peripheral blood mononuclear cells was not inhibited at equivalent concentrations. Moreover, treatment either with extract or the individual compounds induced cell cycle arrest in the S‐phase or the G2/M‐phase. Apoptotic cell death was identified by DNA‐fragmentation, activation of caspase 3/7, and annexin V/propidium iodide staining. Moreover, the extract and individual compounds increased the phosphorylation of the extracellular regulated kinase ERK in the presence of sustained high ROS levels, which are required for the ERK‐mediated cell death. Pretreatment with the ERK ‐ inhibitor U‐0126 was able to inhibit chrysosplenol D induced toxicity towards MDA‐MB‐231 cells. Furthermore, antiproliferative efficacies of the extract and individual compounds were confirmed in vivo using 3D breast cancer xenografts grown on the chorioallantoic membrane of fertilized chick eggs, where the Artemisia annua extract and the isolated substances significantly reduced the MDA‐MB‐231 breast cancer tumor volume as well as the expression of the Ki‐67 proliferation marker. Importantly, no systemic toxic effects on the chick embryos could be detected. This study provides new insights into a potential rational use of Artemisia annua‐derived compounds in anticancer therapy and identifies compounds beside artemisinin with potent anticancer activity. Furthermore, these findings provide evidence for the implication of the MEK‐ERK‐pathway in apoptotic cell death induced by chrysosplenol D.Support or Funding InformationThis work was supported by the Academic Center for Complementary and Integrative Medicine (AZKIM), State Ministry of Baden‐Württemberg for Sciences, Research and Arts.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Pharmacological inhibition of Human Antigen R (HuR) blunts fibroblast activation and cardiac fibrosis
Authors: Green Lisa C , Anthony Sarah R , Slone Samuel , Lanzillotta Lindsey , Lorenz John , Tranter Michael 
Published: June 2021

Heart failure is one of the leading causes of death globally, and the physiology of a failing heart is often worsened by the presence of cardiac fibrosis. Cardiac fibrosis is marked by the chronic activation of fibroblasts into myofibroblasts and excessive extracellular matrix (ECM) protein deposition, leading to decreased contractility and compliance of the myocardium. In this work we show that Human Antigen R (HuR), a stabilizing RNA binding protein, plays a critical role in the activation of fibroblasts and the subsequent fibrosis. In vivo, we used the transverse aortic constriction (TAC) model of heart failure and started treatment with a small molecule inhibitor of HuR four weeks after TAC surgeries. At the end of the eleven‐week study, the mice treated with the HuR inhibitor had markedly reduced fibrosis shown by Masson's Trichrome staining. Western blot and qRT‐PCR analysis also revealed a decreased expression of fibrotic markers such as fibronectin and periostin in the hearts from mice treated with HuR inhibitor. In vitro, we also saw that pharmacologically inhibiting HuR in fibroblasts isolated from adult mouse hearts blunts the activation of fibroblasts using assays such as the scratch assay. To identify a potential underlying mechanism for direct reduction in fibrosis via HuR inhibition, we interrogated the connection between HuR and TGFβ, a well‐known driving factor in fibrotic initiation and progression. Using an Actinomycin D stability assay on isolated primary myocytes, we showed a decrease in TGFβ mRNA stability upon inhibition of HuR. We also performed RNA immunoprecipitation and the results support a direct binding of HuR to TGFβ mRNA. Taken together, these suggest HuR may play a central role in the regulating the stability of TGFβ, thereby affecting pro‐fibrotic signaling. In conclusion, these results suggest that HuR is a promising therapeutic target for the reduction of cardiac fibrosis.Support or Funding InformationThis work was partially funded by an AHA Scientist Development Grant (MT), NIH R01 HL132111 (MT), and T32HL125204‐4 (JM, EK)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Screening of Metabolic Markers of Qi‐Yin deficiency in Chronic Heart Failure Mice by Untargeted Metabolomics Method
Authors: Lai Qiong , Li Fang , Liu Liang Ze , Yu Yang Bo 
Published: June 2021

Qi‐Yin deficiency is the main syndrome of chronic heart failure that one of the major heart disease that threatening human health. Clarifying the time‐history of Qi‐Yin deficiency in chronic heart failure disease, and further exploring the metabolic markers of Qi‐Yin deficiency can help to explain the pathological mechanism and Precise treatment of Qi‐Yin deficiency in chronic heart failure. This study adopted chronic heart failure model induced by coronary artery ligation in mice. We investigated the changes in the appearance of clinical indications corresponding to symptoms of Qi‐Yin deficiency at different time points, which aimed to clear the time‐history of Qi‐Yin deficiency appeared in chronic heart failure model mice. Positive (Yiqifumai) and negative (Danhong) control group were set up to assistant define the time‐history of Qi‐Yin deficiency. We found that Chronic heart failure mice appeared significant weight loss, decreased food intake, spontaneous activity , accelerated heart rate, and increased T wave amplitude in the first 2–3 weeks after modeling. What's more, Yiqifumai had better effects than Danhong in the improvement of Qi‐Yin deficiency in the first 2–3 weeks, which assistant proved that this period was Qi‐Yin deficiency. The coagulation function test results also suggest that it may turn into blood stasis syndrome around 4–5 weeks. In addition, LC‐MS/MS technology was used to determine the endogenous metabolites in serum and urine of mice on the positive ion mode. The data of accurate molecular weight and fragment ion peak were checked on HMDB database, and the identification methods of PCA and OPLS‐DA were used to analyze the multivariable data. Metabolites of the chronic heart failure model mice during the period of Qi‐Yin deficiency and non Qi‐Yin deficiency were compared to explore specific metabolite groups significant related to Qi‐Yin deficiency. The untargeted metabolomic workflow is showed in Figure 1. The results of metabolomic analysis of serum and urine of model mice showed that metabolites were well‐differentiated between the two groups at 2–3 weeks and 4–5 weeks. 29 significant difference metabolites in serum and 19 significant difference metabolites in urine were identified in this study. Hence, these results demonstrated that ischemia‐induced chronic heart failure may be mainly manifested as Qi‐Yin deficiency syndrome in the first 2–3 weeks, followed by conversion to blood stasis syndrome. Metabolites of chronic heart failure mice also changed as the syndrome transformed.Support or Funding InformationNational Natural Science Foundation of China (NO. 81774150)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Towards Identifying the Binding Site for 125I‐Angiotensin 1‐7
Authors: Stoyell‐Conti Filipe Fernandes , Itty Sarin , Stewart Hanna , Asif Mahnoor , Abraham Christy , Vadhan Jason , West Crystal A , Speth Robert C 
Published: June 2021

Angiotensin 1–7 (Ang 1‐7) is a product of the renin‐angiotensin system (RAS) that exhibits central actions to reduce blood pressure and improve baroreflex sensitivity. For more than a decade Mas has been considered the receptor for Ang 1‐7, however, to date, no published studies have characterized Mas receptor radioligand binding in tissue membrane preparations. Our laboratory has demonstrated high affinity (low nanomolar KD) binding of a preparation of 125/127I‐Ang 1‐7 in liver membranes, however, this binding is not pharmacologically specific in that the IC50 of Ang 1‐7 is in the micromolar range and all angiotensin peptides compete for 125/127I‐Ang 1‐7 binding equivalently. 125/127I‐Ang 1‐7 binding to liver, kidney, brain and testes membrane preparations from both rats and mice has also failed to demonstrate a high affinity, pharmacologically specific binding site for 125/127I‐Ang 1‐7. Considering that rapid degradation of Ang 1‐7 could be adversely affecting the binding assay, we tested different peptidase inhibitors (JMV‐390, O‐phenanthroline and a proprietary combination of peptidase inhibitors Biotools®) to prevent degradation of the radioligand. Although we were able to reduce Ang 1‐7 degradation with these inhibitors (~80% preservation), we also reduced the total and specific binding of 125I‐Ang 1‐7. Indeed, competition binding assays for 125I‐Ang‐(1–7) with JMV‐390, O‐phenanthroline and Biotools® showed that 125I‐Ang‐(1–7) and all the inhibitors compete for the same binding site with JMV‐390 having a nanomolar IC50. It is important to note that the radioligand used for receptor binding studies contains an iodine125 on the tyrosine in position four of Ang 1‐7, and may have different characteristics for enzymatic metabolism. To assess this possibility, we determined the metabolism of 127I‐Ang 1‐7 (non‐radioactive iodine isotope) and indeed, the metabolism of Ang 1‐7 and 127I‐Ang 1‐7 in liver appear to differ, with 127I‐Ang 1‐7 being more resistant to metabolism. In addition, competition binding assays showed that 127I‐Ang 1‐7 is ~1000‐fold more potent than Ang 1‐7 in competing for 125I‐Ang 1‐7 binding. In conclusion, our results suggest that 125I‐Ang‐(1–7) may be binding to a peptidase rather than a receptor and the addition of the iodine molecule to the tyrosine in position four attenuates its degradation. Studies are underway to identify this peptidase and determine its potential therapeutic importance as a druggable target.Support or Funding InformationCardiovascular Neuroscience Research Fund 337667 Speth (PI) 03/31/15‐12/31/18 ‐ Nova Southeastern Univ.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Interindividual Variation in Sunitinib Metabolism in Primary Human Hepatocytes
Authors: Abouda Arsany A. , Bissada Jennifer E. , Jackson Klarissa D. 
Published: June 2021

Sunitinib is a multitargeted tyrosine kinase inhibitor associated with idiosyncratic hepatotoxicity. The mechanisms of this toxicity are unknown. Sunitinib is metabolized primarily by cytochrome P450 (CYP) 3A4/5 to form the major active metabolite N‐desethylsunitinib (M1). Recent studies from our lab have also shown that sunitinib undergoes oxidative defluorination via CYP1A and CYP3A enzymes to form a chemically reactive, potentially toxic quinoneimine metabolite. We hypothesized that P450 genetic variations contribute to individual differences in sunitinib metabolism and bioactivation, which may play a role in development of sunitinib‐induced hepatotoxicity. The purpose of this study was to characterize the metabolism of sunitinib in CYP3A5‐genotyped primary human hepatocytes, and evaluate the influence of genetic variation and other individual factors on metabolite formation. Commercially available human hepatocytes from individual donors were purchased from BioIVT. CYP3A5 genotype and P450 enzyme activities for each donor were provided by the company. Donor genotypes were reported as: CYP3A5*1/*1, CYP3A5*1/*3, and CYP3A5*3/*3. Sunitinib (10 uM) was incubated with the hepatocytes in suspension for 2 h, and drug metabolites were analyzed by liquid chromatography‐tandem mass spectrometry. In experiments with individual genotyped hepatocytes, M1 formation varied 13.5‐fold between donors but did not significantly differ by CYP3A5genotype. However, M1 formation was significantly higher in female donors compared to male donors. Generation of defluorinated sunitinib (M3) varied 5.6‐fold between donors but did not significantly differ by CYP3A5genotype or sex. These results suggest that donor sex, but not CYP3A5genotype, may influence the metabolism of sunitinib to the major metabolite M1. Additional studies are needed to identify the individual factors that impact the bioactivation of sunitinib to form the reactive quinoneimine product. Studies are currently underway to determine the relationship between P450 enzyme activity and sunitinib metabolite formation in individual human hepatocytes.Support or Funding InformationThis research was supported by the American Association of Colleges of Pharmacy, Lipscomb University College of Pharmacy and Health Sciences, and the National Cancer Institute of the National Institutes of Health (K01CA190711). Research reported here is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Intestinal HIF‐1α Deletion Exacerbates Alcoholic Liver Disease through Inducing Intestinal Dysbiosis and Barrier Dysfunction
Authors: Feng Wenke 
Published: June 2021

ObjectivesAlcoholic liver disease (ALD) is characterized by gut dysbiosis and increased gut permeability. Hypoxia Induced Factor 1α (HIF‐1α) has been implicated in transcriptional regulation of intestinal barrier integrity and inflammation. We aimed to test the hypothesis that HIF‐1α plays a critical role in gut microbiota homeostasis and the maintenance of intestinal barrier integrity in a mouse model of ALD.MethodsWide type (WT) and intestinal epithelial‐specific HIF‐1α knockout mice (IEhif‐1α−/−) were pair‐fed modified Lieber‐DeCarli liquid diet containing 5% (w/v) alcohol or isocaloric maltose dextrin for 24 days. Serum levels of ALT and endotoxin were determined. Fecal microbiota were assessed. Liver steatosis and injury, and intestinal barrier integrity were evaluated.ResultsAlcohol feeding increased serum levels of ALT and LPS, hepatic triglyceride concentration, and liver injury in the WT mice. These deleterious effects were exaggerated in IEhif‐1α−/− mice. Alcohol exposure resulted in greater reduction of the expression of intestinal epithelial tight junction proteins, claudin‐1 and occludin, in IEhif‐1α−/− mice. In addition, cathelicidin‐related antimicrobial peptide (CRAMP) and intestinal trefoil factor (ITF) were further decreased by alcohol in IEhif‐1α−/− mice. Metagenomic analysis showed an increased gut dysbiosis with a significantly decreased firmicutes/bacteroidetes ratio in IEhif‐1α−/− mice compared to the WT mice exposed to alcohol. An increased abundance of Akkermansia and a decreased level of Lactobacillus in IEhif‐1α−/− mice were observed. Non‐absorbable antibiotics treatment reversed the liver steatosis in both WT and IEhif‐1α−/− mice.ConclusionIntestinal HIF‐1α is essential for the adaptation response to alcohol exposure‐induced changes in intestinal microbiota and barrier function associated with elevated endotoxemia and hepatic steatosis and injury.Support or Funding InformationNIHThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.