bluuebunny/crossref_metadata_2025_split huggingface dataset:
Bioengineered let‐7c is Effective at Reducing Orthotopic Hepatocellular Carcinoma Tumor Burden and is Well Tolerated in Mouse Models
Authors: Jilek Joseph Lionel , Zhang Qianyu , Ho Pui Yan , Tu Meijuan , Duan Zhijian , Yu Aiming 
Published: June 2021

Given pleiotropic molecular targeting mechanisms, restoration or inhibition of endogenous microRNAs (miRNA) is a promising pharmacological strategy. Recently, our group has bioengineered a collection of miRNA agents in live cells using a non‐coding carrier RNA (nCAR) based technology. In vitro screening identified nCAR/let‐7c as the most potent at inhibiting hepatocellular carcinoma (HCC) viability; this was found to be processed to the mature let‐7c molecule and suppressed target gene expression. As such, in vivo efficacy and safety of this agent was characterized in the present study using polyethyleneimine (PEI) and liposomal‐polyplex (LPP) RNA formulations. Huh7 cells stably expressing luciferase and green fluorescent protein were orthotopically engrafted into immunocompromised mice. To evaluate efficacy, 40 μg of nCAR/let‐7c or a truncated control agent was administered systemically every‐other day for two weeks, followed immediately by necropsy. Live animal bioluminescent and ex vivo fluorescent imaging suggested that LPP‐formulated nCAR/let‐7c reduced tumor burden to a greater degree than all controls and PEI‐formulated nCAR/let‐7c. Additionally, both nCAR/let‐7c and truncated control agents were well‐tolerated compared to untreated mice, as evidenced by gross evaluation and similar bodyweights throughout the study; however, further studies are warranted in immunocompetent animal models to elucidate the complete toxicological profile and immunogenic potential of nCAR/let‐7c. These data are the first to demonstrate that let‐7c restoration using a biologically‐derived agent can serve as a potent antineoplastic agent, presenting a new, clinically‐relevant strategy for HCC therapy.Support or Funding InformationThis work was supported by National Institutes of Health grants [R01GM113888 and U01CA175315 to A.‐M.Y.] and an institutional Pharmacology Training Program grant [T32GM099608 to J.L.J.].This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Targeted Osmotic Lysis of Highly Invasive Carcinomas Using a Pulsed Magnetic Field and Pharmacological Blockade of Voltage‐Gated Sodium Channels
Authors: Paul Dennis , Maggi Paul E. , Minkoff Lawrance , Piero Fabio Del , Gould Harry J. 
Published: June 2021

Simultaneous electrical or physiological stimulation of voltage‐gated sodium channels (VGSCs) and concomitant pharmacological blockade of sodium, potassium‐ATPase (sodium pumps) causes lysis of cells that overexpress VGSCs. Lysis does not occur in tissues that express normal levels of VGSCs. Metastatic carcinomas overexpress VGSCs. Pulsed magnetic fields (PMFS) will also activate VGSCs. Accordingly, we hypothesized that a PMF would lyse invasive carcinomas concomitantly treated with the sodium pump blocker, digoxin, without affecting noncancerous cells or tissue. MDA‐MB‐231 breast cancer cells cultured in DMEM, or MCF‐10a normal breast epithelial cells cultured in MEGM, were dissociated with Cellstripper and resuspended in microfuge tubes with or without 5–500 nM digoxin. Microfuge tubes were placed in an 8″l × 2.5″dia. electromagnetic solenoid with 697 turns of 12 ga copper wire. A Tektronix AFG 3021B waveform generator provided a 2 Vpk – 5 Vpk, 25 Hz pulse. The signal was fed into an AE Techron 7224 DC amplifier connected between the solenoid and the function generator, producing 9 Apk – 22 Apk monophasic DC current pulses into the solenoid, resulting in a 36 to 90 mT PMF inside the solenoid. After stimulation, cells were fixed, stained with cresyl violet and percent nonviable cells counted. Digoxin treated MDA‐MB‐231 cells lysed dose‐ and stimulation‐dependently within the 10 min stimulation interval, whereas drug‐only and stimulation‐only controls were unaffected. Neither digoxin‐treated nor untreated MCF‐10a cells lysed. For in vivo experiments, J/Nu mice with MDA‐MB‐231 xenografts were injected with 5 mg/kg digoxin or saline 5 times at 1 hr intervals on days 1, 3 and 5. This protocol achieves steady‐state pharmacokinetics. On each of the stimulation days, mice were confined to the solenoid and exposed to the 25 Hz, 90 mT PMF for 15 min starting 30 min after the last injection. Mice were euthanized and fixed with paraformaldehyde 24 hrs after the last treatment. Subcutaneous tumors from mice treated with digoxin and PMF were characterized by 60–100% tumor lysis. No lysis was observed in any of the normal tissues of treated mice. Drug‐only and stimulation‐only controls did not differ from untreated controls. These results are evidence that targeted osmotic lysis using a PMF warrants further study as a treatment for metastatic carcinomas.Support or Funding InformationSupported by the LSUHSC Depts. of Pharmacology and Neurology and a research contract with Oleander Medical Technologies.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
High Dose Ascorbate Effect on Certain Cancer Cell Lines
Authors: Bennett Lunawati Lo 
Published: June 2021

In recent years, the use of natural dietary antioxidants in cancer treatment has gained popularity due to their minimal cytotoxic effects with no evidence of causing damage to normal cellular tissues. Since the discovery of vitamin C (ascorbic acid, ascorbate), this antioxidant has a controversial history in relation to cancer treatment. Oral ascorbate had no or little effect on survival of the cancer patients. However, the discovery that intravenous ascorbate can provide 100‐fold higher plasma levels as compared to oral intake has renewed interest on the role of ascorbate. One hypothesis to support the use of high‐dose ascorbate is the finding that at high dose (pharmacological concentrations); it may act as a prodrug to deliver extracellular H2O2 that is selectively toxic to cancer cells. High dose ascorbate is non‐toxic and well tolerated to normal cells.In the present study, several cell lines such as HepG2 (human liver carcinoma cells), MCF7 (human breast cancer), SaOS2 (human osteosarcoma), and CaCO2 (human colorectal adenocarcinoma cells) were used. Ascorbate was buffered to pH 7 with sodium hydroxide and prepared immediately before use. Cells viability measured by MTT assay (3‐[4, 5‐dimethylthiazol‐2‐yl]‐2, 5‐diphenyl‐tetrazolium bromide), Hoechst33342, H2DCFDA, and Rhodamine123 staining using fluorescent microscope were conducted to determine cell viability, chromatin inducing apoptosis, intracellular generation of ROS, and mitochondrial membrane potential, respectively. Western Blot to detect different signaling protein expressions such as Poly (ADP‐Ribose) polymerase (PARP), Casp‐3, Casp‐9, P‐53, Bax, Bcl2, Apaf‐1, MMP2, MMP 9, BRCA1, BRCA2, Bim, P‐Map kinases were conducted to test the hypothesis that ascorbate decreased cell proliferation, increased intracellular ROS generation and apoptotic cells, and decreased mitochondrial membrane potential. The concentration of ascorbate that causing 70–80% death of the cells ranged from 0.8 mM in SaOS2, 1.5 mM in CaCO2, 6 mM in HepG2, and 7 mM in MCF7, respectively. This finding suggests that high dose ascorbate has a potential candidate as effective anti‐neoplastic in several cancer types.Support or Funding InformationUnion University College of PharmacyThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Biomarker Discovery for Cancer Sensitivity to CDK8 Inhibitors
Authors: Lu Zhixin , Spear Jensen E. , Russu Wade A. 
Published: June 2021

BackgroundA novel piperazinylpyrimidine compound, Q12, was synthesized and tested for its potential to selectively inhibit the growth of certain tumor cell lines within the NCI‐60 cell line panel. Q12 is an ATP competitive inhibitor of cyclin dependent kinase 8 (CDK 8). We studied Q12 in the context of sensitive cell lines MDA‐MB‐468 (GI50 0.06 μM) and HCT‐116 (GI50 0.27 μM) and a non‐sensitive cell line COLO 205 (GI50 30 μM). The MDA‐MD‐468 cell line represents triple negative breast cancer, a difficult type of cancer to treat because of lacking HER2, estrogen and progesterone receptors. HCT‐116 and COLO 205 are human colon cancer cell lines, which harbor β‐catenin and APC mutations respectively. We hypothesized that Q12 is a promising molecule that can be used to probe biomarker discovery for CDK8 inhibitor sensitive tumor types.Material and MethodsIn vitro studies were performed by using of MDA‐MB‐468 breast cancer cell line, HCT‐116 and COLO 205 colon cancer cell lines. GI50 was measured and apoptosis assay was performed. The growth inhibitory effect of Q12 was assessed by Muse Cell Viability Kit. To evaluate the effects of Q12 on β‐catenin and STATs, Western Blotting were used.ResultsThe cell viability essay shows Q12 potently inhibited the growth and viability of MDA‐MB‐468 cells and HCT‐116 cells, but COLO 205 cell line was resistant. Western blotting shows the reduction of STAT1 phosphorylation, a robust indicator of CDK8 target engagement, in all three cell lines upon Q12 treatment. Western blotting also showed a reduction in β‐catenin in both HCT‐116 and COLO 205 cells, but unchanged in MDA‐MB‐468 cell line, upon treatment with Q12.ConclusionOur data shows that active β‐catenin is not necessarily a driver of colon tumor cell proliferation and viability nor is it a marker for CDK8 inhibitor sensitivity. Both HCT‐116 and COLO205 cells harbor mutations that stabilize β‐catenin (β‐catenin and APC respectively), and both experience reductions in β‐catenin upon treatment with CDK8, but have vastly different proliferation and viability responses. The MDA‐MD‐468 cell line is very sensitive to Q12, but does not experience a reduction in β‐catenin. What is interesting to note is that both HCT‐116 and MDA‐MB‐468 harbor mutations related to RB1 (CDKN2A and RB1 respectively). Disparate E2F1 transcriptional activity may hold the key to discovery of a biomarker of CDK8 inhibitor sensitivity.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Lysosomal Ca2+ Release via TRPML1 Channels Regulated by Acid Ceramidase and Associated Sphingolipids in Podocytes
Authors: Li Guangbi , Li Ningjun , Li Pin‐Lan 
Published: June 2021

Transient receptor potential mucolipin 1 (TRPML1) channel has been reported to mediate lysosomal Ca2+ release that is involved in Ca2+‐dependent lysosome trafficking and autophagic flux. However, this regulatory mechanism of lysosomal TRPML1 channel activity in podocytes remains poorly understood. In the present study, we tested the hypothesis that acid ceramidase (AC) plays a pivotal role in regulation of TRPML1 channel activity in podocytes. To detect TRPML1 channel‐mediated Ca2+ release, cultured murine podocytes were transfected via nucleofection with GCaMP3‐ML1, a TRPML1‐targeted genetically encoded Ca2+ indicator. To characterize GCaMP3 expression in lysosomes, confocal microscopy confirmed the colocalization of GCaMP3‐ML1 and Lamp‐1 suggesting its successful transfection and localization in lysosomes. ML‐SA1 as a specific cell‐permeable TRPML1 inducer remarkably increased GCaMP3 fluorescence intensity in podocytes. For dynamic recording of Ca2+ release, a high speed wavelength switching fluorescent microscopic imaging system was used to continuously monitor GCaMP3 fluorescence signal in living podocytes. It was found that in the presence of a lysosome‐disrupting cathepsin C substrate, GPN that depleted lysosomal Ca2+, GCaMP3‐detected Ca2+ release response to ML‐SA1 was substantially attenuated. Pre‐treatment of podocytes with carmofur, a specific AC inhibitor almost totally blocked ML‐SA1‐induced Ca2+ release in podocytes. Sphingosine as the AC product of ceramide hydrolysis was found to increase GCaMP3 fluorescence by 3.95 folds in podocytes. Whole‐lysosome patch clamp recording using a Port‐A‐Patch system was performed to study the effects of various AC‐associated sphingolipids on TRPML1 channel activity. After characterization of integrity and purity of isolated lysosomes, we detected ML‐SA1‐induced Ca2+ currents through the isolated lysosome membrane in a dose‐dependent manner. Sphingomyelin, the substrate of ceramide, blocked Ca2+ release induced by ML‐SA1, ceramide had no effects on ML‐SA1‐induced Ca2+ currents, but sphingosine as a product of ceramide hydrolysis via AC remarkably increased ML‐SA1‐induced Ca2+ release by 2.35 folds. Furthermore, interactions of lysosome and autophagosome in podocytes indicated by colocalization of LC3B‐GFP and Lamp‐1‐RFP were inhibited by carmofur. These results suggest that normal function of lysosomal AC and associated production of sphingosine are important for TRPML1 channel activity and Ca2+‐dependent lysosome trafficking.Support or Funding InformationThis study was supported by NIH grants DK54927 to Pin‐Lan Li and HL89563 to Ningjun Li.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Structure‐Activity Relationship Studies of SK Channel Modulation
Authors: Orfali Razan , Nam Young‐Woo , Viegas Adam , Zhang Miao 
Published: June 2021

The small‐ and intermediate‐conductance Ca2+ activated K+ (SK/IK) channels play a fundamental role in the regulation of neurons in the central nervous system. In animal models, SK/IK channel positive allosteric modulators (PAMs) have been shown to be effective in reducing the symptoms of neurological diseases such as ataxia. Ataxia is a lethal neurological disease characterized by lack of balance and incoordination of muscle movements, often as a result of cerebellar or spinocerebellar neurodegeneration. Despite these progresses, current available SK/IK channel PAMs often suffer from low potency and/or poor pharmacokinetic properties. Lack of knowledge about the binding site for the compounds is a major reason that hinders the development of more effective therapeutics targeting SK channels. We previously determined the crystal structures of the PAMs such as 1‐EBIO and NS309 in complex with their binding pocket. We found a strong correlation between the potency of the PAMs and their interaction energy within the binding pocket. These structure‐activity relationship studies of SK/IK channel modulation by PAMs might facilitate drug discovery targeting SK/IK channels.Support or Funding InformationAmerican Heart Association SDG Grant and National Ataxia Foundation YI‐SCA Grant to M.Z.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Characterization of the Cardiovascular and Renal Effects of Synthetic Nociceptin/Orphanin FQ Receptor Partial Agonists
Authors: Denys Ian B. , Gao Juan , Zaveri Nurulain T. , Kapusta Daniel R. 
Published: June 2021

Following discovery of the endogenous opioid‐like peptide, nociceptin/orphanin FQ (N/OFQ) and the N/OFQ peptide receptor (NOP), numerous peptide and non‐peptide NOP receptor ligands have been developed for potential treatment of disease states including Parkinson's, depression, anxiety, and chronic pain. In addition, our laboratory has demonstrated that NOP receptor peptide ligands produce changes in cardiovascular and renal function. However, we have yet to determine whether non‐peptide NOP receptor ligands have similar cardiovascular and renal effects. Therefore, the aim of the present study was to characterize the cardiovascular and renal profile of the non‐peptide NOP receptor compounds AT‐039, AT‐127, AT‐090, and AT‐403 (Astraea Therapeutics).MethodsMale Sprague‐Dawley rats were surgically implanted with a venous, arterial, and bladder catheter and continuously infused intravenously (i.v.) with isosaline. After stabilization, conscious rats received an i.v. bolus injection of AT‐039, 127, 090, or 403 (30, 100, or 300 nmol/kg) while mean arterial pressure (MAP), heart rate (HR), and urine flow rate (V) were measured for 90 minutes. Urine samples were analyzed for urinary sodium excretion (UNaV) and urinary potassium excretion (UKV).ResultsFollowing i.v. bolus administration all AT compounds produced a dose‐dependent diuresis with a decrease in MAP, HR, MAP, UNaV, and UKV. Each ligand also produced sedation with the exception of AT‐039. Further studies with AT‐039 demonstrated that i.v. infusion of this ligand produced dose‐dependent effects on water and electrolyte excretion and blood pressure, but did not alter HR or produce sedation.ConclusionsThese findings indicate that i.v. injection of all non‐peptide NOP receptor AT compounds produced a significant diuretic and hypotensive effects. However, sedation and bradycardia were absent with AT‐039 i.v. infusion treatment. Further studies are needed to explore the mechanisms by which these compounds produce their cardiorenal responses.Support or Funding InformationFunding for this work was supported by Astraea Therapeutics (NZ), R01DA027811 (NZ), R01014026 (NZ), and NIH P30GM106392 (DRK).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Impaired Mesenteric Arterial Function of Male UC Davis Type 2 Diabetes Mellitus (UCD‐T2DM) Rats: Possible Involvement of Small Conductance Calcium‐activated Potassium Channels (SKca)
Authors: Razan Md Rahatullah , Akther Farjana , Wang Der Yu , Shaligram Sonali , Graham James L. , Stanhope Kimber L. , Havel Peter J. , Rahimian Roshanak 
Published: June 2021

UC Davis type‐2 diabetes mellitus (UCD‐T2DM) model is a novel and validated model of type‐2 diabetes exhibiting etiology very similar to clinical T2DM seen in humans. This model is characterized by polygenic adult‐onset obesity and diabetes observed in both sexes along with development of insulin resistance, impaired glucose tolerance, and subsequent β‐cell decompensation. We have previously shown that vascular reactivity in mesenteric arteries (MA) of male UCD‐T2DM is altered. Endothelium‐dependent hyperpolarizing factors (EDHF) is an important regulator of vascular tone, especially in small vessels such as MA. The objective of this study was to investigate the contribution of small or intermediate conductance calcium‐activated potassium channels (SKca & IKca) in modulating vascular function of MA in this model. Endothelium‐dependent vasodilation (EDV) to acetylcholine (ACh, 10−8 to 10−5 M) in pre‐contracted MA with phenylephrine (PE, 2μM) was measured before and after pretreatment with indomethacin (Indo, 10μm), a cyclooxygenase inhibitor, followed by addition of L‐NAME (200uM), a nitric oxide synthase inhibitor, and then Apamin (1μm), a selective inhibitor of SKca; and TRAM‐34 (1μm), a selective inhibitor of IKca. We demonstrated that the sensitivity to ACh‐induced relaxation was significantly reduced in MA from male UCD‐T2DM rats compared to controls. Addition of L‐NAME reduced Indo‐insensitive vasorelaxation in both control and diabetic animals. However, when compared with that of control, the effect of L‐NAME was much greater in MA of UCD‐T2DM, indicating a reduced contribution of EDHFtype relaxation or an enhanced role of NO in this disease model. The remaining EDV to ACh which is referred to as the L‐NAME/Indo‐insensitive component, or EDHF‐type relaxation was subsequently blocked by inhibiting intermediate (but not small) conductance calcium‐activated potassium channels in MA of diabetic model. In control animals, however, incubation of MA with apamin (a SKca inhibitor) led to a further reduction of EDV. The subsequent addition of TRAM‐34 eventually blunted the EDHF‐type relaxation in this group. These data, for the first time, show that the contribution of EDHF in EDV is reduced in the MA of UCD‐T2DM group, and the down regulation of SKca may be involved. Clearly, further studies will be needed to document the underlying mechanism(s) of altered vascular function in male UCD‐T2DM rats.Support or Funding InformationSupported by NIH/NHLBIThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Plasminogen Activator Inhibitor‐1 after Acute Myocardial Infarction in Obstructive Sleep Apnea Patients
Authors: Svatikova Anna , Singh Prachi , Kuniyoshi Fatima , Gami Apoor , Somers Virend K. 
Published: June 2021

BackgroundAcute cardiac and vascular events, including myocardial infarction (MI) exhibit a peak onset between 6 AM and 11 AM. Plasminogen activator inhibitor‐1 (PAI‐1) is a main inhibitor of tissue‐plasminogen activator and a key indicator of prothrombotic state. Elevated levels of PAI‐1 have been linked to increased cardiac and vascular risk. Obstructive sleep apnea (OSA) has also been associated with increased risk of MI. Hypoxemia, as well as OSA, increase PAI‐1. We tested the hypothesis that the early morning after awakening is associated with higher PAI‐1 activity in subjects with acute MI who also have OSA.MethodsWe studied 56 subjects after acute myocardial infarction (mean age 60±2 years; BMI 28±1 kg/m2). All subjects underwent complete overnight sleep polysomnography within 2 weeks after MI to assess for presence and severity of OSA. PAI‐1 activity was measured in all 56 subjects, first before sleep (at 9 PM), and then immediately after awakening in the morning (at 6 AM). A third measurement of PAI‐1 was obtained in all subjects at 11 AM.ResultsFor the group, PAI‐1 activity almost doubled from 12.5±1.6 U/mL at 9 PM to 22.5±2.5 U/mL after waking at 6 AM and decreased to 14.2±2.4 U/mL by 11 AM (P<0.0001). Fifty seven percent of patients were observed to have OSA, with apnea hypopnea index of >10 events/hour. Sleep apnea did not contribute to further elevation of morning PAI‐1 activity level in acute MI patients (9 PM, 6 AM and 11 AM PAI‐1 activity respectively: no OSA 11.6±2.5 U/mL, 24.1±3.8 U/mL and 15.2±3.5 U/mL; OSA 13.3±2.1 U/mL, 21.1±3.3 U/mL and 13.3±3.3 U/mL; P=0.8 for between group comparison).ConclusionsIncreased PAI‐1 activity in acute MI patients may be an important mechanism underlying the early morning peak in cardiovascular events. The presence of OSA however does not appear to further accentuate the already elevated PAI‐1 activity in acute MI patients. In patients after acute MI, the diurnal variation in PAI‐1, rather than apnea, may drive the impaired fibrinolysis.Support or Funding Information‐‐R01 grant HL65176‐‐This study was also supported in part by a gift to the Mayo Foundation by the Philips Respironics Foundation.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.