bluuebunny/crossref_metadata_2025 huggingface dataset:
G‐protein Coupled Receptor Kinase 4 gamma (GRK4γ) Mediated Activation of NF‐κB
Authors: Kanassatega Rhye‐Samuel , Andresen Bradley T
Published: November 2023

Polymorphisms within GRK4γ strongly associate with hypertension, and recently A142V GRK4γ was implicated in NF‐κB‐mediated expression of the angiotensin type 1 receptor (AT1R). Luciferase assays driven by the 2x κB pGL3 vector, nuclear fractionation, and pull‐down assays were utilized to investigate the mechanism via which wild‐type (wt) GRK4γ and single nucleotide polymorphisms (SNPs) R65L, A142V, and A486V activate NF‐κB. Luciferase assays were conducted in growth media and serum‐free media. Only in growth media did the SNPs increase NF‐κB activity; wtGRK4γ decreased NF‐κB activity compared to the vector control. Utilizing kinase‐dead mutants of GRK4γ demonstrated that only A142V GRK4γ‐mediated NF‐κB activity is dependent on the kinase activity. The luciferase assays demonstrate that GRK4γ SNPs act as an amplifier, but do not constitutively activate NF‐κB. Nuclear fraction confirmed the luciferase data and demonstrated that RelB is the NF‐κB isoform activated through the GRK4γ pathway. Pull‐down affinity assays with S‐tagged wtGRK4γ and GRK4γ SNPs showed significantly increased interaction between SNP GRK4γ and NEMO (IKKγ). No interaction between the other IKK isoforms (α, β, ɛ) and wtGRK4γ or GRK4γ SNPs was detected. Currently, the IκBs are being examined to complete the NF‐κB‐mediated signaling pathway that is activated by SNPs of GRK4γ. Collectively, the data indicates that polymorphic GRK4γ amplifies serum‐mediated activation of the canonical NF‐κB pathway through a kinase‐mediated (A142V) and protein‐protein interaction‐mediated (R65L & A486V) mechanism. Further elucidation of these mechanisms may allow for the development of drugs that block polymorphic GRK4γ‐mediated NF‐κB activity and consequently reduce AT1R expression and thus blood pressure.

Support or Funding Information

This work was funded from Internal funds from Western University of Health Sciences and American Indian Graduate Center STEM Student Fellowship.
Tankyrases Are Druggable Targets for the Treatment of Metabolic Disorders
Authors: Obianom Obinna , Yang Wei , Xue Fengtian , Shu Yan
Published: November 2023

Purpose

Tankyrases (TNKS) catalyze the poly‐ADP‐ribosylation of axin, targeting it for ubiquitination and proteasomal degradation. Along with adenomatous polyposis coli (APC), glycogen synthase kinase 3 (GSK3) and caseine kinase 1α (CK1α), axin makes up the β‐catenin destruction complex that promotes the degradation of β‐catenin, a critical effector in the canonical WNT signaling pathway that has been recently recognized to play a role in glucose homeostasis and the pathogenesis of metabolic disorders. This study sought to understand the relevance of TNKS1/2 inhibition and subsequent axin stabilization for the treatment of metabolic disorders.

Methods

We investigated the effect of a TNKS1/2 inhibitor, XAV939, on WNT signaling and gluconeogenesis pathway. TNKS1/2 inhibition leading to axin stabilization was measured by repression of WNT signaling pathway using dual luciferase assay. Western blots and quantitative PCR analysis were employed for the assessment of gene expression. Glucose production assay was used to measure direct effect on gluconeogenesis pathway.

Results

Cell based dual‐luciferase assay in HEK293 cells transiently transfected with a TCF/LEF‐responsive reporter showed potent inhibition (IC50 = 34 nM) of Wnt signaling as a result of TNKS1/2 inhibition by XAV939. Western blot analysis with lysates from XAV939 treated hepG2 cells confirmed increase in phospho‐β‐catenin and axin1 protein levels. More so, mRNA and protein expression of the Wnt signaling targets c‐Myc and cyclin D1 were repressed. In the gluconeogenesis pathway, gene expression of glucose 6‐phosphatase was significantly decreased by XAV939, the anti‐diabetic metformin, and a hybrid drug containing both metformin and XAV939 (YW1083). Glucose production assays using hepG2 and mouse primary hepatocytes showed that XAV939 was able to decrease glucose production even more potently than metformin.

Conclusion

This study adds to the meager scientific information that stabilization of axin via inhibition of tankyrase1/2 is valuable in combating metabolic disorders such as type 2 diabetes.

Support or Funding Information

the National Institute of General Medical Sciences of the National Institutes of Health (NIH) under Award R01GM099742
The Effect of PHLPP2 Removal on Cardiac Hypertrophy
Authors: Purcell Nicole , Yeh Szu‐Tsen , Koch Walter J , Zambrano Cristina M
Published: November 2023

Crucial cellular events such as death, growth, metabolism, proliferation, and hypertrophy are regulated by phosphorylation and dephosphorylation of proteins. PH domain leucine‐rich repeat protein phosphatase (PHLPP) is a serine/threonine phosphatase that has been shown to directly dephosphorylate several members of the AGC family of kinases. Preliminary data suggests that removal of PHLPP2 in neonatal rat ventricular myocytes (NRVMs) activates fetal gene re‐expression at baseline and potentiates phenylephrine (PE) induced gene expression 2 fold over siControl. Since we have previously published that removal of PHLPP2 in NRVMs does not affect Akt phosphorylation as previously reported in other cells, we hypothesize that PHLPP2 must have the ability to repress cellular hypertrophy through the dephosphorylation of other unknown targets. G‐protein coupled receptor kinase 5 (GRK5), an AGC kinase, has been shown to regulate cardiac hypertrophy through de‐repression of gene transcription and by directly binding DNA. Upon stimulation of NRVMs with the hypertrophic agonist phenylephrine (PE), GRK5 translocates to the nucleus to regulate hypertrophic gene expression. We wanted to determine whether PHLPP2 directly affects GRK5 phosphorylation and localization to augment cardiomyocyte hypertrophy. We found that removal of PHLPP2 increased GRK5 translocation to the nucleus at baseline and following PE treatment. Conversely, overexpression of PHLPP2 blocked nuclear translocation of GRK5 following PE treatment. Immunoprecipitation experiments confirm an interaction between PHLPP2 and GRK5 and removal of PHLPP2 increased phosphorylation of GRK5 at baseline and following PE treatment. Ongoing studies using GRK5 phosphorylation mutants will determine whether the phosphatase activity of PHLPP2 is necessary for the inhibition of GRK5 translocation. Our preliminary data is the first to uncover GRK5 as a novel PHLPP2 target in cardiomyocytes. Since little is known about the non‐canonical regulation of GRK5, understanding whether phosphorylation and localization is regulated within the cardiomyocyte by PHLPP has potential for new therapeutic targets in the treatment of cardiac hypertrophy and failure.

Support or Funding Information

This work is supported by the National Heart, Lung and Blood Institute grant HL114949
Functions of Human RGS2 Missense Mutations in Hypertension
Authors: Phan Hoa , SjÖgren Benita , Neubig Richard
Published: November 2023

RGS2 attenuates Gq/11 a signaling which mediates the action of a majority of vasoconstrictors. The association between RGS2 deficiency and hypertension has been demonstrated by genetic knock‐out mouse models and RGS2 mutations identified in Japanese hypertensive patients. Though biochemical functions of RGS2 are well characterized, the actual role of RGS2 in human hypertension is not known. 1.6% of individuals from the NHLBI exome sequencing project have missense mutations in RGS2, however, the functional significance of these mutations is unknown. The objective of this study is to examine the effect of human RGS2 missense mutations on protein expression levels, subcellular localization and how they differ from wild‐type RGS2 in their ability to regulate GPCR‐mediated signaling. Wild‐type and 14 RGS2 mutants were investigated by transient transfection in HEK293T and CHO cells. RGS2A4V showed reduced protein expression (30% of WT, n=10, p<0.01) and impaired inhibition of AT1R‐mediated calcium release (57% of WT vs 23% of A4V, n=5, p<0.01). In contrast, RGS2Q2L showed reduced expression (40% of WT, n=10, p<0.05) but did not exhibit a significant loss of function (57% of WT vs 51% of Q2L, n=5). In this functional model, RGS2R44H did not exhibit loss of function as previously reported. Preliminary data on other mutants suggesting functional deficits are currently being assessed as are potential differences in actions of RGS2 mutants on different GPCRs. Together, these studies will provide a molecular understanding of a number of human RGS2 mutant alleles in vitro. This will identify key candidate alleles for further study as contributors to human hypertension.

Support or Funding Information

Supported by AHA predoctoral fellowship to H.P. and NIH R01GM110195 to R.R.N.
Using NMR‐Based Fragment Screening to Probe the Druggability of Regulator of G Protein Signaling 17
Authors: Hayes Michael Patrick , Fowler C Andrew , Yu Liping , Roman David L
Published: November 2023

Regulator of G protein Signaling 17 (RGS17), as a GTPase accelerating protein (GAP), temporally controls G protein‐coupled receptor activity by accelerating the intrinsic rate of GTP hydrolysis of inhibitory Gα subunits, thus terminating signaling. Recent evidence has shown that RGS17 expression is drastically increased in malignancies in various tissues, including lung, breast, prostate, and liver, and inhibition of RGS17 via RNA interference is sufficient to slow cancer growth and progression. This makes RGS17 an attractive target for the development of small molecule inhibitors that could serve as chemotherapeutics with a novel mechanism of action. In order to identify novel druggable sites on the RGS homology domain of RGS17, we performed triple resonance NMR experiments using 15N, 13C doubly labeled protein samples to assign the protein. We then screened a library of 1000 fragments and assessed compound binding via chemical shift perturbation in protein‐detected 1H‐15N HSQC spectra. Seven hits were identified with affinities ranging from 530 μM to 5.2 mM and ligand efficiencies from 0.18 to 0.31. Interestingly, none of these identified hits bound directly to the region of RGS17 that forms the Gα interaction interface. Six fragments bound between α6 and α7 in the helical bundle domain, a site distant from the Gα interface. Additionally, one hit, an isothiazaolone, interacts with RGS17 in a covalent manner, showing that RGS17, like other RGS family members RGS4 and RGS8, is subject to inhibition via cysteine‐modification. Subsequent experimental and computational efforts will focus on structure‐activity relationship optimization of these hits to increase their affinities for RGS17 to further explore the druggabiliy of this allosteric site, as well as to facilitate the determination of the structure of the RGS17:fragment complex with atomic resolution.

Support or Funding Information

This work was supported by NIH 5R01CA160470 (DLR) and an American Foundation for Pharmaceutical Education Predoctoral Fellowship (MPH).
A series of substituted isoindolinones as selective small molecule modulators of the Gβγ‐SNARE interaction
Authors: Zurawski Zack P , Temple Kayla Jo , Han Changho , Delbove Claire , Senter Timothy J , Hyde Karren , Lindsley Craig W , Zhang Qi , Stauffer Shaun R , Hamm Heidi E
Published: November 2023

The interaction of G betagamma (Gβγ) subunits with its effectors is a key mechanism for G protein coupled receptor‐mediated signal transduction. One such effector is the soluble N‐ethylmaleimide‐sensitive attachment protein receptor (SNARE) complex, to which Gβγ binds to inhibit exocytosis downstream of calcium release. Starting from a single hit from a focused high‐throughput screen of a library of small molecules designed to block protein‐protein interactions, we have generated relatively potent and selective modulators of Gβγ‐effector interactions, in particular, the interaction of Gβγ with the SNARE protein SNAP25. From this hit, a series of substituted isoindolinones was developed. It was then determined that these compounds were also able to inhibit a second effector of Gβγ subunits, the G‐protein coupled inwardly rectifying potassium channel (GIRK). Utilizing structure‐activity relationships, we were able to further refine the potency and selectivity of these compounds, generating Gβγ‐binding small molecules that were selective for blocking interaction with an individual effector. Finally, we show that these compounds are able to abolish the inhibitory effect of the α2 adrenergic receptor on vesicle release in neurons. Together, these studies demonstrate the ability to generate selective small molecule modulators of Gβγ‐effector interactions and the suitability of such molecules for investigating mechanisms of Gi/o‐coupled GPCR‐mediated inhibition of exocytosis.

Support or Funding Information

This work was supported by the National Institutes of Health National Eye Institute [R01‐EY010291]; and the National Institutes of Health National Institute of Mental Health [MH101679‐ 01A1]. ZZ was supported by the T32 Training in Pharmacological Sciences, T32 GM07628.
Structure‐Dependent β2 Adrenergic Receptor Agonists Induce Mitochondrial Biogenesis through the Gβγ‐Akt‐eNOS‐cGMP Pathway
Authors: Cameron Robert , Beeson Craig , Schnellmann Rick
Published: November 2023

Acute kidney injury (AKI) occurs in hospitalized patients and carries a high morbidity and mortality with no treatments beyond renal replacement therapy. AKI is characterized by mitochondrial dysfunction, particularly in the renal proximal tubule cell (RPTC). The induction of mitochondrial biogenesis (MB) is a therapeutic target for AKI. Our group has shown that formoterol, a βγ2 adrenoceptor agonist, can induce MB in vitro and in vivo, and stimulate recovery of mitochondrial and renal function following AKI in mice. In contrast the β2 adenoceptor agonist clenbuterol does not induce MB. Our goal was to elucidate the signaling pathway by which formoterol induces MB and examine why clenbuterol does not. RPTC were treated with 30 nM formoterol or 30 nM clenbuterol following pretreatment with DMSO, 100 nM gallein, 10 mM L‐NAME, 100 nM MK2206, or 5 mM ODQ for 30 min. Oxygen consumption rates (OCR) were measured using the Seahorse XF‐96 analyzer. Protein phosphorylation was assessed by immunoblot analsis. Formoterol, but not clenbuterol increased Akt phosphorylation in RPTC at 30 min, and this increase was attenuated by pretreatment with the Gβγ inhibitor gallein. Pretreatment of RPTC with gallein also attenuated formoterol‐induced increases in FCCP‐uncoupled oxygen consumption rate (FCCP‐OCR), a biomarker of MB. eNOS is a downstream target of Akt and was measured following formoterol or clenbuterol treatment with and without the Akt inhibitor MK2206 (100 nM). Formoterol, but not clenbuterol increased eNOS phosphorylation at 1 hr, and this increase was attenuated by pretreatment with MK2206. Additionally, pretreatment with either the NOS‐inhibitor L‐NAME or the guanylate cyclase inhibitor ODQ attenuated formoterol‐induced increases in FCCP‐OCR. These results show that formoterol induces MB through a Gβγ‐Akt‐eNOS‐cGMP‐dependent pathway, while clenbuterol does not activate Akt to initiate this pathway. This study is the first to associate Gβγ‐and cGMP‐dependent signaling with β2‐adrenoceptor induced MB.

Support or Funding Information

This study was supported by R01 GM084147 to RGS, F30 DK104550 to RBC, and by the Biomedical Laboratory Research and Development Program of the Department of Veterans Affairs (BX‐000851).
Phospholipase C‐epsilon Mediates Inflammatory Responses in Experimental Autoimmune Encephalomyelitis Progression through its Role in Astrocyte GPCR Signaling
Authors: Purcell Nicole H , Dusaban Stephanie , Ryu Jae K , Akassoglou Katerina , Smrcka Alan V , Brown Joan Heller
Published: November 2023

Phospholipase C‐epsilon (PLCɛ) is a novel signaling molecule that integrates upstream G‐protein coupled receptor (GPCR) activation to downstream biological processes. We previously demonstrated that PLCɛ is important in mediating inflammatory gene expression in astrocytes in response to GPCR ligands that are released at sites of Central Nervous System (CNS) injury. Multiple Sclerosis (MS) is a CNS disease characterized by inflammation, breakdown of the blood brain barrier (BBB), demyelination, and axonal damage. Because astrocytes play a role in MS progression, and since PLCɛ is important for astrocytic inflammatory responses, we sought to identify the role of PLCɛ in the Experimental Autoimmune Encephalomyelitis (EAE) animal model of MS. Loss of PLCɛ resulted in attenuated clinical scores from days 17 to 30 after EAE induction compared to wild‐type (WT) mice. Spinal cords from knockout (KO) mice also show attenuated iNOS and IL‐1β expression as well as decreased inflammation as assessed by hematoxylin/eosin staining. We also observe S1P3 receptor and sphingosine kinase‐1 (Sphk1) up‐regulation in spinal cords of WT mice after EAE induction. Further studies demonstrate S1P3 and Sphk1 mediate inflammatory gene expression in astrocytes. Use of GFAP‐Cre to knock out PLCɛ specifically in astrocytes also demonstrate the role of PLCɛ is primarily through astrocytes. Lastly, GPCR profiling and receptor knockdown including several orphan receptors implicate an importance of receptor coupling to PLCɛ in astrocyte inflammation. Thus, the data presented here demonstrate the importance of PLCɛ in mediating neuroinflammatory responses, which ultimately affects MS progression.
Crystallographic Studies of Human Acetylcholinesterase Reactivation by Oximes: Towards a Neutron Structure
Authors: Gerlits Oksana , Fajer Mikolai , Cheng Xiaolin , Blumenthal Donald , Taylor Palmer , Radic Zoran , Kovalevsky Andrey
Published: November 2023

Human acetylcholinesterase (hAChE) is responsible for degrading neurotransmitter acetylcholine at synapses of the nervous system. Organophosphate (OP) nerve agents and pesticides inactivate hAChE through chemical modifications of the enzyme's active site leading to a life‐threatening increase of acetylcholine concentration. Exposure to OPs is fatal if not treated with antidotes. However, the current generation of antidotes is not highly efficient, and the rates of reactivation are far slower than the catalytic rate of acetylcholine hydrolysis.

Insights into the molecular structures of Torpedo (1) and mammalian AChEs (2) as well as other cholinesterases reveal the underlying limitations in enhancing reactivation rates but are limited in their ability to resolve positions of protons and hydrogens important for understanding of proton transfer in reactivation of organophosphate inhibited cholinesterases. The only experimental method that can provide this information is macromolecular neutron crystallography. In combination with X‐ray crystallography it is capable of providing insights into the active sites of enzymes with the atomic resolution.

In preparation for the neutron diffraction studies we have obtained several new high resolution X‐ray structures of hAChE at room and low temperatures in the apo form and in complex with oxime reactivators and other ligands. The enzyme crystallizes in a novel crystal form with the unit cell amenable to neutron crystallography (space group P31, a=b=124.3, c=129.1 Å). Detailed analysis of the reactivators and ligand interactions at the enzyme active will be presented.

Support or Funding Information

This work was supported by the CounterACT Program, National Institutes of Health Office of the Director, Grant 1U01NS083451 from NINDS.
Inhibitory Effect of 4‐hydroxysattacin, Extract of Soil Bacteria, on Melanogenesis Using B16F10 Melanoma Cells And 3D Human Skin Model
Authors: Kim Kyuri , Park Hyeonji , Nam Sangjip , Lim Kyung‐Min
Published: November 2023

Epidermal or dermal pigmentation is a skin disorder produced from accumulated melanins in hyperactivated melanocyte. Various chemicals or natural compounds have been reported as a therapeutic agent of hyperpigmentary disorders that manifests, post‐inflammatory, melanoderma, solar lentigo, or freckles. Here, we investigated melisma inhibitory effect of 4‐hydroxysattabacin, crude extract of soil bacteria, Bacillus sp, on melanogenesis in murine melanoma cell, B16F10, and UVB‐induced hyperpigmentation on 3D‐human skin model. Using alpha‐melanocyte stimulating hormone (a‐MSH) as triggering factor of pigmentation, 4‐hydroxysattabacin treatment decreased melanin contents in dose dependent manner. The Real‐Time Polymerase Chain Reaction result demonstrated 48 hour treatment of 4‐hydroxysattabacin produced down‐regulation of several melanogenic genes that encompass Tyrosinase, Tyrosinase‐related protein 1 (TRP‐1), and Tyrosinase‐related protein 2 (TRP‐2) while melanogenic enzyme activities were slightly affected. Moreover, diminution of Tyrosinase protein expression was confirmed by immunoblotting assay as well as anti‐melanogenic effects of 4‐hydroxysattabacin was proved on 3D human epidermal skin model, which supplant animal test for cosmetic testing. Finally, inhibitory effect of 4‐hydroxysattabacin was clarified both in vitro and 3D human skin model that is to say 4‐hydroxysattabacin would be a potential therapeutic agent for cutaneous hyperpigmentation.
Role of Blueberry in modulating suicide gene SKA2 in an animal model of Posttraumatic stress disorder (PTSD)
Authors: Ebenezer Philip J , Nair Anand R , Francis Joseph
Published: November 2023

Background and Rationale

Post‐traumatic stress disorder (PTSD) has been related to suicidal thoughts and behavior. The principal origin and the mechanism contributing to the suicidal thoughts in PTSD is not known. Understanding molecular mechanisms and identifying novel genes that might contribute to suicide in the context of PTSD might be vital in the prevention of suicide. Recently, SKA 2 (spindle and kinetochore associated complex subunit 2) a novel genetic and epigenetic target was shown to decrease in patients that attempted or committed suicide. In addition, it has been shown that SKA2 genes chaperone the glucocorticoid receptors from the cytoplasm to the nucleus, thereby possibly assimilating and protecting against stress. It has been well established that blueberries (BB) attenuate stress factors and inflammation. In addition, we recently showed that BB modulates serotonin levels in the brain of PTSD animals. Due to these beneficial properties of BB, in this study we explored the possible role played by BB in modulating SKA2 genes in an animal model of PTSD

Methods

Rats were fed with a blueberry‐enriched (2%) or a control diet. Rats were exposed to cats for one hour on days 1 and 11 of a 31‐day stress schedule to simulate traumatic conditions. At the end of the study, the rats were euthanized and prefrontal cortex (PFC), hippocampus (HC) and plasma were isolated. We measured plasma SKA2 levels in the experimental animals by ELISA. In addition SKA2 level was also measured in PFC and HC regions of the brain were quantified by western blotting and RT‐PCR.

Results

We found that our PTSD animals had a decreased levels of SKA2 in the plasma, hippocampus and prefrontal cortex compared to control (non‐PTSD) animals. In contrast, treatment with blueberry in the PTSD animals increased SKA2 levels both in the blood and in the brain regions.

Conclusions

The finding that SKA2 gene is down regulated in the blood and brain indicates a possible target for further investigating into suicide prevention using our animal model of PTSD. Furthermore, the finding that non pharmacological agents like blueberry is able to modulate SKA2 gene in context of PTSD is novel. Further studies to understand the molecular mechanism of protection by BB might provide a potential therapeutic target for the treatment of PTSD.

Support or Funding Information

This work was supported by U.S. Highbush blueberry council (USHBC) grant to Dr. Francis.
Arsenic silences liver PDK4 expression through activation of histone H3K9 methylatransferase G9a
Authors: Zhang Xi , Choiniere Jonathan , Bennett Jason , Wang Li
Published: November 2023

It is well established that the increased liver cancer incidence is strongly associated with epigenetic silencing of tumor suppressor genes; the latter is contributed by the environmental exposure to arsenic. The pyruvate dehydrogenase kinase 4 (PDK4) is a mitochondrial protein that regulates TCA cycle. However, little is known about its role in arsenic‐mediated tumor progression. In the present study, we show that the inactivation of PDK4 in hepatocellular carcinoma (HCC) cells is resulted from aberrant repressive histone modification. Histone methyltrasferase G9a‐ and Suv39H‐mediated histone H3 lysine 9 (H3K9) methylations contribute to the PDK4 silencing in HCC cells. The PDK4 expression is induced by G9a inhibitor BRD4770 (BRD) and Suv39H inhibitor Chaetocin. In contrast, arsenic exposure increases G9a protein expression and the global levels of H3K9 di‐ and tri‐methylations (H3K9me2/3). Compared to BRD treated cells, arsenic exposure restores the decreased H3K9me2/3 enrichment in the PKD4 promoter. Our results also show that the PDK4 expression is refractory to BRD‐mediated transcriptional activation upon arsenic exposure. When C57Bl/6 mice receive arsenic in drinking water alone or in combination with BRD treatment, the hepatic expression of PDK4 is induced by BRD which is markedly reversed by arsenic. Based on these observations, we conclude that arsenic disrupts the epigenetic restoration of PDK4 expression in both HCC cells and in mouse liver.

Support or Funding Information

NIEHS 1R01ES025909
Anti‐invasive activity of capsaicin and natural capsaicin‐like compounds in human NSCLC
Authors: Nolan Nicholas A , Hurley John D , Williams Cathryn , Rollyson William D , Brown Kathleen C , Akers Austin T , Eskew Matthew N , Perry Haley E , Egleton Richard D , Dasgupta Piyali
Published: November 2023

Non small lung cancer (NSCLC) accounts of 80% of all lung cancer cases. About 30–40% of NSCLC patients present with metastatic disease at the time of their diagnosis. The average survival time of such NSCLC patients is about 7 months. The invasion of neoplastic cells to the surrounding stroma is a vital step of cancer metastasis. Recent studies have shown that nutritional compounds (by themselves or in combination with standard chemotherapy) can suppress metastasis of several human cancers. Capsaicin is the spicy ingredient of chili peppers. Recent studies have shown that capsaicin inhibits the invasion and metastasis of several types of human cancers including melanoma, prostate cancer and cholangiosarcoma. We investigated the anti‐invasive properties of capsaicin and two capsaicin‐like compounds, capsiate and capsiconiate, found in select variety of chili peppers on NSCLC. We measured the anti‐invasive activity of these compounds by the Boyden chamber assay. We found that capsaicin and capsiate displayed equivalent anti‐invasive activity in NSCLC cells. In contrast, capsiconiate did not suppress the invasion of NSCLC cells. The anti‐invasive activity of capsaicin, capsiate and capsiconiate in human NSCLC was found to be independent of their growth‐inhibitory activity. Currently, we are testing the anti‐metastatic activity of capsaicin in syngenic mouse models of metastasis. The results of our studies may foster the hope of nutrition‐based interventions in NSCLC metastasis.

Support or Funding Information

Funding for our study was supported by the an NIH R15‐AREA Grant (1R15HL113681‐01A1), AICR Investigator Grant, and a NASA Undergraduate Fellowship to NAN
Effect of Acidic Tumor Microenvironment on Motility and Stemness of Non‐Small Cell Lung Cancer Cells
Authors: Arikatla Swetha , Livesey John C
Published: November 2023

Ineffective tumor angiogenesis and uncontrolled proliferation of cancer cells leads to hypoxia and an acidic tumor microenvironment (TME). Acidosis, caused by the accumulation of lactic acid in solid tumors, is due to the induction of glycolysis in cancer cells by hypoxia. Tumor cells also maintain a high rate of aerobic glycolysis, also known as the Warburg effect. A better understanding of the effect of an acidic TME on motility and stemness of non‐small cell lung cancer (NSCLC) cells may allow the development of new treatments targeting this TME. A substantial alteration in phenotype was observed in NCI‐H292 and NCI‐H1650 cells propagated in media at acidic pH (a‐pH; pH ~ 6.5 to 6.7) compared to cells propagated in media at neutral pH (n‐pH; pH ~ 7.2 to 7.4). H292 cells propagated at a‐pH showed a significant decrease in motility compared to these cells propagated in n‐pH while no difference in motility was observed when H1650 cells were propagated in a‐pH or n‐pH. A significant decrease in cell proliferation was observed in H292 cells and H1650 cells propagated in a‐pH media. No significant difference was observed in mRNA and protein expression of E‐cadherin when either H292 or H1650 cells were propagated in a‐pH and n‐pH media. Vimentin mRNA expression was not observed in the H292 cell line while vimentin mRNA expression was observed in the H1650 cell line but no protein expression was observed in either cell line. Minimal Sox2 and OCT4 mRNA expression was observed in H292 cells and H1650 cells and was unaffected by acidity. These data indicate that as a single TME variable, a‐pH has little effect on EMT and stemness while reducing motility in only one of these two NSCLC cell lines.

Support or Funding Information

Supported by University of the Pacific's Holmok Cancer Research Fund.
MITO‐APOCYNIN, A NOVEL MITOCHONDRIA‐TARGETED DERIVATIVE OF APOCYNIN, INHIBITS NLRP3 INFLAMMASOME ACTIVATION IN PRIMARY MURINE MICROGLIA IN DOPAMINERGIC NEUROTOXIC MODELS
Authors: Sarkar Souvarish , Panicker Nikhil , Neal Matthew Layne , Harischandra Dilshan , Jin Huajun , anantharam Vellareddy , Kalyanaraman Balaraman , Kanthasamy Arthi , Kanthasamy Anumantha
Published: November 2023

The NLRP3 inflammasome signaling pathway has recently been recognized as a major player in neuro‐inflammation in the CNS. Oxidative stress and mitochondrial dysfunction are known to play key roles in the pathophysiological processes of many neurodegenerative diseases, including Parkinson's disease (PD). To date, the relationship between mitochondrial defects and neuro‐inflammation is not well understood. In the present study, we show that neurotoxic pesticide‐induced mitochondrial impairment induces the NLRP3 inflammasome's pro‐inflammatory cascade in primary microglia. Treating primary mouse microglia with LPS induced NLRP3 and pro‐IL1β expression. Interestingly, exposing LPS‐primed microglial cells to the mitochondrial complex I inhibitory pesticides rotenone and tebufenpyrad potentiated activation of the NLRP3 inflammasome and processing of pro‐IL‐1β in a time‐ and dose‐dependent manner, indicating that mitochondrial impairment heightened the pro‐inflammatory response in microglia. Morphological analysis revealed that rotenone and tebufenpyrad significantly increased mitochondrial circularity and solidity in LPS‐primed microglia as well as decreased mitochondrial perimeter relative to unprimed microglia, thereby augmenting mitochondrial fragmentation during neurotoxic pesticide‐induced inflammasome activation. Immunocytochemistry (ICC) revealed enhanced mitochondrial superoxide production in rotenone‐ and tebufenpyrad‐treated LPS‐primed microglia via the co‐localization of NLRP3 and mitochondrial superoxide in these cells. Finally, mito‐apocynin, a novel mitochondria‐targeted derivative of apocynin, was able to significantly reduce neurotoxic pesticide‐induced NLRP3 inflammasome activation through attenuation of mitochondrial superoxide generation from damaged mitochondria. Collectively, our studies demonstrate for the first time that mitochondrial impairment resulting from neurotoxic pesticide exposure can activate NLRP3 inflammasome signaling in microglia, further augmenting pro‐inflammatory events in the brain. Also, for the first time, we show that reducing mitochondrial superoxide generation by mitochondria‐targeted pharmacological agents can attenuate the inflammasome activation.

Support or Funding Information

Supported by NS088206 and ES19267
Preclinical In‐vivo Safety Evaluation of Concanavalin A‐based Nanomicrobicide in Female Mouse Reproductive Tract
Authors: Ezoulin Miezan JM , Coulibaly Fohona , Dim Daniel
Published: November 2023

Objective

Tenofovir (TFV) loaded Concanavalin A nanoparticles (ConA‐NPs) was recently synthesized as mannose sensitive nanomicrobicide template for the prevention of HIV/AIDS transmission. To advance into clinical trials, such nanomicrobicide must be evaluated in preclinical safety and efficacy models. This study aims at examining the mucosal safety of these specific ConA‐NPs in the female mouse reproductive tract.

Method

TFV loaded ConA‐NPs (~ 670 ± 234 nm, −15 ± 0.55 mV) were prepared via the “layer‐by‐layer” technology by coating a core template, calcium carbonate (CaCO3) crystal, with Con A crosslinked with glycogen. Pre‐clinical safety studies of intravaginally administered TFV loaded ConA‐NPs (100 mg/kg) were performed using a female mouse model C57Bl6. Mouse cervicovaginal lavage (CVL) and tissues (CVT) were collected 24h after a single application. Changes in the levels of inflammatory cytokines (IL‐1l, IL‐1β, IL‐6, IL‐7, IP‐10, MKC, and TNF‐l) were quantitated by a high‐sensitivity multiplexed bead‐based immunoassay. The histopathology of the genital tract (vagina, cervix, uterus, and ovary), rectum and other major organs (spleen, lung, liver, kidney, heart, brain) was studied using Hematoxylin & Eosin staining. The vaginal tissues were evaluated for localized immune cells (lymphocytes) infiltration by immunohistochemical staining for CD45.

Results

After 24h exposure to these NPs, the basal levels of proinflammatory cytokines (IL‐1l, IL‐1β, IL‐6, IL‐7, IP‐10, MKC, and TNF‐l) in mouse CVL and CVT increased 3 – 56 fold and 1.2 – 5.2 fold, respectively when compared to phosphate buffered saline (PBS) used as a negative control. The induced inflammation was associated with the infiltration of immune cells to the vaginal epithelium. The histological analysis of the reproductive tract revealed signs of epithelial disruption in the vagina. However, all other tissues were histologically similar to control tissue at 24 h post application.

Conclusion

These experiments showed that the present ConA‐NPs are cytotoxic to the genital tract epithelial cells at 100 mg/kg. These data suggest the need for further preclinical studies to determine the maximum tolerable dose of the nano formulation for vaginal application.

Support or Funding Information

The project was supported by Award Number RO1 AI087304 from the National Institutes of Allergy and Infectious Diseases (Bethesda, MD, USA). The content is the sole responsibility of the authors and does not necessarily represent the official views of the National Institutes of Allergy and Infectious Diseases or the National Institutes of Health.
17β‐Estradiol Up‐regulates UGT1A9 Expression via Estrogen Receptor α
Authors: Cho Sung‐joon , Ning Miaoran , Zhang Yan‐Yan , Rubin Leah , Jeong Hyunyoung
Published: November 2023

UGT1A9 is a major phase II enzyme responsible for elimination of drugs and endogenous molecules. Clinical data have shown increased elimination of UGT1A9 substrates in pregnant women or oral contraceptive users, but the role of estrogen in the regulation of UGT1A9 expression remains unknown. In this study, we investigated the effect of 17β‐estradiol (E2) on UGT1A9 expression and the role of estrogen receptor (ER) α in the transcriptional regulation of UGT1A9. E2 significantly increased UGT1A9 promoter activity in HepG2 cells in the presence of ERα. UGT1A9 induction by E2 was abrogated by antiestrogen ICI182,780 in HepG2 cells constitutively expressing ERα. Results from UGT1A9 promoter reporter assays in HepG2 cells transfected with different ERα mutants showed that DNA‐binding domain of ERα is essential in UGT1A9 induction by E2. Deletion and mutation assays of UGT1A9 promoter revealed a putative ERE located within −2262/−1987 region, but electrophoretic mobility assays demonstrated no direct ERα binding to a DNA probe harboring the region. Examination of healthy human liver tissues revealed statistically significant increases in UGT1A9 expression in women (of age < 45 years) as compared to men. Together, these results indicate an important role of estrogen in the regulation of UGT1A9 expression. This finding may shed a light on prediction of potential drug‐drug interaction involving UGT1A9 substrates as well as on identifying sources for inter‐individual variability in UGT1A9‐mediated drug metabolism
In Vivo Treatment with Natriuretic Peptide Receptor Type C Agonist Attenuates Overexpression of Cell Cycles Proteins and Hyperproliferation of Vascular Smooth Muscle Cells from SHR : Molecular Mechanisms
Authors: RAHALI Sofiane , ANAND‐SRIVASTAVA Madhu B
Published: November 2023

Vascular remodeling due to hyper‐proliferation of vascular smooth muscle cells (VSMCs) is associated with hypertension. We previously showed that in vivo treatment of spontaneously hypertensive rats (SHR) with NPR‐C specific agonist C‐ANP4–23 attenuates the hyper‐proliferation of VSMC. We undertook the present study to investigate if the anti‐proliferative effect of C‐ANP4–23 treatment is mediated through the inhibition of the over‐expression of cell cycle proteins and explore the signaling molecules contributing to this effect. For this study, one‐week‐old SHR and age‐matched WKY rats were injected intraperitoneally with C‐ANP4–23 twice per week for 6 weeks and sacrificed at the end of the 8th week, blood pressure measurements were done by tail cuff method, proliferation of VSMC was determined by thymidine incorporation and MTT assay, and Western blotting was used to measure the levels of proteins. VSMC from SHR exhibited enhanced proliferation as compared to Wistar Kyoto rats (WKY) and C‐ANP4–23 treatment attenuated the hyper‐proliferation to control levels. In addition the overexpression of cyclin D1, cyclin A, cyclin E, cyclin dependent kinase 2 and 4 (cdk2, cdk4), phosphorylated retinoblastoma protein (pRb) and Giα proteins exhibited by VSMC from SHR was attenuated to control levels. Furthermore, the enhanced phosphorylation of ERK1/2, AKT, EGF‐R, PDGF‐R, IGF‐R and c‐Src was significantly decreased by C‐ANP4–23 treatment. Moreover, the enhanced levels of superoxide anion (O2 −), NADPH oxydase activity, and the enhanced expression of NOX4 and P47phox in SHRs were attenuated by C‐ANP4–23. These results indicate that in vivo NPR‐C activation attenuates the over‐expression of cell cycle proteins through the inhibition of the enhanced oxidative stress, c‐Src and EGF‐R, PDGF‐R, IGF‐R activation, MAPK signaling and over‐expression of Giα proteins resulting in the inhibition of the hyper‐proliferation of VSMC from SHR. Thus, it can be suggested that C‐ANP4–23 may be used as a therapeutic agent for the treatment of vascular complications associated with hypertension and atherosclerosis.

Support or Funding Information

Supported by grants from Canadian Institutes of Health Research (CIHR) and Heart and Stroke Foundation of Canada (HSFC).
Enhanced endothelial permeability by free fatty acid due to lysosomal cathepsin B‐mediated activation of Nlrp3 inflammasome
Authors: Li Xiang , Wang Lei , Nguyen Thaison , Zhang Yang
Published: November 2023

Recent studies have demonstrated that free fatty acid (FFA) palmitate induces insulin resistance leading to Type 2 diabetes via the activation of Nlrp3 inflammasome with a focus on inflammatory cells. Our findings demonstrated that enhanced vascular permeability in the myocardium in mice on the high fat diet (HFD) compared to mice on the normal diet, whereas such HFD‐induced impairment of vascular integrity was significantly attenuated in Nlrp3−/− mice. In microvascular endothelial cells (MVECs), FFA markedly induces Nlrp3 inflammasome complex formation leading to caspase‐1 activation and IL1β production. However, FFA has no effects on the transcription level of Nlrp3 inflammasome genes including Nlrp3, Asc, and caspase1. By fluorescence microscopy and flow cytometry, we observed that such FFA‐induced Nlrp3 inflammasome activated was accompanied by a reduction in interendothelial tight junction proteins ZO1/2. Such FFA‐induced decrease of ZO1/2 was also correlated with an increase in the permeability of endothelial monolayers treated with FFAs. Moreover, FFA‐induced alterations in ZO1/2 or permeability were significantly reversed by an inflammasome activity inhibitor, YVAD, or a high mobility group box 1 (HMGB1) activity inhibitor. Lastly, blockade of a lysosomal protease, cathepsin B, with its inhibitor Ca‐074Me significantly abolished FFA‐induced activation of Nlrp3 inflammasomes, down‐regulation of ZO1/2, and enhanced permeability in MVECs or their monolayers. Taken together, these data strongly suggest that activation of endothelial Nlrp3 inflammasomes due to increased FFAs produces HMGB1, which disrupts inter‐endothelial junctions and increase paracellular permeability of endothelium contributing to early onset of endothelial injury during metabolic disorders such as obesity.

Support or Funding Information

NIH grants HL122769 and HL122937
Retinoic Acid Metabolites and Isomers have Differential Effects on Lipid Metabolism
Authors: Tripathy Sasmita , Han Chang Yeop , Isoherranen Nina
Published: November 2023

All‐trans retinoic acid (atRA) and 13‐cisRA, and their metabolites 4‐oxo‐atRA and 4‐oxo‐13‐cisRA are found in circulation in humans as endogenous compound and following clinical doses of atRA and 13‐cisRA. Of these four compounds, only atRA is believed to be biologically important due to its high affinity towards RARs and activation of PPARδ. 13‐cisRA and 4‐oxo‐atRA also activate RARs but less is known about the 4‐oxo‐13‐cisRA. The activation of PPARs by 13‐cisRA and 4‐oxo‐metabolites has not been studied. The aim of this study was to determine whether atRA, 4‐oxo‐atRA, 13‐cisRA and 4‐oxo‐13‐cisRA have biological effects via RAR and other non‐RAR mediated pathways in human hepatocytes, HepG2 cells and adipocytes. At 1 μM concentration atRA, 4‐oxo‐atRA, 13‐cisRA and 4‐oxo‐13‐cisRA each significantly increased the mRNA levels of RARβ demonstrating that all four compounds activate RARs. atRA treatment increased expression of PPARδ targeted genes PGC1α, PGC1β and NRF1 and fatty acid oxidation, but had no effect on fatty acid and triglyceride synthesis genes SREBP1, MTTP, DGAT2 and PPARγ. In contrast, 13‐cisRA, 4‐oxo‐atRA, and 4‐oxo‐13‐cisRA had no effect on PPARδ targeted genes PGC1α, PGC1β and NRF1 and fatty acid oxidation, but significantly increased the expression of SREBP1, MTTP, DGAT2 and PPARγ. This suggests that 13‐cisRA, 4‐oxo‐atRA, and 4‐oxo‐13‐cisRA increase triglyceride synthesis and secretion. In addition, 4‐oxo‐atRA antagonized the induction of PGC1α, PGC1β and NRF1 and fatty acid oxidation by atRA demonstrating an independent activity of the 4‐oxo‐atRA when compared to atRA. Collectively, these results indicate that atRA promotes lipid catabolism via PPARδ, and 13‐cisRA and 4‐oxo‐RAs increase triglyceride synthesis, possibly via SREBP1 and PPARγ. Our findings regarding 13‐cisRA and its main circulatory metabolite 4‐oxo‐13‐cisRA explain the clinical data of increased incidence of hypertriglyceridemia and hypercholesterolemia following 13‐cisRA dosing. In addition, our data supports the mouse findings that atRA increases fatty acid oxidation in vivo. This study demonstrates that the 13‐cisRA isomer and 4‐oxo‐metabolites of RA found in circulation regulate lipid metabolism independent of atRA and RAR activation, and may play an important role in vivo in modulating lipid homeostasis in human tissues.

Support or Funding Information

NIH R01 GM111772, R01 GM081569, R01 GM081569‐S1 and P30 DK035816
Inhibition of in vivo oxidation of native low‐density lipoprotein by N‐acetylcysteine is associated with decreased atherosclerosis
Authors: Liu Patrick Z , Cui Yuqi , Narasimhulu Chandrakala A , Li Xin , Xiao Yuan , He Guanglong , Liu Zhenguo , Parthasarathy Sampath
Published: November 2023

Background

Low‐density lipoprotein (LDL) is not atherogenic itself. Oxidized LDL (ox‐LDL) is a potent oxidative agent, produces reactive oxidative species (ROS), and critical to the development of atherosclerosis. N‐acetylcysteine (NAC) has anti‐atherosclerotic effect. The mechanisms for its anti‐atherosclerotic actions remain largely unknown.

Objective

The present study aimed to determine if NAC could attenuate in vivo oxidation of LDL and inhibit atherosclerosis.

Methods and results

A single dose of human native LDL (50 μg) was injected intravenously into male C57BL/6 mice with and without NAC treatment. Human ox‐LDL levels in the blood were measured at different time after LDL injection using ELISA kit. Measurable serum level of human ox‐LDL was detected 30 min after the injection, reached the peak in 3 hours, started to decline afterwards, and became undetectable in 12 hours. NAC treatment significantly reduced serum ox‐LDL level with no detectable ox‐LDL in blood 6 hours after injection. No difference in ox‐LDL clearance was observed in NAC‐treated animals. Intracellular and extracellular ROS production was significantly increased in the animals treated with ox‐LDL and in hyperlipidemic LDL receptor knockout (LDLR−/−) mice that was completely prevented with NAC treatment. NAC also significantly reduced atherosclerotic plaque formation in hyperlipidemic LDLR−/− mice.

Conclusion

Native LDL was converted to ox‐LDL in vivo. NAC attenuated in vivo oxidation of LDL and ROS formation from ox‐LDL that might contribute to decreased atherosclerotic plaque formation in hyperlipidemia.

Support or Funding Information

NIH
Investigation of the cardiovascular toxicity of oxidized cholesterols using the zebrafish as an animal model
Authors: Kurogi Katsuhisa
Published: November 2023

Zebrafish has been used as an animal model for the investigation of cardiovascular disorders. One of the major pathogenic factors for cardiovascular diseases, such as atherosclerosis, is oxidized cholesterols. The cytotoxicity of oxidized cholesterols for the vascular endothelial cells has been proposed to be an etiological factor for atherosclerosis. However, it is unclear which type(s) of oxidized cholesterol may exhibit more serious pathogenic effects. In the current study, the zebrafish was employed to verify the cardiovascular toxicities of oxidized cholesterols.

Zebrafish larvae at 48 hours postfertilization (hpf) were exposed to cholesterol and 10 different oxidized cholesterols by the soaking method or by microinjection into yolk sac. At every 24 hours during exposure up to 7 dpf, heart beats, blood flow, and mortality were examined. To investigate further the effect on the cardiovascular system, o‐dianisidine staining was performed.

5,6α‐Epoxycholesterol and 5,6β‐epoxycholesterol, tested using both the soaking and the microinjection methods, showed the strongest cardiovascular toxicities, including lower heart beats, decreased blood flow, and higher mortality rate, among the oxidized cholesterols tested. Intriguingly, 22‐hydroxycholesterol exhibited a severe cardiovascular toxicity when tested using the soaking method, but not the microinjection method. Collectively, these results indicated that epoxycholesterols caused stronger cytotoxicity toward vascular endothelial cells than other oxidized cholesterols such as 7β‐hydroxycholesterol and 7‐ketocholesterol. Our results suggested that the zebrafish larva may serve as a convenient and useful model for investigating the pathogenicity and/or prevention of atherosclerosis caused by oxidized cholesterols.