bluuebunny/crossref_metadata_2025 huggingface dataset:
Structural Insights into Phospholipase Cɛ: A Multi‐Method Biophysical Approach
Authors: Garland‐Kuntz Elisabeth E. , Vago Frank S. , Sieng Monita , Chakravarthy Srinivas , Jiang Wen , Lyon Angeline M.
Published: November 2023

Phospholipase C (PLC) enzymes hydrolyze phosphatidylinositol lipids to produce second messengers, including inositol‐1,4,5‐triphosphate and diacylgycerol, which increase intracellular calcium and activate protein kinase C, respectively. PLCɛ contributes to cardiac hypertrophy, where it is chronically activated by the small G protein Rap1A, as well as to oncogenic Ras signaling pathways downstream of receptor tyrosine kinases. Specifically, Rap1A and Ras proteins are proposed to bind to the C‐terminal Ras‐association (RA) domain of PLCɛ, promoting conformational changes that increase lipase activity and/or membrane association. However, the role of the RA domains in PLCɛ activity is poorly understood. We are using small‐angle X‐ray scattering, electron microscopy, and biochemical approaches to determine the roles of the RA domains in basal regulation and activity upon G protein activation. These results provide the first structural insights into this essential enzyme and improve understanding of its roles in cardiovascular disease and cancer.

Support or Funding Information

This work is supported by the Showalter Foundation, Purdue Center for Cancer Research, and the American Heart Association (Grant 16SDG29920017 to AML).
Universal, homogenous and bioluminescent assay to monitor the activity of wide range of methyltransferases
Authors: Goueli Said , Hsiao Kevin
Published: November 2023

Methylation/demethylation of DNA, RNA, Proteins and small molecules play major roles in modulation of the epigenome, transcriptome, neurotransmitter uptake and metabolic regulation, and thus, they are believed to be involved in a wide variety of human diseases. Recent biochemical and biological data suggest that altered enzymatic activities of several of these enzymes have pathogenic roles in cancer, inflammation, and neurodegenerative diseases. In addition to chromatin modulation, altered methylation of DNA and more recently mRNA have been recognized to regulate the transcriptome and the rate of message translation and stability. Furthermore, methylation of small molecules such as catechols and nicotinamide play critica roles in neurotransmitters uptake and function, and metabolic regulation, respectively. Thus, pharmacological modulation of these enzymes by small molecules will be beneficial in developing novel therapeutics for multiple unmet medical needs. Towards this goal of searching for activators/inhibitors of these enzymes for the development of next generation of drugs, screening assays for these modulators are urgently needed. To address these unmet needs, we have developed a novel ay hat monitors the activities of these enzymes and their modulation by small molecules. The assay is bioluminescent based, HTS formatted and highly sensitive. A unique feature of this assay is its universality since it is based on monitoring the formation of the universal product S‐adenosylhomocysteine (SAH), i.e., capable of detecting changes in activity of a broad range of methyltransferases such as DNA, RNA, protein, and small molecules. In addition, the assay has been validated for all classes of protein methyltransferases (Lysine and Arginine), and with different types of substrates (small peptides, large proteins, or even nucleosomes). This enables determining the specificity of these enzymes and their substrate requirements. The assay has high signal to background, low C.V., robust (Z′ value > 0.7), and has been validated using various plate densities such as 96‐, 384, and 1536‐well plates. A strong feature of this assay is its utility with broad range of substrates with no limitations on the use of high concentrations of substrates or the composition of the substrates (short vs. long peptides), thus enabling the generation of kinetic data and determining the mechanism of action of various modulators of methyltransferases of interest.

Support or Funding Information

Promega Corp.
mRNA Structural Modelling Predicts Variability in Efficacy of Post‐transcriptional Gene Silencing Between Species
Authors: Trujillo Alexandria J , Sullivan Jack M
Published: November 2023

Post‐transcriptional Gene Silencing (PTGS) agents, such as hammerhead ribozymes, face unique barriers when designed against an mRNA target. Hammerhead ribozymes(hhRzs) are able to cleave ‘NUH’ (N= any nucleotide H= U, A, C) sites in target mRNA. Along with sequence specificity, the NUH site must be located in a region of the mRNA that is accessible after secondary and tertiary structural folding. Our lab has developed a streamlined RNA Drug Discovery (RDD) platform and designed hhRzs that target the 266 and 1362 regions in Rhodopsin(RHO) mRNA, to develop a treatment for Autosomal Dominant Retinitis Pigmentosa, a disease that causes progressive vision loss. Bioinformatic analysis using mRNA structure prediction programs MFold, SFold, and RNA structure was conducted on fourteen species representatives to determine if the 266 and 1362 regions have similar accessibility for our ribozymes. The analysis shows that even in cases of sequence homology and close evolutionary relationships, access to the target region varies among species compared to human RHO. This suggests that during RDD efficacy and toxicity studies of PTGS agents in vitro and in vivo should be conducted against human RHO and transgenic animals expressing human RHO for the most accurate predictions.

Support or Funding Information

This work was supported majorly by NIH/NEI R01 grant EY013433 (JMS), and partially by the Department of Veterans Affairs (VA), Veterans Health Administration, Office of Research and Development (Biomedical Laboratory Research and Development) (VA Merit Grant 1I01BX000669 (JMS)), and partially by an Unrestricted Grant from Research to Prevent Blindness to the Department of Ophthalmology at the University at Buffalo‐SUNY, and also by the Interdisciplinary Science and Engineering Partnership (NSF 1102998 PI: Joseph Gardella (AJT)). The study was conducted in its entirety at the Veterans Administration Western New York Healthcare System (Buffalo, NY). Contents do not represent the views of the Department of Veterans Affairs or the United Stated Government.
Exercise Preconditioning as A Means To Protect The Kidney Against Doxorubicin‐Induced Oxidative Stress
Authors: Awwad Issam M. , D'Lugos Andrew C , Carroll Chad C , Gonzales Rayna J , Sweazea Karen L , Dickinson Jared M , Angadi Siddhartha S , Hale Taben M.
Published: November 2023

It is well known that anthracycline‐based neoadjuvant chemotherapeutic agent doxorubicin (DOX) successfully reduces recurrences and improves survival in patients with breast cancer. However, DOX produces significant adverse effects which are permanent, increase morbidity/mortality and reduce overall quality of life. DOX, is highly toxic to the kidney, with damage resulting in part from increased oxidative stress. DOX has been shown to both increase reactive oxygen species production and decrease antioxidant enzyme expression. We have recently demonstrated that high intensity interval exercise pre‐conditioning was effective in reducing mortality associated with DOX treatment in young adult ovariectomized female Sprague‐Dawley rats. Exercise has been shown to be effective in increasing antioxidant capacity in various organs and may serve as a means to protect against DOX‐induced renal toxicity. The purpose of this study was to determine the extent to which high intensity interval exercise pre‐conditioning in ovariectomized female rats attenuated DOX‐induced reduction in renal antioxidant enzyme expression. Rats were divided into 3 groups (sedentary vehicle, sedentary DOX, exercise DOX). DOX (4mg/kg) or Veh (saline) were administered intraperitoneally bi‐weekly for a total of 3 injections resulting in a cumulative dose 12mg/kg. High intensity interval training at 85–95% VO2 peak (4×4 min bouts) was performed using a treadmill for 5 days per week starting 1 week prior to the first injection and continued throughout study duration. Animals were euthanized 5 days following the last injection, kidneys removed and tissue lysate prepared for western analysis to determine the relative expression of the antioxidant enzymes superoxide dismutase (SOD)‐1 and −2, as well as catalase. DOX tended to increase kidney weight relative to tibial length in both sedentary and exercise rats. In addition, DOX significantly reduced SOD‐1 expression in sedentary rats by approximately 40% relative to vehicle (p<0.05), and tended to reduce SOD‐2 and catalase expression. Exercise preconditioning did not prevent DOX‐induced reduction in the expression of these antioxidant enzymes in the kidney. In conclusion, DOX treatment significantly increased renal mass and reduced antioxidant enzyme expression. Although exercise has been shown to increase antioxidant enzyme expression in the heart and skeletal muscle, the present findings reveal that similar effects are not observed in the kidney. Future studies will evaluate the functional and structural impact of DOX treatment on the kidney.

Support or Funding Information

Valley Research Partnership Award P1201603
Identification of ROS‐induced Poly(ADP‐ribose)‐Associated Proteins by LC‐MS/MS
Authors: Islas‐Robles Argel , Munoz Frances , Lau Serrine , Monks Terrence
Published: November 2023

Hydroquinone occurs ubiquitously found in the environment, is metabolized via oxidation and glutathione conjugation to 2,3,5‐tris‐(glutathion‐S‐yl)hydroquinone (TGHQ). TGHQ is nephrotoxic, nephrocarcinogenic and generates ROS, which cause DNA strand breaks, hyperactivation of poly(ADP‐ribose) polymerase‐1 (PARP‐1), increase in intracellular calcium concentration ([iCa2+]), and ultimately cell death. PARP‐1 is an abundant nuclear protein involved in DNA repair, transcriptional regulation, intracellular trafficking and is an epigenetic mediator. PARP‐1 catalyzes the attachment on target proteins of a posttranscriptional modification, consisting of multi branched ADP‐ribose polymers (PAR). We have shown that ROS stress promotes PARP‐1 hyperactivation and elevations in [iCa2+] which are reciprocally coupled, resulting in renal tubule cell death. The molecular mechanism of this interaction is unclear. Using LC‐MS/MS we have identified proteins associated with PAR that have a potential role in ROS mediated toxicity. Kidney proximal tubule cells (HK‐2) were treated with 400 μM TGHQ creating a ROS cell stress environment leading to PARP‐1 hyperactivation. PAR‐associated proteins were immunoprecipitated using an antibody against PAR, followed by subsequent separation by SDS PAGE. Tryptic peptides were obtained from the protein Gel sections and analyzed by LC‐MS/MS. Scaffold was used to validate MS/MS based peptides, protein identification, and perform the relative abundance analysis by spectral counts. Protein‐protein interaction modeling and gene ontology (GO) analysis were performed in Network Analyst. Coomassie stained protein gels showed differentially PARylated proteins between treated and control samples, the former displaying far more abundant bands. The spectral counting analysis allowed us to obtain fold‐changes in protein abundance relative to control. 361 PAR‐associated proteins were confidently identified as modified by TGHQ treatment. From this cohort, 276 proteins have increased PAR association while 85 are decreased. Protein network analysis revealed 2 subnetworks on the PAR‐increased proteins data set, the functions of which, are related to RNA metabolism and cellular organization. Cellular compartmentalization functions were prominently identified on the PAR‐decreased proteins. 13 proteins from the whole cohort contain GO annotations related to calcium. Importantly, Neuroblast Differentiation‐Associated Protein (AHNAK), Calcium Homeostasis Endoplasmic Reticulum Protein (CHERP) and General Transcription Factor II‐I (GTF2I) are directly involved in modulation of [iCa2+]. Currently we are investigating the possible role of these PAR‐associated proteins in ROS induced cell death.
The Role of DNA Methylation and Histone Acetylation in Bromate‐Induced Alteration of p21 Expression in Human and Rat Kidney Cells
Authors: Kolli Ramya T , Kaur Sukhneeraj P , Glenn Travis C , Cummings Brian S
Published: November 2023

Bromate (BrO3−) is a drinking water disinfection by product formed from naturally occurring bromide during the ozonation of source water. We previously showed that BrO3−‐induced nephrotoxicity in vitro and in vivo correlated to increased expression of the cyclin‐dependent kinase inhibitor p21. We also showed that the DNA methyltransferase inhibitor 5‐aza‐2‐deoxycytidine (5‐Aza) and the histone deacetylase inhibitor trichostatin A (TSA) increased p21 expression indicating the role of both DNA methylation and histone acetylation in its regulation. In this study we used next‐generation sequencing (NGS) to determine the percent methylation at the sis‐inducible element (SIE‐1), a transcription factor binding site in the promoter region of human embryonic kidney (HEK293). Treatment of the cells with 5‐Aza decreased DNA methylation by 35%, however BrO3− failed to significantly alter this value, suggesting that promoter demethylation at the SIE‐1 site is not a major mechanism by which BrO3− increased p21 expression in kidney cells. In contrast, exposure of normal rat kidney (NRK) cells to similar concentrations of BrO3− altered histone acetylation at the promoter region spanning 200bp upstream from the start site, as determined using chromatin immunoprecipitation (ChIP) assays, in a concentration‐ and time‐dependent manner. Changes in histone acetylation correlated to changes in p21 protein expression. These data suggest the novel finding that BrO3− regulates renal expression of p21 using mechanisms that alter histone acetylation, and not by inducing demethylation of its promoter.
Resveratrol Protection of Mitochondrial Function in HK‐2 Cells Exposed to Cisplatin.
Authors: Valentovic Monica , Brown Katie , Lamyaithong Andre Benja , Dial Mason
Published: November 2023

Cisplatin is a cancer chemotherapeutic agent widely used to control cancer to the testes, ovaries and cervix. Cisplatin is nephrotoxic and with a potential incidence of 33%. Resveratrol (RES) is a polyphenolic compound that possesses anticancer activity. Part of the mechanism for reducing cisplatin toxicity by RES may be mediated by preserving mitochondrial function and diminishing cisplatin induced oxidative stress. Our hypothesis is that RES will prevent mitochondrial impairment by cisplatin in HK‐2 cells. HK‐2 cells were selected as they are a human proximal tubular kidney cell line (HK‐2). Hk‐2 cells were plated for 48 followed by a 1 h pretreated with RES or 1% DMSO (vehicle). Renal cells were subsequently exposed to cisplatin at a final concentration of 0–50 uM for 24 h. Viability was assessed using MTT release and cell count with an n=6/group and repeated 3 separate times. Cells pretreated with 10 uM RES were protected from cisplatin cytotoxicity at 24 h. Cisplatin caused a concentration dependent decline in MTT viability after 24 h exposure to 0, 7.5 and 15 uM cisplatin. RES pretreatment for 1 h with 10 or 15 uM RES increased HK‐2 viability relative to cells exposed only to RES Vehicle (DMSO). Mitochondrial respiration was measured using a Seahorse XFp analyzer and expressed as oxygen consumption rate (OCR) and extracellular acidification rate (ECAR). Mitochondrial function was further probed by monitoring ATP levels and cytochrome c leakage. Basal and uncoupled (FCCP) mitochondrial respiration were monitored in RES and cisplatin treated cells. Additional studies examined oxidative stress as increased protein carbonylation and 4‐hydroxynonenal adduction (4‐HNE) following cisplatin exposure of HK‐2 cells pretreated with DMSO or RES. Western analysis detected an increase in protein carbonylation with 24 h cisplatin exposure which was reversed by RES. Mitochondrial function was diminished by cisplatin and protected by RES. RES protects human proximal tubular epithelial cells from cisplatin cytotoxicity, preserves mitochondrial integrity and mitochondrial respiration.

Support or Funding Information

(Supported by NIH Grants INBRE 3P20RR016477‐09S4; 5P20RR016477 and 8P20GM103434 and a WV NASA Undergraduate Research Fellowship to MD.
Altered Irinotecan Pharmacokinetics During Inflammation in Mice
Authors: Chityala Pavan Kumar , Gao Song , Hu Ming , Ghose Romi
Published: November 2023

Irinotecan is an anti‐cancer drugprimarily used in the treatment of colorectal cancer and its primary metabolite, SN‐38, is an active compound that also causes intestinal toxicity. It is widelyknown that inflammation downregulates the expression of many drug metabolizingenzymes, resulting in changes in drug disposition. Our aim is to investigate the role of inflammation on pharmacokinetics of irinotecan and its metabolites, SN‐38 and SN‐38G. Mice (C57BL6, male) were treated with either saline or LPS(2mg/kg) I.P and Irinotecan (10 mg/kg) was administered 16 hours later via oralgavage. Blood samples were collected by snipping the tail at different time points from 0–24 h and the blood concentrations of CPT‐11, SN‐38, and SN‐38G were measured by LC‐MS/MS. The concentrations of SN‐38 were found to be significantly higher in LPS treatment group compared to saline group. Our findings show that the toxic metabolite SN‐38 blood concentrations were increased during inflammation, which suggests that patients with inflammation may have higher risk of adverse effects of irinotecan due to higher SN‐38 concentrations.
Effects of MicroRNA‐34a on the Pharmacokinetics of Cytochrome P450 Probe Drugs in Mice
Authors: Jilek Joseph Lionel , Tian Ye , Yu Ai‐Ming
Published: November 2023

MicroRNAs (miR) including miR‐34a have been shown to regulate nuclear receptor, drug‐metabolizing enzyme and transporter gene expression in cell models. However, the degree to which miRNAs affect pharmacokinetics (PK) at the systemic level remains unknown. Additionally, miR‐34a replacement therapy represents a new cancer treatment strategy, however it is undefined if miR‐34a therapeutics elicit any drug‐drug interactions (DDI). To address these questions, we developed a practical single‐mouse PK approach and investigated the effects of a bioengineered miR‐34a prodrug on the PK of five cytochrome P450 (CYP) probe substrates (midazolam, dextromethorphan, phenacetin, diclofenac, and chlorzoxazone) administered as a cocktail to mouse models. This approach utilized manual serial blood microsampling from a single mouse and a sensitive LC‐MS/MS assay. This approach was validated by the sharp changes in midazolam PK by ketoconazole and pregnenolone 16α‐carbonitrile as well as phenacetin PK by a‐napthoflavone and 3‐methylcholanthrene. Surprisingly, 3‐methylcholanthrene also decreased systemic exposure to midazolam, while both pregnenolone 16a‐carbonitrile and 3‐methylcholanthrene largely reduced the exposure to dextromethorphan, diclofenac and chlorzoxazone. Finally, miR‐34a had no significant effects on the PK of dextromethorphan, phenacetin and chlorzoxazone, and caused a marginal (45–48%) increase in systemic exposure to midazolam and diclofenac in mice. These findings from the single‐mouse PK model suggest that therapeutic miR‐34a might have minor or no effects on the PK of CYP‐metabolized drugs co‐administered.

Support or Funding Information

This study was supported by grants [R01GM133888] and [U01CA175315] from NIH. J.L.J. was supported by a NIGMS Pharmacology Training Program [T32GM099608].
CCR1 as a Target for Metastatic Breast Cancer
Authors: Cao Minh‐An Dinh , Zhang Zhong , Gilchrist Annette
Published: November 2023

Breast cancer is the leading cancer in women. In ~30% of cases, breast cancer becomes metastatic, with the primary preferred site being the bone. Several investigators have found the chemokine receptor, CCR1, is upregulated in metastatic breast cancer. Using MCF7 and MDA‐MB‐231 breast cancer cell lines, we have assessed the ability of pharmacologic CCR1 antagonists to alter 125I‐CCL3 binding, cell proliferation, and chemotaxis. MCF7 is a HR+, PR+ luminal A subtype breast cancer cell line while MDA‐MB‐231, is an aggressive triple negative (ER−, PR−, HER2−) basal subtype breast cancer cell line. We found that CCR1 was present at equivalent levels in both breast cancer cell lines. However, there were distinct differences between the two cell lines for some of the compounds in their ability to reduce binding of 125I‐CCL3, inhibit proliferation, and diminish chemotactic activity. Our results will be used going forward to select compounds for in vivo studies.
Targeting the Anaphase Promoting Complex to Prevent Lung Cancer Cell Growth
Authors: Wainscott Nathan W , Hoffman J. Mason , Taylor B. Frazier , Trent John O. , States J. Christopher
Published: November 2023

Background

Current chemotherapeutic agents widely used for the treatment of lung cancer include mitotic inhibitors such as taxanes. Taxanes stabilize the mitotic spindle, maintaining the activity of the Spindle Assembly Checkpoint (SAC), thus preventing the metaphase to anaphase transition. Prolonged mitotic arrest results in cell death. Non‐small cell lung cancer (NSCLC) accounts for 80–85% of lung cancer cases. NSCLC may intrinsically be SAC deficient or acquire defects, making them resistant to these drugs. New treatments are needed for SAC deficient tumors. One potential new target for mitotic inhibition is the Anaphase Promoting Complex/Cyclosome (APC/C), an E3 ubiquitin ligase that drives the transition from metaphase to anaphase. The catalytic core of the APC/C consists of the subunits ANAPC2 and ANAPC11.

Hypothesis

Targeting ANAPC2 to prevent binding of ANAPC11 to ANAPC2 will inhibit the Anaphase Promoting Complex/Cyclosome in lung cancer cells and induce cell death.

Methods

Four lead compounds (3, 8, 10, and 11) were predicted by in silico analyses to bind the ANAPC2 subunit, and thus inhibit the APC/C by preventing binding of the catalytic subunit, ANAPC11. Compounds 8, 10 and 11 are 2‐amino‐propanol derivatives while compound 3 is a triazaspiro derivative. The compounds were tested for their ability to induce cytotoxicity in two human NSCLC cell lines (H460, A549) and a non‐transformed human bronchial epithelial cell line (HBEC). Cell viability following treatment was evaluated by morphological change by microscopy, Alamar Blue fluorescence assays, and colony forming assays.

Results

Morphological assessment of cell viability indicated a concentration‐ and time‐dependent increase in the number of mitotic cells with increasing concentration for compounds 3, 8 and 10 in all three cell lines, and evidence of cell death at higher concentrations in A549 and H460 cells. The Alamar Blue assays and colony forming assays confirmed that these compounds lead to a concentration–dependent decrease in A549 and H460 cell viability and proliferation. Compounds 3, 10 and 11 were more effective at inducing cell death than compound 8. Compound 11 was toxic at all concentrations tested in all three cell lines.

Conclusions

These results indicate that the APC/C could be a viable target for development of novel chemotherapeutic drugs to treat NSCLC.

Support or Funding Information

ASPET‐Summer Undergraduate Research Fellowship (NCW); KY Lung Cancer Research Program (JCS,); KY Science and Engineering Foundation (JCS); Kosair Charities Pediatric Oncology Research Program (JOT); NIH P30GM106396 (JOT)
Aggressive Breast Cancer Cell Line and Role of Translocator Protein
Authors: Mukherjee Shyamali , Sharan Chakradhari , Mitra Piyali , Gopal Srirupa Hari , Das Salil K
Published: November 2023

We have already established that translocator protein (TSPO) is a breast cancer biomarker. It is also established that translocator protein shows low expression in ER positive breast cancer cells than ER negative breast cancer cells. It is also reported that African American (AA) women have higher grade breast tumors, higher incidence of triple negative breast cancers (TNBCs), higher proliferation indices, and node positive disease than their Caucasian (C) counterparts. Mortality rate is also higher in AA than C women. Based on these facts our hypothesis is that expression of TSPO is higher in TNBC than non‐TNBC and that higher mortality rate among breast cancer in AA women is mechanistically linked to higher incidence of TNBC cells. To test this hypothesis we have studied four TNBC (AA: HCC‐1806 and MDA‐MB‐468; C: BT‐549 and MDA‐MB‐231) and five non‐TNBC (C: MCF‐12A, MCF‐12F, MCF‐7, T47D and ZR‐75‐1) human cell line obtained from ATCC for TSPO expression. We analyzed the cultured cells for gene expression by RT‐PCR and protein by western blot. Both gene expression and protein levels were significantly higher in TNBC cells than non‐TNBC cells. We did not find any significant variation in either protein level or gene expression in TNBC cells of AA and C. TSPO Binding capacity (Bmax) was significantly higher in TNBC cell lines than normal cell lines. Proliferation of both TNBC and non‐TNBC cell lines is inhibited by PK 11195, antagonist to TSPO, in a dose‐dependent manner. However, inhibition efficiency was higher in TNBC cell line than non‐TNBC cell line.

Support or Funding Information

MeTRC NIH grant 5U54MD007593
Structure‐Activity Relationship Studies of Oxypropanolamines: The Importance of Hydrophobicity About the Amine for Maintaining Anti‐Proliferative Effects in Lung Cancer Cells
Authors: Hoffman J. Mason , Wainscott Nathan W , Taylor B. Frazier , Burlison Joseph A. , Trent John O. , States J. Christopher
Published: November 2023

Background

Mitosis‐inhibiting chemotherapeutics are frequently used to treat cancer. Successful therapy is often hindered by drug‐resistant tumors, neurotoxicity, and limited supply. There is high demand for new drugs with diverse cellular targets. The anaphase promoting complex/cyclosome (APC/C) is a multi‐subunit E3 ubiquitin ligase that promotes cell cycle progression, especially mitosis, by marking key proteins for degradation. Thus, the APC/C offers great potential as an alternative target for anti‐mitotic drug development. The APC/C catalytic core consists of an ANAPC2/ANAPC11 dimer. Homology models of the ANAPC2/ANAPC11 subunits were analyzed in silico to identify lead compounds predicted to bind the ANPAC11‐binding domain of ANAPC2, and prevent ANAPC11 association and inhibit APC/C function. Among these leads were oxypropanolamine‐containing compounds. Preliminary studies indicate several of these compounds possess anti‐proliferative effects in various cancer cell lines. Based on this finding, a series of amine derivatives were synthesized to probe the effect of steric bulk and hydrophobicity in this region.

Objective

We aim to determine how modification of the amine substituent groups influences the ability to inhibit proliferation in several lung cancer cell lines in vitro.

Methods

To quantify the anti‐proliferative effect of compounds, AlamarBlue and colony forming assays were performed in A549 and H460 lung cancer cells and HBEC‐3KT immortalized non‐transformed bronchial epithelial cells.

Results

Outcomes of AlamarBlue and colony forming assays indicate that derivatives containing a secondary amine, but not tertiary, cause a decrease in cell proliferation. This anti‐proliferative effect is strengthened by increased steric bulk and hydrophobicity of the substituent amine.

These results provide structural insights to guide development of ANAPC2‐targeting compounds and are consistent with the hypothesis that the APC/C is a promising target for development of novel chemotherapeutics.

Support or Funding Information

KY Lung Cancer Research Program (JCS,); KY Science and Engineering Foundation (JCS); ASPET Summer Undergraduate Research Fellowship (NCW); Kosair Charities Pediatric Oncology Research Program (JOT); NIH P30GM106396 (JOT)
Screening of a Small Flavonoid Library for Anti‐Cancer Activity Against Glioblastoma
Authors: Faler Ashton D. , Stang Christopher R.T. , Khupse Rahul S. , Schneider Ryan A.
Published: November 2023

Flavonoids, including chalcones and flavanones, have been highly studied for their multitude of activities including antioxidant and anti‐cancer effects. We previously demonstrated that several of our chalcone derivatives had IC50 values in the low micromolar range in multiple distinct human cancer cell lines, but appeared to be most potent against A‐172 glioblastoma cells. The poor prognosis of glioblastoma necessitates further investigations to identify more effective treatment options. To determine if the activity of these chalcones was comparable in additional glioblastoma lines, our small library of flavonoids was screened against T98G and U87 MG glioblastoma cells. Our library consisted of eight flavonoid derivatives; six of which were chalcones and two flavanones. IC50 values, as measured by the CyQuant NF or XTT cell proliferation assay, were noted to be in the low micromolar range for three of these compounds; RK6, RK7, and RK15. RK6 had an IC50 of 9 μM and 20 μM against the T98G and U87 MG cell lines respectively. RK7 displayed IC50 values of 33 μM and 51 μM against the T98G and U87 MG cell lines respectively. RK15 was the most potent chalcone derivative with IC50 values of 3 μM in T98G cells and 5 μM in U87 MG cells. A Caspase‐Glo 3/7 assay was utilized to investigate the mechanism by which these compounds exhibited their cytotoxic effects. A significant increase in effector caspase activity was noted following treatments with RK6, RK7, and RK15 in both T98G and U87 MG cells. Furthermore, partial reversal of apoptosis by the pan‐caspase inhibitor, Z‐VAD‐FMK, suggests that caspases are involved in the observed cell death by RK6, RK7, and RK15 in these glioblastoma cell lines. Further investigations are ongoing with these chalcones to determine the molecular mechanisms of cell death in glioblastoma cells and to optimize the structure activity relationship.

Support or Funding Information

This work was supported in part by an Individual Summer Undergraduate Research Fellowship sponsored by ASPET for the first author.
Opposite angiogenic outcome of curcumin against ischemia and lung cancer: in silico, in vitro and in vivo studies
Authors: Li Xuejun
Published: November 2023

Since many diseases are caused by complex factors, we try to apply the network pharmacology techniques to study the targets and networks of drugs for complex diseases. We investigated the angiogenic effects of curcumin on an ischemia and lung cancer model. To induce ischemia combined with lung cancer models, unilateral femoral arteries of C57BL/6 mice were disconnected on one side of the mouse and Lewis lung carcinoma (LLC) cells were xenografted on the opposite side. Angiogenic effects and underlying mechanisms associated with curcumin were investigated. Molecular target(s), signaling cascades and binding affinities were detected by Western blot, two‐dimensional gel electrophoresis (2‐DE), computer simulations and surface plasmon resonance (SPR) techniques. The results showed that Curcumin promoted post‐ischemic blood recirculation and suppressed lung cancer progression in inbred C57BL/6 mice via regulation of the HIF1α/mTOR/VEGF/VEGFR cascade oppositely. Inflammatory stimulation induced by neutrophil elastase (NE) promoted angiogenesis in lung cancer tissues, but these changes were reversed by curcumin through directly reducing NE secretion and stimulating α1‐antitrypsin (α1‐AT) and insulin receptor substrate‐1 (IRS‐1) production. Meanwhile, curcumin dose‐dependently influenced endothelial cells (EC) tube formation and chicken embryo chorioallantoic membrane (CAM) neovascularization. In conclusion, Curcumin had opposite effects on blood vessel regeneration under physiological and pathological angiogenesis, which was effected through negative or positive regulation of the HIF1α/mTOR/VEGF/VEGFR cascade. Curcumin had the promise as a new treatment modality for both ischemic conditions and lung cancer simultaneously in the clinic.

Support or Funding Information

This work was supported by the National Natural Science Foundation of China (No. 81673453, 81473235, 91129727, 81673486) and Research Fund from Ministry of Education of China (111 Projects No. B07001)
Macrophage‐derived extracellular vesicles transfer inflammasome components to endothelial cells and induces endothelial injury
Authors: Zuo Ping , Lin Xingguang , Li Xiang , Zhang Yang
Published: November 2023

Background and aims

Recent studies have demonstrated an important role of Nlrp3 inflammasome in the development of endothelial dysfunction and injury under various pathological conditions. Extracellular vesicles (EVs) play an important role in cell‐cell communication and functional regulation. The role of macrophage‐derived EVs in endothelial homeostasis remains largely unknown. Here, we test whether or not the inflammasome components can be secreted into EVs derived from macrophages and then EVs transfer these proteins into endothelial cells and causes endothelial injury.

Methods

Exosomes and microvesicles, two major subtypes of EVs, were isolated from the cell culture medium from J774 macrophages by sequential ultracentrifugation. Exosomes were detected by Western blotting analysis of marker protein CD63. Inflammasome components Nlrp3, ASC, and caspase‐1 were detected in EVs by Western blotting. Endothelial injury was examined by analyzing the endothelial cell death using LDH assay and flow cytometry analysis.

Result and conclusion

In macrophages, lipopolysaccharide (LPS) treatment dose‐dependently increased the expression of inflammasome component Nlrp3 and pro‐caspase‐1 in isolated exosomes or microvesicles. Co‐stimulation of macrophages with palmitate and LPS did not further enhance the secretion of Nlrp3 or pro‐caspase‐1 into these EVs. Incubation of isolated exosomes or microvesicles from LPS‐treated macrophages with naïve microvascular endothelial cells (MVECs) resulted in up‐regulation of the protein expression for Nlrp3 and pro‐caspase‐1 in these cells. LPS alone did not increase Nlrp3 expression in MVECs. These data indicate that the EV‐induced increases in inflammasome components in naïve MVECs may be primarily due to a protein transfer between EVs and the naïve MVECs but not up‐regulation of inflammasome gene expression. Lastly, incubation of naïve MVECs with microvesicles or exosomes derived from macrophages treated with LPS‐ or LPS plus palmitate induced significantly cell death in MVECs, whereas LPS alone had no effects on these naïve cells. Together, these results suggest that intercellular EV transfer of inflammasome components from macrophages to endothelial cell may exacerbate inflammasome‐mediated endothelial injury during vascular inflammation in obesity (Supported by NIH grants HL122769 and HL122937).

Support or Funding Information

Supported by NIH grants HL122769 and HL122937
Psychedelics As A New Anti‐Inflammatory Therapeutic For Atherosclerosis
Authors: Nichols Charles D , Sebastian Melaine , Flanagan Thomas
Published: November 2023

Background and Objective

We previously discovered that serotonin 5‐HT2A receptor activation with psychedelics has potent anti‐inflammatory activity in both cell culture and whole animals, which indicated potent anti‐inflammatory effects in vascular tissues among others. More recently we found that the psychedelic (R)‐DOI potently prevents the development of allergic asthma in a mouse model. The effects of (R)‐DOI were found to not result from a generalized anti‐inflammatory process, but due to specific inflammatory pathways inhibition in both innate and Th2 cells. In this work, we have examined the therapeutic effects of the psychedelic (R)‐DOI in the ApoE −/− high‐fat model of atherosclerosis.

Methods

Osmotic minipumps were used to deliver very low doses of (R)‐DOI to male ApoE −/− mice that were divided into four treatment groups [Saline, normal chow; (R)‐DOI, normal chow; Saline, hi‐fat diet; (R)‐DOI, hi‐fat diet]. After 16 weeks, mice were euthanized and tissues collected for analysis

Results

Calculated steady state levels of ~0.0013 mg/kg (R)‐DOI resulted in a significant reduction of mRNA expression for inflammatory markers like Il6 in vascular tissue, reduced levels of glucose, and a reduction in circulating cholesterol in the high fat fed animals. Additional ongoing studies are examining arterial plaque size and heart pathology.

Extremely low levels of the psychedelic (R)‐DOI were sufficient to significantly block the development of vascular inflammation, normalize glucose homeostasis, and prevent the increase in cholesterol associated with a hi‐fat ‘western’ high diet. Activation of serotonin 5‐HT2A receptor therefore represents a powerful new strategy to treat inflammatory‐related vascular disease.

Support or Funding Information

NIH/NIGMS P30GM106392
Nuclear PTEN maintains vascular homeostasis by stabilizing the expression of ETS transcription factor, ERG
Authors: Avin BR Vijay , Mohammad Tauseef , Farazuddin Mohammad , Yue Lili , Mehta Dolly
Published: November 2023

Loss of endothelial barrier integrity leads to the formation of protein‐rich edema in the interstitial tissue, a hallmark of tissue inflammation that if left untreated, causes fatal diseases like acute lung injury (ALI). Phosphatase and tensin homologue (PTEN), a tumor suppressor, is well‐known to negatively regulate angiogenesis in part by blocking PI3‐kinase signaling. Intriguingly, we found that tamoxifen‐inducible deletion of PTEN in the endothelium of mice (EC‐PTEN−/− mice) induced microvascular injury. FACS analysis showed marked reduction in CD31+/CD45− cell population as well as VE‐cadherin expression in EC‐PTEN−/− lungs relative to control lungs indicating loss of PTEN impaired endothelial cell (EC) regeneration. PTEN is known to localize in the cytosol, plasma‐membrane and nucleus. Members of ETS (E26 Transformation Specific) family of the transcription factors play a primary role in vascular development and angiogenesis. ERG, an ETS family members, is constitutively expressed in ECs and drives transcription of genes involved in endothelial homeostasis and angiogenesis. Intriguingly, we identify a novel role of nuclear PTEN in dynamically maintaining lung vascular homeostasis by stabilizing the expression of ERG. Furthermore, PTEN localized in the nucleus and interacted with ERG. Nuclear localized PTEN increased ERG expression and promoter activity leading to de novo synthesis of VE‐cadherin thereby rapidly resolving lung vascular injury. Thus, endothelial‐PTEN maintains lung vascular homeostasis by serving as a co‐factor of ERG that induces key ECs genes including VE‐cadherin resulting in ECs regeneration and vascular barrier formation.
NACHT, LRR and PYD Domains‐containing Protein 3 (NLRP3) Crucial Role of the Cavernous Tissue Tone Modulation in Mice
Authors: Fais Rafael Sobrano , Rodrigues Fernanda Luciano , Tostes Rita Cassia , Carneiro Fernando Silva
Published: November 2023

INTRODUCTION

Erectile function depends on the tone of the cavernous tissue (CT), and increased contraction and/or decreased relaxation responses contribute to erectile dysfunction. Erectile dysfunction and cardiovascular diseases share several mechanisms, like inflammation. NLRP3 is an intracellular receptor of the innate immune system, responsible for the release of pro‐inflammatory cytokines that induce inflammation and has been implicated in vascular dysfunction. However, the role of NLRP3 in the modulation of vascular and CT tonus is unknown.

HYPOTHESIS

NLRP3 modulates the basal tonus of the CT.

AIM

To determine the role of NLRP3 in the modulation of contraction and relaxation pathways in CT.

METHODS

CT strips were obtained from male C57BL/6 (WT), deficient to NLRP3 (NLRP3−/−) and caspase1/11 (caspase1/11−/−) mice, with 10 to 12 weeks. Reactivity of CT and western blot were performed to evaluate the function of the contraction and relaxation pathways.

RESULTS

There was no difference in contraction to phenylephrine (PE) (Emax WT: 836,5 ± 93,3, n=5; NLRP3−/−: 813.0 ± 75,4, n=5; caspase1/11−/−: 925,4 ± 73.7, n=4), to electric field stimulation (EFS, frequencies from 0,2 to 32 Hz) and relaxation to acetylcholine (Ach) (Emax WT: 71,6 ± 7,84, n=5; NLRP3−/−: 75,9 ± 6,8 n=5; caspase1/11−/−: 91,25 ± 8,8, n=4) or non‐adrenergic non‐cholinergic (NANC) stimulation. However, L‐NAME and atropine incubation promoted a significant increase in the contraction to PE (Emax WT: 865,2 ± 76,6, n=5; NLRP3−/−: 1202,0 ± 85,9, n=5; caspase1/11−/−: 1160,0 ± 70, n=4) and EFS (mean WT: 329,2 ± 24,2, n=5; NLRP3−/−: 705,7 ± 130,1, n=5; caspase1/11−/−: 594,9 ± 73,1, n=4, at 16 Hz) and (mean WT: 554,0 ± 50,4, n=5; NLRP3−/−: 847,5 ± 157,1, n=5; caspase1/11−/−: 985,7 ± 124,8, n=4, at 32 Hz) Hz in the CT strips of NLRP3−/− and caspase1/11−/− and reduced relaxation to sodium nitroprusside in NLRP3−/− strips (Emax WT: 82,0 ± 2,75, n=5; NLRP3−/−: 63,9 ± 3,78 n=5; caspase1/11−/−: 91,5 ± 7,8, n=4). The eNOS activity was increased in the NLRP3−/− and caspase1/11−/− CT strips. In addition, there was an increase in tyrosine hydroxylase expression. Furthermore, there was an increase in the activity of MYPT‐1 in the NLRP3−/−, and PKCα in the caspase1/11−/− strips. The expression of the soluble guanylate cyclase alpha was reduced in NLRP3−/− and caspase1/11−/− CT strips.

CONCLUSION

The NLRP3 constitutively modulates the CT tonus, and its absence leads to a series of compensatory alterations in the contraction and relaxation pathways.

Support or Funding Information

FAPESP, CNPQ, CAPES, CRID, FAEPA
Doxorubicin Dose Dependently Attenuates Cyclooxygenase‐2 in Female Human Coronary Vascular Smooth Muscle Cells
Authors: Tat Trinh , So Me , Vijayavel Nirmal , Raman Puneet , Dickinson Jared M , Angadi Siddartha S , Gonzales Rayna J
Published: November 2023

Doxorubicin (Dox), an anthracycline‐based chemotherapeutic agent, is highly effective at reducing recurrence and mortality in breast cancer patients. However, this anti‐cancer drug elicits dose‐dependent cardiotoxicity. Multiple mechanisms have been implicated in its pathogenesis, one of which involves the development and progression of inflammation mediated by activation of the TLR4/NFkappaB pathway. However, we recently demonstrated that chronic in vivo Dox treatment reduced expression of the NFkappaB downstream proinflammatory mediator, cyclooxygenase‐2 (COX‐2), in cerebral and peripheral vasculatures isolated from ovariectomized female rats. Therefore, in the current study we tested whether Dox similarly alters vascular proinflammatory enzyme expression in the human vasculature and whether this response is dose dependent. We further investigated whether Dox alters upstream mediators of COX‐2 expression by assessing levels of expression and activation of NFkappaB and an additional downstream gene product, TLR4. Primary human coronary vascular smooth muscle cells of female origin were studied at passage 6 and treated with vehicle or Dox (0.3, 1.0, and 5 μM). Following drug treatment, cells were isolated, homogenized, and lysates (whole cell; nuclear and cytosolic) analyzed for NFkappaBp65, COX‐2, and TLR4 protein levels via immunoblotting. Our results demonstrate that while total NFkappaB levels were unaffected following Dox, enhanced translocation of NFkappaB to the nucleus was observed. However, even in the presence of Dox‐induced NFkappa translocation, basal expression of its downstream mediator, COX‐2, was dose dependently attenuated. Similar to COX‐2, a trend for a decrease in TLR4 levels was observed. Dox had no effect on cell morphology or density, as visualized via light microscopy. In conclusion, although others have suggested the involvement of NFkappaB activation during the development and progression of Dox‐induced inflammation in cardiac muscle, our studies demonstrate a possible novel action for the anticancer agent targeting important downstream mediators of inflammation in the female vasculature. Future studies will evaluate the impact of Dox on COX‐2 at the transcriptional or translational level to determine the mode of molecular regulation.

Support or Funding Information

University of Arizona Sarver Heart Center (RG and MS); Mayo Clinic Dept. of Medicine Extramural Chair Award in collaboration with Arizona State University (SA)
Expression and Characterization of Transcription factor EB in Coronary Arterial Smooth Muscle Cells
Authors: Bao Renyue , Zuo Ping , Li Xiang , Li Pin‐Lan , Zhang Yang
Published: November 2023

Background

We recently demonstrated that lysosome injury associated with abnormal sphingolipid metabolism causes impaired autophagic flux in coronary arterial smooth muscle cells (CASMCs). Under atherogenic stimulation, such impaired autophagic flux accelerates dedifferentiation of CASMCs to a synthetic phenotype. In the present study, we hypothesized that transcription factor EB (TFEB), a master regulator of lysosome biogenesis and autophagy signaling is involved in lysosome injury‐associated CASMC dedifferentiation.

Methods

CASMCs were primarily cultured from mouse coronary arterial arteries and used in this study. The mRNA and protein expression of TFEB in CASMCs were detected by real time quantitative PCR and Western blot, respectively. The activation of TFEB was determined by nuclear translocation of TFEB using immunofluorescence microscopy.

Results and conclusion

Lysosome functional inhibitors increased TFEB expression and activation in CASMCs, which was associated with up‐regulation of lysosome and autophagy markers. These results indicate that TFEB are expressed and functioning in CASMCs. Genetic deficiency of acid sphingomyelinase (ASM) results in enhanced cell proliferation and migration of CASMCs upon platelet‐derived growth factor (PDGF) stimulation. Such phenotypic changes in ASM‐deficient CAMSCs were associated with decreased nuclear translocation of TFEB compared to that in wild‐type cells. These results suggest that ASM/TFEB signaling axis is involved in the regulation of cell cycle in CASMCs implicating a novel role of TFEB in vascular smooth muscle plasticity.

Support or Funding Information

Supported by NIH grants HL057244, HL122769 and HL122937
Impact of Sex and GPER on the Cardiovascular Effects of the Environmental Estrogen Bisphenol A
Authors: Spooner Hallie Marie , Lindsey Sarah H.
Published: November 2023

We previously found that the membrane‐bound G‐protein coupled estrogen receptor (GPER) attenuates blood pressure. Environmental estrogens, such as Bisphenol A (BPA) compete for binding at GPER, potentially reducing its beneficial vascular effects. Recent clinical data shows that exposure to BPA is associated with higher blood pressure and cardiovascular disease. The goal of the current study was to assess the effects of BPA on blood pressure in a mouse model and determine the impact of sex and GPER. We hypothesized that BPA administration in female mice would induce a larger increase in blood pressure in comparison to males because of its interference with estrogen's protective vascular effects. Further, we hypothesized that the response to BPA in GPER knockout mice would be diminished. Female (f) and Male (m) wild type (WT) and knockout (KO) mice were administered 5 mg/kg body weight/day of BPA via the drinking water for eight weeks. Blood pressures were measured via tail cuff by volume pressure recording biweekly. Two‐way ANOVA revealed a significant effect of sex (P<0.001) but not GPER deletion (P=0.19) on systolic blood pressure, with females having lower pressures (mWT 148 ± 2.8 vs. fWT 131± 4, P<0.01; mKO 142±3 vs. fKO 128± 3, P<0.05). Diastolic pressures were not statistically different by sex (P=0.08) or GPER deletion (P=0.82). Although the study is ongoing, two weeks after the administration of BPA, there was no significant impact of BPA on blood pressure in any group (P>0.05). In conclusion, since GPER mediates some of the beneficial cardiovascular actions of estrogen, environmental estrogens such as Bisphenol A may act as a “hormonal block” of GPER. Increasing amounts of environmental estrogens may promote cardiovascular problems, especially in aging postmenopausal women.

Support or Funding Information

Tulane University Newcomb College Lurcy GrantAmerican Heart Association 15GRNT25890021