bluuebunny/crossref_metadata_2025 huggingface dataset:
Anti‐tumor Activity of PCAIs in 3D Cell Culture and Mice Models
Authors: Salako Olufisayo O. , Nkembo Augustine T. , Ntantie Elizabeth , Lamango Nazarius S.
Published: November 2023

PURPOSE

Of the four molecular subtypes of breast cancer, TNBC is the most difficult to treat due to its lack of biomarkers and targeted therapies. Metastasis, which is characterized by cancer cell migration and invasion of distal organs, is highly prevalent in TNBC, making the disease harder to manage while lowering survival rates. The PCAIs, developed and modeled around the post‐translational modifications of regulatory proteins such as K‐Ras, were designed to inhibit the interactions of overactive monomeric G‐proteins. These proteins operate downstream of EGFR which is overexpressed in 60% of TNBC cases and are vastly involved in regulating cell survival, motility, apoptosis, and ultimately cancer progression. Previous work in our lab has shown that treatment of various cancer cell lines with PCAIs leads to cell cycle arrest, suppression of cell viability, inhibition of monolayer cell migration and invasion, and induction of caspase 3/7‐mediated apoptotic death. The purpose of this study was to determine the effectiveness of PCAIs at impeding the trademarks of cancer progression in 3D cell culture and in in vivo models.

METHODS

MDA‐MB‐231 cells were used in 3D cell culture assays to assess the PCAIs' ability to inhibit spheroid formation and invasion while inducing apoptosis. TNBC mice xenografts were used to determine the efficacy of PCAIs in vivo.

RESULTS

Compounds NSL‐BA‐055 and NSL‐BA‐040 were the most potent of the PCAIs, halting the formation of compact spheroids at 1.0 and 2.0 μM while significantly inhibiting spheroid invasion at 0.2 and 1.0 μM, respectively. Both compounds induced apoptosis and completely obliterated compact TNBC spheroids at 5.0 μM. Five and 10 mg/kg NSL‐BA‐040 inhibited the growth of TNBC xenograft tumors by 60.1 and 71.2%, respectively.

CONCLUSION

The PCAIs' ability to prevent spheroid formation and invasion while inducing spheroid apoptosis suggests their ability to prevent metastasis, tumor formation, growth and potential as effective therapies for TNBC.

Support or Funding Information

The research reported in this publication was supported by the National Cancer Institute (NCI) and National Institute of General Medical Sciences (NIGMS) of the National Institutes of Health (NIH) under Grant SC1CA190505 and by the National Institute On Minority Health and Health Disparities (NIMHD) of the NIH under Award Number G12 MD007582 (previous award NIH/NCRR/RCMI G12 RR03020). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Arginase Inhibition Suppresses Breast Cancer Cell Proliferation
Authors: Shatanawi Alia , Qasrawi Heba
Published: November 2023

Breast cancer is the second most common cause of cancer worldwide. It is recognized to be the most common cancer in women. It is also considered as the second leading cause of cancer death in women in the United States. There is accumulating evidence of increased arginase activity in patients with breast cancer. Arginase, an enzyme of the urea cycle, catalyzes the hydrolysis of L‐arginine to urea and L‐ornithine. L‐ornithine is converted to polyamines by the enzyme ornithine decarboxylase (ODC). Polyamines such as spermidine and spermine have an important role in cell proliferation. Hence, polyamines are also found to have an important role in tumor development and growth. Levels of polyamines are found to be 2–3 times higher in breast cancer cells than in normal surrounding tissue.

We studied the effects of arginase inhibition on breast cancer cells and endothelial cells in vitro. Our experiments utilized MCF‐7 breast cancer cells or bovine aortic endothelial cells. Arginase activity was measured in cells treated with or without the novel arginase inhibitor; 2(S)‐amino‐6‐boronohexanoic acid (ABH). Additionally, the effect of ABH treatment on cells proliferation in vitro was determined using cell proliferation assay.

Our results show that arginase inhibition caused a concentration dependent suppression of MCF‐7 cell proliferation with a maximum inhibitory concentration of 500 μM. This dose of ABH resulted in a 30% decrease in cell proliferation versus untreated controls. Arginase activity was measured in cells treated with ABH and was decreased by 60% versus controls. Interestingly, while ABH inhibited arginase activity in endothelial cells, it did not have any effect on the proliferation rate of endothelial cells. Our results indicate that arginase can be considered a new therapeutic target for suppressing breast cancer cell growth. Arginase inhibitors represent a potential cancer therapy in tumors with elevated arginase activity.

Support or Funding Information

The University of Jordan Deanship of Research
Involvement of Polo‐like kinase 1 (Plk1) in tumor growth and epithelial‐mesenchymal transition of tamoxifen‐resistant breast cancer
Authors: Jeong Sung Baek , Kang Keon Wook
Published: November 2023

The most common therapy for estrogen receptor‐positive breast cancer is anti‐hormone therapy such as tamoxifen treatment. However, one‐third of the patients acquires tamoxifen resistance and become a serious clinical problem. Polo‐like kinase 1 (Plk1) is a key regulator for the completion of G2/M phase cell cycle. When we assessed Plk1 expression in six chemo‐resistant cancer cell sets, we found that Plk1 and its downstream phosphatase, Cdc25c were selectively overexpressed in tamoxifen‐resistant MCF‐7 (TAMR‐MCF‐7) breast cancer cells. Real‐time monitoring of cell proliferation also showed that BI2536 (ATP‐competitive Plk1 inhibitor)‐mediated inhibition of cell proliferation was more sensitive in TAMR‐MCF‐7 cells than the parent MCF‐7 cells. Moreover, we observed that BI2536 suppressed the expression of epithelial‐mesenchymal transition (EMT) marker proteins and 3D‐spheroid formation in TAMR‐MCF‐7 cells. By using TAMR‐MCF‐7 cells‐implanted xenograft and spleen‐liver metastasis models, we observed that BI2536 inhibited tumor growth and metastasis in vivo. Our results propose that Plk1 could be a novel target for the treatment of tamoxifen‐resistant breast cancer.

Support or Funding Information

This work was supported by a grant (2014M3A9A9073788) from National Research Foundation of Korea, Republic of Korea.
A Novel Hydrogen Sulfide Donor, JK1 Protects the Heart Against Cardiac Hypertrophy and Heart Failure
Authors: Li Zhen , Organ Chelsea L. , Kang Jianming , Xian Ming , Lefer David J.
Published: November 2023

Background and Hypothesis

Hydrogen sulfide (H2S) protects against acute myocardial ischemia/reperfusion injury and heart failure by ameliorating oxidative stress, improving mitochondrial function, and attenuating apoptosis. One major limitation in the development of H2S‐based therapy is a lack of evidence supporting its cardioprotective effects in a clinically relevant scenario that mimics heart failure in humans. A second limitation related to the development of H2S therapeutics for heart failure is the very poor pharmacokinetic profile of most H2S‐donating agents. In the present study we tested the efficacy of a novel H2S donor, JK1, with controlled H2S release and increased half‐life compared to traditional H2S‐releasing agents. We evaluated JK1 in a clinically relevant murine model of transverse aortic constriction (TAC) induced heart failure (HF). We hypothesized that delayed administration of JK1 would protect the myocardium against TAC‐induced heart failure.

Methods

Male C57/BL6J at 10 weeks (wks) of age were subjected to TAC (27G needle) for 12 wks. Optimal dosing of JK1 was determined from preliminary studies of in vivo myocardial ischemia and reperfusion in which 100 μg/kg JK1 treatment reduced myocardial infarct size from 48.19% to 17.44%. JK1 (100 μg/kg/d, n=16) or control compound (100 μg/kg/d, n=15) were administered (i.p.) starting at 3 wks post TAC. Echocardiography was performed at baseline and following TAC to assess cardiac function.

Results

Delayed treatment of JK1 significantly preserved left ventricular (LV) ejection fraction (LVEF 49.7% vs. 36.2%, p < 0.001), reduced left ventricular dilation (LVEDD/LVESD in mm): 3.77/2.81 vs. 4.34/3.50, p < 0.001), and attenuated eccentric hypertrophy (IVSD in mm: 0.84 vs. 1.00, p < 0.01. LVPWd in mm: 0.78 vs. 0.89, p < 0.05) at 12 wks post TAC as compared to Control. Complete data for LVEF and % LV fractional shortening (FS) are shown below.

Conclusion

These results demonstrate that delayed administration of JK1 significantly preserves LV function and attenuates adverse remodeling in HF. Studies are currently underway to define the molecular mechanisms involved in JK1 mediated cardioprotection.

Support or Funding Information

Acknowledgement: This work was supported by the National Institutes of Health (R01HL116571).
SRY‐box 9 (SOX9): A Novel Binding Partner of Cardiac Muscle‐specific A‐Kinase Anchoring Protein (mAKAP) Signalosome
Authors: Suryavanshi Santosh , Bark Steven , McConnell Bradley K
Published: November 2023

Background

By definition, A‐kinase anchoring proteins (AKAPs) bind to protein kinase A and various other signaling proteins at specific subcellular compartments to regulate downstream signal transduction. Heart specific muscle AKAP (mAKAP) is present on the nuclear envelope of cardiomyocytes and regulates cardiac hypertrophy by forming a signalosome of its binding partners. It was previously reported that hypoxia‐inducible factor 1 alpha (HIF‐1α) physically binds to mAKAP. Also, it was previously published that HIF‐1α physically interacts with SRY‐box 9 (SOX9). Chromatin immunoprecipitation (ChIP) studies showed that HIF‐1α binds to SOX9 promoter region. Interestingly, both mAKAP and SOX9 have been demonstrated to play significant role in cardiac differentiation and endocardial cushion formation, respectively. Hence, we were interested to know whether SOX9 is a part of mAKAP scaffold on the nuclear envelope.

Objective

The aim of the current work is to confirm that SOX9 unambiguously interacts with mAKAP; either directly or indirectly.

Methods

As HEK 293T cells express SOX9 endogenously and not mAKAP, we chose these cells for immunoprecipitation (IP) studies. Myc‐DDK tagged human mAKAP cDNA was purchased from Origene and overexpressed in HEK 293T cells using Lipofectamine 2000, according manufacturer's instructions. IP was performed using M2 affinity gels (Sigma) and immunoblotting was performed using mAKAP and SOX9 antibodies. HepG2 cell lysate was used as positive control for SOX9 expression. In another set of experiment, a Coomassie Blue stained gel was used for in‐gel digestion to perform MALDI‐TOF Mass Spectrometry, followed by data analysis.

Summary of Results

IP samples of mAKAP transfected HEK cells displayed SOX9 expression; whereas, untransfected cells did not show expression. mAKAP expression was used as a loading control and remained unchanged in all samples. Mass Spectrometric data validated our findings by demonstrating the expected mass peak for SOX9 in the IP samples.

Conclusion and Future Implications

SOX9 was identified as a novel binding partner of mAKAP. As mAKAP and SOX9 already have a role in cardiac developmental biology, we suggest that mAKAP‐SOX9 interaction has substantial role in cardiac physiology and pathophysiology. The significance of this mAKAP‐SOX9 interaction is a part of our ongoing investigations.

Support or Funding Information

R15HL124458 Scaffolding Protein Interaction Network Regulates Cardiac Cell Signaling
Regulation of neuroinflammatory cytokines by Angiotensin and Cannabinoid systems in SHR astrocytes
Authors: Haspula Dhanush , Clark Michelle
Published: November 2023

Objective

To understand the distinct regulatory roles of astroglial cannabinoid and Angiotensin systems, in altering neuroinflammatory states in hypertension.

Background

Neuroinflammation in the brainstem is deemed as a major contributor to neurogenic hypertension. An imbalance in the levels of pro‐ and anti‐inflammatory cytokines, has been reported in the brains of spontaneously hypertensive rats (SHRs). These rats are a model of hypertension and Attention Deficit Hyperactivity Disorder (ADHD). Although cannabinoids exert potent neuroprotective and anti‐inflammatory effects, their role in regulation of cytokines in hypertension has not been investigated.

Methods

Astrocytes were isolated from the brainstem and cerebellum of Wistar rats and Spontaneously Hypertensive Rat (SHRs) pups. treatments were done with 100 nM Angiotensin (Ang) II or 10 nM of the potent Cannabinoid Type 1 receptor (CB1R) agonist‐ACEA, both alone and in combination, for varying time periods, the mRNA and secreted protein levels of interleukin 1 (IL1) and IL10 were measured using qPCR and ELISA, respectively.

Results

Both IL1 and IL10 basal levels were significantly higher in brainstem astrocytes, but not cerebellar astrocytes, of SHRs when compared to normotensive Wistar rat counterparts. Ang II treatment resulted in predominantly elevated IL1 and reduced IL10 levels in both brainstem and cerebellar astrocytes of SHRs and Wistar rats. However, only the Ang II‐mediated alterations in IL10 levels was significantly greater in brainstem astrocytes of SHRs when compared to Wistar rats. ACEA treatment on the other hand, was associated with a significant increase in IL10, and a slight reduction in IL1 levels, in both brainstem and cerebellar astrocytes of Wistar rats and SHRs. The effect of ACEA on IL10 was greater in brainstem astrocytes of Wistar rats when compared to SHRs. Co‐treatment of Ang II and ACEA resulted in a greater reduction of ACEA‐mediated increase in IL‐10 in brainstem astrocytes of SHR when compared to Wistar rats.

Conclusion

Significant differences in basal cytokine levels in brainstem astroglial cultures from SHRs and Wistar rats, indicates a dysregulated neuroinflammatory state in brainstem astrocytes under hypertensive conditions. Greater IL10 elevation, by ACEA, in brainstem astrocytes of Wistar rats when compared to SHRs, is indicative of endocannabinoid hypofunction in hypertension. The inability of CB1R to elicit a protective role in brainstem astrocytes under hypertensive states, could well be an important facet in the etiology of essential hypertension.

Support or Funding Information

NSU HPD: Grant # 335585
Regulation of the Functional Properties of Xeroderma Pigmentosum Complementation Group A (XPA) Protein through Lysine Acetylation
Authors: Njeri Catherine , Kaur Jasmeet , Turchi John , Balakrishnan Lata
Published: November 2023

Nucleotide Excision Repair (NER) is the main repair pathway through which DNA damages occurring on account of ultraviolet (UV) or ionizing radiation (IR) are repaired. Xeroderma pigmentosum complementation group A (XPA) protein is a crucial part of the NER multi‐protein complex that plays an indispensable role in recognizing and binding the damaged DNA, as well as positioning other NER proteins optimally for efficient repair of the injury. Studies have shown that XPA can be dynamically acetylated and deacetylated impacting its involvement in the NER repair pathway. Deacetylation of XPA by SIRT1 enhances its interaction with the single strand DNA (ssDNA) binding protein, replication protein A (RPA). Our current research is focused on evaluating the effects of lysine acetylation on the structural and functional properties of XPA. Our mass spectrometry analysis revealed that XPA is in vitro acetylated at multiple sites by p300 and CBP acetyltransferases. We evaluated the effects of acetylation on the DNA binding properties of XPA protein using electrophoretic‐mobility shift assays (EMSA) and bio‐layer interferometry (BLI) technology. Interestingly, our results show that acetylation of XPA significantly lowers its binding affinity for cisplatin damaged DNA. Similar to reports in vivo, analysis of protein‐protein interactions between XPA and RPA, revealed that acetylation of XPA reduces its ability to interact with RPA. Our results suggest that acetylation of XPA negatively affects its role in recognizing, binding and consequently repairing damaged DNA, which in turn may impact subsequent recruitment of RPA. By application, our results present a mechanistic approach towards negatively modulating the effectiveness of the NER pathway, with the aim of improving the efficacy of current chemotherapeutics.

Support or Funding Information

This work was supported by a grant from the National Institutes of Health GM0983289 (to LB)
G protein specificity of inhibitory adrenergic a2a receptor‐mediated modulation of synaptic transmission
Authors: Yim Yun Young , Betke Katherine , McDonald W. Hayes , Gilsbach Ralf , Chen Yunjia , Hyde Karren , Wang Qin , Hein Lutz , Hamm Heidi
Published: November 2023

Modulation of neurotransmitter exocytosis by activated Gi/o‐type G‐protein coupled receptors (GPCRs) is a universal regulatory mechanism used both to avoid overstimulation and to influence circuitry. One of the known modulation mechanisms is Gβγ and soluble N‐ethylmaleimide‐sensitive factor attachment protein receptor (SNARE) complex interaction. There are 5 Gβ and 12 Gγ subunits, but specific Gβγs activated by a given GPCR in vivo are not known. Presynaptic α2a‐adrenergic receptors (α2a‐ARs) in both adrenergic (auto α2a‐ARs) and non‐adrenergic neurons (hetero α2a‐ARs) inhibit neurotransmitter release and affect various physiological function such as anesthetic sparing and working memory enhancement. Here, we investigate whether auto α2a‐ARs in sympathetic neurons use the same Gbg subunits as hetero α2a‐ARs in other neuronal types to inhibit exocytosis by interacting with SNARE. Using several mice models including transgenic Flag‐α2a‐ARs, knock‐in HA‐α2a‐ARs, co‐immunoprecipitation, mass spectrometry analysis, we have determined the Gb and Gg subunits that interact with α2a‐ARs and SNARE complexes. So far, we find Gβ2 preferentially interacting with activated auto α2a‐ARs. We also see a basal Gβγ‐SNARE interaction and the 2 fold enhancement of this interaction upon the auto α2a‐ARs activation. Further understanding Gβγ specificity and Gβγ‐SNARE interaction may offer new insights into the normal functioning of the brain, as well as better understanding of disease progression.

Support or Funding Information

This work was supported by the National Institutes of Health (EY10291, MH101679, DK109204, and T32)
An internalization assay for Dopamine D2(short) Receptors using Bioluminescence Resonance Energy Transfer (BRET) technology
Authors: De Vries Luc , Finana Frederic , Cathala Claudie , Ronsin Brice , Cussac Didier , Gatti‐McArthur Silvia
Published: November 2023

Internalization and subsequent degradation of GPCRs located at the plasmamembrane represent a desired therapeutic approach to eliminate those receptors whose activation leads to unwanted cellular behaviour. In order to follow the trafficking of endocytosed receptors and thus the efficacy of agents directing these receptors into the internalization/degradation pathway, markers for their localisation at the plasmamembrane (K‐Ras), early (Rab5), recycling (Rab4) and late (Rab7) endosomes, and lysosomes (Rab7) have been described previously. We reasoned that such markers could be used in BRET biosensor assays (live cells) to time‐dependently follow the destination of internalized receptors and quantify the efficacy of agents that force the receptors into lysosomes. We therefore started by coexpressing the Renilla luciferase8‐tagged D2 dopamine receptor (D2‐R), with GFP2‐tagged K‐Ras, Rab5, Rab4 or Rab7 in CHO cells. We observed a basal BRET signal between D2‐R and K‐Ras that was robustly diminished by a 1 h agonist treatment (1 μM dopamine) and completely antagonized by 10 μM haloperidol treatment. At the same time point the BRET signals between D2‐R/Rab5, D2‐R/Rab4 and D2‐R/Rab7 were enhanced by the agonist and again neutralized by the antagonist. Using this technology we were thus able to follow the trafficking of the D2‐R from the plasmamembrane to the endosomal/lysosomal compartments. It also allowed us to perform a comparative pharmacological/functional study for different antipsychotic and anti‐Parkinson agents with affinities for the D2‐R receptor.
Glycosaminoglycans Inhibit Substance P‐ and IL‐33‐stimulated IL‐8 and TNF Release from Human‐Cultured Mast Cells
Authors: Gross Amanda Rae , Theoharides Theoharis C.
Published: November 2023

Glycosaminoglycans (GAGs) are long, linear, highly negatively charged carbohydrate chains that are known for their functional role in connective tissue, joint lubrication, and anticoagulant activity. Mast cells (MC) are hematopoietic tissue immune cells, involved in allergic and inflammatory reactions, which contain high amounts of GAGs, such as chondroitin sulfate (CS) and heparin, in their numerous secretory granules. In fact, heparin is synthesized and stored exclusively in MC granules and is thought to electrostatically store biogenic amines, such as histamine. A few previous studies reported that CS and heparin can inhibit the release of histamine from activated connective tissue rat and canine MC, respectively. However, the inhibitory potential of GAGs on human MC remains unknown. Here we report that CS had no effect on MC degranulation, as measured by beta‐hexosaminidase release, from LAD2 human‐cultured MC stimulated by the neuropeptide substance P (SP; 2 μM for 1 hr); however, pre‐incubation with CS did inhibit SP (1 μM for 24 hrs)‐induced IL‐8 and TNF release in a dose‐dependent manner. Specifically, CS 100 μg/mL inhibited IL‐8 and TNF release from SP‐stimulated LAD2 MC by 32% and 40%, respectively. Moreover, dermatan sulfate (DS) 5 μg/mL and heparin 40 μg/mL inhibited IL‐8 and TNF release from LAD2 MC stimulated by the cytokine IL‐33 (10 ng/mL for 24 hrs) by approximately 32% (p<0.05) and 39% (p<0.05), respectively. Similarly to heparin, at 37.5 μg/mL CS inhibited IL‐8 and TNF release from IL‐33‐stimulated LAD2 MC by approximately 36% and 43%, respectively. Preliminary, ongoing studies indicate that DS has an inhibitory effect on TNF gene expression at 6 hrs after IL‐33 stimulation. These results are important because IL‐33 is known to be an alarmin involved in allergic inflammation. GAGs from the tissue microenvironment or released by MC, themselves, upon activation, could inhibit the release of mediators from MC in a paracrine or autocrine fashion. It would be interesting to investigate the amount of these GAGs present in certain conditions involving allergic inflammation of the skin, such as atopic dermatitis, cutaneous mastocytosis, and psoriasis. DS may be formulated for local application, alone or in conjunction with other anti‐allergic molecules for the topical treatment of these diseases.

Support or Funding Information

Support for this project comes from the Tufts University Department of Integrative Physiology and Pathobiology (IPP) Fiscal Year 2016 Research Seed Funds.
Joint application of N‐Acetylcysteine and Allopurinol reduces spinal cord TNF‐alpha expression and attenuates mechanical allodynia in diabetic Rats
Authors: Li Sisi , Yan Dan , Gu Pan , Zhu Qiqi , Cheung Chi Wai , Irwin Michael G , Yu Cong , Xia Zhengyuan
Published: November 2023

Diabetic Neuropathy is a frequent and painful complication of diabetes that leads to progressive and length‐dependent Diabetic Peripheral Neuropathic Pain (DPNP). Studies have indicated the involvement of oxidative stress and the activation spinal pro‐inflammatory TNF‐alpha in the development of diabetic neuropathy. Joint application of the antioxidants N‐Acetylcysteine (NAC) and allopurinol (ALP) has been shown to confer superior protection against diabetic cardiomyopathy to their individual administration (Free Radic Biol Med. 2013; 63: 291–303). However, the potential effects and mechanism NAC and ALP combination on DPNP has not been explored. We hypothesized that NAC and ALP may reduce diabetes induced increase in central nervous system (CNS) TNF‐alpha expression and attenuate DPNP.

Control or streptozotocin (STZ, 65mg/kg)‐induced diabetic Sprague Dawley rats (n=5 per group) received vehicle or NAC (1.5g/kg/day) plus ALP (100mg/kg/day) in drinking water for three weeks starting one week after STZ injection. Paw withdrawal mechanical threshold (PWMT) and paw withdrawal thermal latency (PWTL) were measured weekly. At the end of the experiments (4 weeks after STZ injection), L3–L5 segments of spinal cord and somatosensory cortex were extracted for detecting TNF‐alpha protein expression by western blotting assay.

The results showed that the thresholds for PWMT and PWTL were reduced significantly in diabetic rats respectively at 1 and 2 weeks after diabetic induction as compared to non‐diabetic control (all p<0.05), which was associated with significant increase of spinal cord TNF‐alpha protection expression (p<0.05 diabetic vs. control) and moderate increase in cortex TNF‐alpha protection expression (P>0.05). Joint application of NAC and ALP significantly attenuated the reduction of thresholds for PWMT starting at two weeks of treatment and onwards (P<0.01 diabetic vs. Control) that was associated significant reduction and normalization of spinal cord TNF‐alpha expression (P<0.05, NAC+ALP vs. diabetes; p>0.05, NAC+ALP vs. control). However, NAC and ALP combination did not affect diabetic induced reduction in the threshold for PWTL, nor did it affect cortex TNF‐alpha protein expression.

It is concluded that reduction of spinal cord TNF‐alpha may represent a mechanism by which antioxidants NAC and ALP reduce mechanical allodynia in diabetic rats, and that diabetic thermal hyperalgesia may be mediated by mechanisms other than spinal cord TNF‐alpha at a relatively early stage of diabetes.
NHEJ and BER Concurrently Repair Oxidative DNA Damage via Initiation of APE1 in Rat Cortical Neurons
Authors: Yang Jenq‐Lin , Chen Yin‐Ping , Sun Shu‐Fang , Chen Shang‐Der
Published: November 2023

Base excision repair (BER) is the primary mechanism to repair oxidative, alkylated, deaminated, and single‐strand break DNA damage in eukaryotic cells, which is also commonly suggested to be the major machinery repairing oxidative DNA damage in neurons due to highly active metabolism companied with reactive oxygen species production. Many previous studies have suggested that neurons are deficient in other DNA repair pathways, including nucleotide excision repair and homologous recombination repair. However two earlier studies observed that neurons could efficiently repair glutamate‐ and menadione‐induced oxidative DNA damage. The current study has shown that markers of DNA damage and double‐stranded break, γH2AX and phosphorylated p53 binding protein 1 (p53BP1), after menadione‐produced oxidative insults. Moreover, both γH2AX and p53BP1 appeared and highly co‐localized with Apurinic/apyrimidinic endonuclease 1 (APE1) respectively after the oxidative DNA lesion. In nuclei of oxidative insult‐treated neurons, foci of p53BP1 were robustly abolished by suppression of APE1 endonuclease activity. Inhibition of DNA‐dependent protein kinase catalytic subunit (DNA‐PKcs) prolonged the DSB repair after oxidative insults. APE1 plays a key role in BER mechanism, but our results also indicated APE1 diverting BER concurrently works with non‐homologous end join (NHEJ) to repair oxidized DNA. Findings of this study explain the neurons are able to repair their nuclear DNA efficiently to cope with highly oxidative burden. APE1 expression is inducible via CREB‐mediated signaling pathway, which has been reported by many studies. Accordingly, elevating APE1 will enhance repair efficiency in oxidative DNA by both BER and NHEJ, which is a potential strategy for prevention or therapeutic intervention in ischemic stroke and neurodegenerative diseases.

Support or Funding Information

This research is supported by Ministry of Science and Technology Taiwan [grant number 103‐2320‐B‐182A‐011‐MY3 and 103‐2314‐B‐182‐026‐MY2].
Phytopharmacological Evaluation of Chamomile (Matricaria recutita) for Indirect Modulation of the Endocannabinoid System
Authors: El‐Alfy Abir T. , Manlimos Kristine , Patel Nidhi , Kendrek Jennifer , Abourashed Ehab
Published: November 2023

Anecdotal reports and small randomized clinical trials document a robust role of German chamomile (Matricaria recutita L.) in anxiety. However, the mechanism of such anxiolytic action remains unclear. Preliminary studies in our laboratory have shown that total chamomile extract significantly inhibited the endocannabinoid catabolizing enzymes, fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL). The goal of this study was to isolate and characterize the compounds responsible for such activity. With the significant role the endocannabinoid system plays in mood regulation, it is our hypothesis that chamomile extract contains compounds that interact with the endocannabinoid system and hence result in anxiolytic effect. Using a bioassay‐guided fractionation approach, two pure compounds (compounds A and B) were isolated, and spectroscopically and pharmacologically characterized. The compounds were isolated from the hexane fraction that demonstrated highest inhibition of FAAH enzyme (88.3%). Time and concentration inhibition curves were generated for both compounds. The IC50 values for compounds A and B were calculated as 0.32 ± 0.03 and 6.66 ± 2.58 mM, respectively. Time course inhibition studies showed that compound A sustained >50% FAAH inhibition up to 48h post treatment. On the other hand, the percent FAAH inhibition exerted by compound B dropped below 50% following 24h of treatment. The effect of compound A on total locomotor activity was evaluated in CD1 adult male mice, injected i.p. with the compound at doses of 20–120 mg/kg. The compound did not exert any significant effect on total locomotor activity, rearing, or exploratory behavior. In addition, compound A was evaluated in the elevated plus maze (EPM) animal model for anxiety using an automated infrared tracking system. The compound showed a dose‐dependent anxiolytic activity, as evidenced by the significant increase in open arm permanence time at 120 mg/Kg i.p. dose (p<0.05). In conclusion, the results of this study demonstrate that chamomile compounds could indirectly modulate the activity of the endocannabinoid system through inhibition of FAAH enzyme. Such activity sheds light on the potential mechanism of the reputed anxiolytic action of chamomile. Future studies will aim at further evaluation of both compounds in other models of anxiety, as well as isolating compounds responsible for MAGL inhibition activity observed with the total chamomile extract.
Effects of Chronic Pramipexole on AMPA Receptor Trafficking and Akt/GSK‐3β Signaling in a Rat Model of Parkinson's Disease
Authors: Kase Matthew Scott , Persons Amanda , Napier Celeste
Published: November 2023

The D3 receptor (D3R)‐preferring agonist pramipexole (PPX) is an effective treatment for motor symptoms in Parkinson's disease (PD). However, behavioral addictions associated with limbic systems emerge following chronic PPX administration. The D3R is involved in the addiction process, and one D3R‐mediated signaling cascade involves the Akt/GSK‐3β pathway. We previously demonstrated in limbic brain regions that acute PPX dephosphorylated Akt, which prevented phosphorylation of constitutively active GSK‐3β. Un‐phosphorylated GSK‐3β strengthens glutamatergic synapses via insertion of AMPA receptors (AMPAR) in neuronal membranes; a phenomenon associated with addiction. However, the effects of chronic PPX on this pathway remain unknown. Because PPX is often used as a therapeutic in early‐stage PD, clarification of these neuronal processes is highly relevant to clinicians. Here, we tested the hypothesis that chronic PPX can increase AMPAR surface expression in limbic brain regions of PD‐like rats. The neurotoxin 6OHDA was injected bilaterally into the dorsolateral striatum of rats to model PD (4μg/2μl); sham‐lesioned controls were injected with ascorbic acid vehicle (2μl). Twenty‐one days post‐lesion, rats were subcutaneously implanted with osmotic minipumps containing vehicle or PPX (1.2 mg/kg/day). The forelimb akinesia task was used to confirm PD‐like motor deficits and PPX efficacy in lesioned rats. After 14 days of PPX treatment, brain tissue was harvested. Modified Western blot protocols determined surface and intracellular levels of GluA1 (an AMPAR subunit), as well as ratios of pAkt/Akt and pGSK3β/GSK3β. In the nucleus accumbens and ventral pallidum, there was no effect of chronic PPX on pAkt/Akt or pGSK/GSK‐3β. In the medial prefrontal cortex, the ratios of pAkt/Akt and pGSK‐3β/GSK‐3β were significantly increased in PPX‐treated, PD‐like rats (ANOVA; p=0.0161 and p=0.0119, respectively), but surface/intracellular GluA1 was unchanged. Thus, while acute PPX promoted AMPAR trafficking, AMPAR surface expression appeared to normalize with chronic PPX. In addition to AMPAR, adaptation of excitatory synapses can be regulated by GSK‐3β‐linked changes in NMDA receptors. Thus, it is possible that changes in NMDA receptors may contribute to PPX‐induced behavioral addictions. Future studies will make this determination.

Support or Funding Information

Work supported by USPHSG #NS087559
Hyperinsulinemia Induced Changes in Chromatin Acetylation and Gene Expression in Triple Negative Breast Cancer
Authors: Senapati Parijat , Ann David K. , Seewaldt Victoria , Schones Dustin E.
Published: November 2023

Triple negative breast cancer (TNBC), characterized by lack of expression of the estrogen receptor (ER) and progesterone receptor (PR) and absence of HER2 amplification, is a particularly aggressive breast cancer subtype, constituting about 15–20% of all breast cancers. Premenopausal women of African descent have a high risk for TNBC, especially those with higher body mass index (BMI). Moreover, obesity associated hyperinsulinemia or insulin‐resistance is considered as a poor prognostic factor for TNBC patients. Insulin signaling can activate the PI3K/Akt/mTOR pathway that is associated with increased proliferation rate of TNBC cells. However, the mechanistic role of insulin resistance in promoting TNBC is unclear. We are particularly interested in studying how insulin signaling impinges on mitochondrial dysfunction and nuclear gene expression through the mTOR pathway. mTOR signaling activates oxidative phosphorylation by the mitochondria thereby potentially increasing Acetyl‐CoA production through the TCA cycle. Nuclear histone acetyltransferases utilize acetyl‐CoA as a substrate to acetylate histones, leading to increased chromatin accessibility and gene expression. We show here that insulin treatment to the MDA‐MB‐231 TNBC cell line leads to increases in global histone acetylation levels, in particular H3K9ac, and altered expression of several genes. Transcriptome analysis along with H3K9ac ChIP‐sequencing reveal rewiring of cells towards anabolic processes such as DNA and protein synthesis. Insulin treatment furthermore induced cell proliferation and survival related pathways. Future studies will be focused on studying the molecular mechanism of insulin induced chromatin hyperacetylation and possible reversal by metformin.
Nanoceria Dissolution and Carboxylic Acid Stabilization in Aqueous Dispersions
Authors: Yokel Robert A , Hancock Matthew L , Grulke Eric A , Unrine Jason M , Graham Uschi M
Published: November 2023

The objective was to determine whether carboxylic acids stabilize nanoceria in aqueous dispersions. Citric acid is used as a stabilizing agent for hydrothermal syntheses of nanoceria and for nanoceria dispersions. Nanoceria has been shown to have therapeutic potential for conditions with an oxidative stress/inflammation component including cancer, radiation damage, bacterial infection, sepsis, wounds, stroke‐induced ischemia, and retinal degeneration. It is used as an abrasive to prepare integrated circuits by chemical mechanical planarization and as a combustion catalyst in diesel fuel. Citrate‐coated nanoceria (average primary particle size ~ 4 nm (determined by electron microscopy; EM), hydrodynamic diameter of agglomerates in aqueous dispersions ~ 14 nm (determined by dynamic light scattering; DLS)) was incubated in multiple media for months. Nanoceria was introduced into 2 kD MWCO Slide‐A‐LyzerTM dialysis cassettes immersed in ~ 300 mOsm/L media. Samples from within and outside of the cassette were withdrawn for cerium quantitation by inductively coupled plasma mass spectrometry and high resolution electron microscopy (HR‐EM). Cerium quantitation, hydrodynamic diameter determination, and electron microscopic observations were repeatedly conducted. Nanoceria dissolution occurred at pH 4.5, shown by increased cerium in media surrounding the dialysis cassette and decreased primary particle size (1 to 3 nm, HR‐EM) within the cassette. Nanoceria agglomerates were ~ 10 to 15 nm in the presence of citric and malic acids and ammonium but grew to ~ 1 micron in media containing acetic, glutaric, tricarballylic, 3‐hydroxybutyric, adipic, or pimelic acid. In medium containing citrate, the nanoceria primary particle core retained its crystalline structure. Dissolution occurred at the nanoceria particle surface, which was rich in Ce(III); the antioxidant valence state. Cerium‐containing agglomerates (~ 10 nm comprised of 1 to 3 nm primary particles) were seen in the citric acid medium surrounding the dialysis cassette, suggesting diffusion of cerium through the dialysis cassette membrane followed by particle formation in the medium. In contrast, appreciable nanoceria dissolution did not occur in the presence of horseradish peroxidase and hydrogen peroxide at pH 6.2, a condition shown to degrade carbon nanotubes. By extension of the observations in acidic media, acidic environments, as found in phagolysosomes, may degrade nanoceria by dissolution. Carboxylic acids are known to form coordination complexes with metal ions. Formation of cerium‐chelate complexes after nanoceria dissolution may enable cerium redistribution within animals and uptake into plant roots. Ligands that can enable nanoceria dissolution in acidic environments greatly affect nanoceria's fate (dissolution rate and size).

Support or Funding Information

1R01GM109195
Structural and Inflammatory Alterations in the Respiratory Tract Following Chlorine Gas Inhalation in Mice
Authors: Malaviya Rama , Gardner Carol R , Laskin Jeffrey D , Laskin Debra L
Published: November 2023

Chlorine gas is a cytotoxic pulmonary irritant that causes acute damage to the upper and lower respiratory tract. In the present studies we developed a mouse model of chlorine inhalation exposure to investigate inflammatory mechanisms of lung injury. Spontaneously breathing male C57BL/6 mice were exposed to 325 ppm chlorine for 15 min in whole body chambers. Animals were sacrificed 24 h post‐exposure and bronchoalveolar lavage fluid (BAL) and lung tissue collected. Exposure of mice to chlorine resulted in peribronchial and perivascular edema, hemorrhage and marked congestion in the pulmonary parenchyma. Extensive loss of the epithelium was observed in the large bronchi resulting in denuded bronchial surfaces. This was correlated with increases in BAL cell and protein content, and bronchial epithelial expression of phospho‐histone H2A.X, a marker of double strand DNA breaks, indicating alveolar‐epithelial barrier injury. Chlorine exposure also resulted in increased numbers of lung epithelial cells and macrophages expressing cyclooxygenase and inducible nitric oxide synthase, proteins important in generating mediators involved in inflammatory lung injury. An increase in macrophages expressing Ym‐1+, which are important in wound healing and fibrosis, was also observed. These changes closely resemble the pathogenic response observed in humans following exposure to chlorine. Mechanistic understanding of chlorine toxicity in the respiratory tract will be useful in the identification of efficacious countermeasures for mitigating morbidity following exposure.

Support or Funding Information

Supported by NIH Grants U54AR055073, R01ES004738, and P30ES005022.
Inhibitory Role Of BMP4 On The PDGF‐Induced Proliferation And Collagen Synthesis In PASMCs
Authors: Kovacs Laszlo , Cai Pengcheng , Su Yunchao
Published: November 2023

Pulmonary arterial hypertension (PAH) is a severe lung disease characterized by remodeling of pulmonary vascular walls due to the abnormal vascular smooth muscle cell (VSMC) proliferation and accumulation of extracellular matrix proteins such as collagen. Our recent work shows that platelet‐derived growth factor‐BB (PDGF‐BB) induces cell proliferation and collagen synthesis in pulmonary artery SMC (PASMCs) via extracellular signal–regulated kinases (ERK)‐dependent calpain activation in PAH. The aim of the present study to determine the effect of bone morphogenetic protein 4 (BMP4) on PDGF‐stimulated calpain‐Transforming growth factor beta 1 (TGFβ1) signaling in PASMCs. PASMCs from normal and PAH patients were incubated with PDGF‐BB in the presence and absence of BMP4 for 0.5 to 24 h after which protein levels of collagen‐I, p‐Smad2/3, p‐Smad1/5, p‐ERK and intracellular active TGFβ1, calpain activity and cell proliferation were measured. We found that BMP4 augmented the protein levels of p‐Smad1/5 but did not influence the protein contents of collagen‐I, p‐Smad2/3, p‐ERK, active TGFβ1, and calpain activity in normal PASMCs. However, BMP4 attenuated the PDGF‐induced increases in calpain activity, cell proliferation and protein levels of collagen‐I, p‐Smad2/3, and active TGFβ1 in control cells. The results also showed that BMP4 did not alter the PDGF‐induced increases in p‐ERK or p‐Src. In addition, downregulation of Smad1/5 did not prevent the negative effects of BMP4 on the PDGF‐activated signal pathway. Furthermore, BMP4 increased the PKA activity and blocked the PDGF‐induced decrease in calpain‐2 phosphorylation at Ser369 in normal PASMCs. Inhibition of PKA prevented the inhibitory effects of BMP4 on calpain activation and dephosphorylation of Ser369 induced by PDGF. In addition, PKA activator forskolin recapitulated the inhibitory effect of BMP4 on PDGF‐stimulated calpain activation in normal PASMCs. In contrast, the suppressive effects of BMP4 on PDGF‐induced cell proliferation and collagen synthesis were abrogated in PASMCs from PAH patients. BMP4‐evoked PKA activation was diminished in PASMCs from PAH patients and BMP4 was not able to prevent the PDGF‐induced reduction in S369 phosphorylation and activation of calpain. Collectively, these data demonstrate that BMP4 inhibits PDGF‐induced cell proliferation and collagen synthesis via PKA‐mediated inhibition of calpain‐2 in normal PASMCs. Impairment of the BMP4‐PKA signaling may contribute to elevated calpain activation and pulmonary vascular remodeling in PAH.

Support or Funding Information

Supported by FAMRI CIA140083 (YS), Department of Veterans Affairs BX002035 (YS) and AHA 16POST27730023 (LK).
Proteasome activity is required for ribosomal DNA transcription and muscle hypertrophy
Authors: Chandler Paige Nicole , Liu Chang , von Walden Ferdinand , Nader Gustavo A
Published: November 2023

Skeletal muscle hypertrophy requires enhanced ribosome production, which is mediated by increased transcription of ribosomal DNA (rDNA) genes. Additionally, muscle growth necessitates that protein turnover is finely tuned to ensure a net protein accretion. However, how anabolic and catabolic processes coordinate muscle protein turnover remains to be determined.

The objective of this study was to determine the interaction between anabolic and catabolic mechanisms during myotube hypertrophy.

Myotubes were stimulated to undergo hypertrophy in the presence of proteolytic inhibitors for the ubiquitin proteasome (UPS, MG‐132), autophagy (ATG, Chloroquine), and caspase systems (CSP, Z‐VAD‐FMK). Inhibition of UPS, but not ATG or CSP, impaired muscle hypertrophy as evidenced by a 68% reduction in protein synthesis rates (p<0.001). Myotube protein content was also impaired by 25%, reflecting the decrease in UPS activity. Because hypertrophy requires increases in rDNA transcription, we considered the possibility that UPS inhibition impaired hypertrophy by disrupting rDNA transcription. Inhibition of UPS by MG‐132 resulted in impaired rDNA transcription, and subsequent analysis revealed that absence of functioning UPS resulted in altered nucleolar morphology in a pattern typical of nucleolar stress.

In order to identify potential mediator(s) of nucleolar stress, we determined whether inhibition of the UPS was associated with changes in p53 levels. UPS inhibition caused a significant increase in p53 levels, and direct pharmacological activation of p53 in the presence of a functioning UPS decreased myotube protein content by 25% (p<0.05) and impaired rDNA transcription by 25% (p<0.001).

These data indicate that UPS activity impinges in anabolic processes by blocking protein synthesis and rDNA transcription. Furthermore, UPS activity causes nucleolar stress and rDNA transcriptional suppression in a p53‐dependent manner.

Support or Funding Information

Supported by the Swedish Research Council (Vetenskapsradet VRK2008‐67X‐20797‐01‐04), The Pennsylvania State University, and The Huck Institute of the Life Sciences.
The role of IP‐10 in Chronic Fatigue Syndrome.
Authors: McArdle Anne , Gusnanto Arief , Earl Kate , Sakellariou George , Lawton Clare , Owens Daniel , Close Graeme , Beadsworth Michael , Dye Louise
Published: November 2023

Chronic fatigue syndrome (CFS) is a severely debilitating and complex illness of uncertain cause, characterised by prolonged, fatigue triggered by minimal activity. There is evidence that CFS is associated with chronic inflammation. Studies have shown that plasma levels of cytokines are chronically modified in patients with CFS. This study examined physiological, subjective and cognitive factors associated with plasma cytokine concentrations in a cohort of 92 patients compared with age and sex matched healthy controls. A sub‐group of patients and healthy controls (HCs) also underwent more detailed analyses of muscle function, cytokine production and cognitive function. Patients were diagnosed with CFS if they met the Oxford criteria for Chronic Fatigue Syndrome and recommended NICE guidelines. Patients completed a number of validated questionnaires including the Chalder Fatigue Questionnaire (CFQ) which is considered a valid and reliable measure of fatigue in patients with CFS.

Patients with CFS demonstrated a characteristic significant reduction in Maximal Voluntary Contraction Force compared with HCs. Data on plasma concentrations of 27 pro‐ and anti‐inflammatory cytokines were analysed using multiple or logistic regression with age and sex which were significant covariates included in each model. CFS was strongly associated with a limited number of cytokines. Diagnosis of CFS was associated with increased plasma contents of MIP‐1a, MIP‐1b and RANTES (p<0.05) and marginally with Eotaxin (p=0.07) when modelled individually. MVC and self‐reported fatigue both showed particularly strong associations with plasma IP‐10 concentrations. Muscle content of IP‐10 mRNA was significantly elevated, suggesting that, at least in part, muscle was a source of this IP‐10 but not the other cytokines. Pairwise associations between MVC and cytokines demonstrated that the reduced MVC seen in patients with CFS was strongly associated with plasma levels of IP‐10, TNF‐α and IL‐5. Further analyses revealed strong correlations between plasma RANTES and eotaxin levels and poorer verbal recall and RTs of patients with CFS. The consistent association of IP‐10 with the physiological features and of RANTES and eotaxin with the cognitive features of CFS provides compelling evidence for a role of these cytokine/chemokines in the physiological and cognitive pathology of CFS. This work was funded by the Medical Research Council.

Support or Funding Information

This work was funded by the Medical Research Council.