Intracellular Signaling Proteins Research Articles

Off-target activity of NBOMes and NBOMe analogs at the μ opioid receptor

New psychoactive substances (NPS) are introduced on the illicit drug market at a rapid pace. They are usually poorly pharmacologically characterized, leaving users unaware of the potential harms they could be exposed to. Apart from the unknown effects and potencies of these new substances, their exact molecular targets are often inadequately elucidated. Guided by recent literature, we investigated the μ opioid receptor (MOR) activation potential of a large set of psychedelics, which typically activate their target receptor, the serotonin (5-HT2A) receptor. Screening for MOR activity of a panel of psychedelics was done using a cell-based NanoBiT® bioassay. The NanoBiT® assay monitors the recruitment of the intracellular signaling protein β-arrestin2 to the ligand activated MOR receptor. The principle exploits functional complementation of a split nanoluciferase enzyme of which the inactive subunits are either fused to the receptor and βarr2. Receptor activation causes the 2 subunits to come in close proximity, resulting in restoration of the nanoluciferase activity. Upon addition of the substrate, a luminescent signal can be detected using a luminometer. Based on the results of this initial screening, MOR activity of a selected panel of NBOMes and NBOMe analogs was evaluated using the both the NanoBiT® and AequoScreen® assay, the latter measuring the rapid intracellular calcium flux caused by receptor activation, a more downstream effect caused by G protein signaling. Using the NanoBiT® assay, 5 NBOMes showed MOR activation potential. Based on these findings, a panel of NBOMe analogs was selected and evaluated using 2 distinct assays. Comparing both assays, the same overall trends regarding MOR activity could be determined. The iodo analog 25I-NBOMe was more potent than the bromo substituted 25B-NBOMe, which was more potent than the chloro analog 25C-NBOMe, signifying that MOR activity increased with an increased size of the halogen atom. Similarly, MOR activity increased with increasing size of the alkyl side chain, with the ethylated 25E-NBOMe being more active than the methylated 25D-NBOMe. However, a propyl or isopropyl side chain might be too long or bulky, as the 25P-NBOMe and 25iP-NBOMe showed a substantial decrease in activity. NBOMe compounds were more active at MOR than their NBOH counterparts, whereas earlier data showed that they had a similar activity at their designated target receptor 5-HT2A. Moving the methoxy group from position 2 to position 3 or 4 resulted in an important decrease in MOR activity. Furthermore, the effects at MOR of the analyzed compounds could be blocked by the opioid antagonist naloxone, suggesting that these NBOMes occupy the same common opioid binding pocket as conventional opioids. Using the AequoScreen® assay, aspecific, receptor independent effects were found for the alkylated 25iP-NBOMe, 25E-NBOMe and 25D-NBOMe, indicating that these may directly interact with other targets in the downstream signaling pathway. A limited potential to activate the MOR was found for 10 NBOMes and NBOMe analogs, which was confirmed using 2 distinct cell-based assays. Structure-activity relationships could be determined, and additional experiments revealed that the effects of these psychedelics at MOR could be antagonized by naloxone, signifying that these substances occupy the same binding pocket as common opioids. Some compounds showed an aspecific effect in the AequoScreen® assay, which was not observed using the NanoBiT® assay. As MOR activity of these psychedelics was only noticed at high concentrations, it is unlikely that these will contribute to pronounced opioid toxicity at physiologically relevant concentrations. However, small modifications to the original NBOMe structure may result in a panel of more efficacious and potent MOR agonists.

A Broad-Based Characterization of a Cell-Penetrating, Single Domain Camelid Bi-Specific Antibody Monomer That Targets STAT3 and KRAS Dependent Cancers.

STAT3 and KRAS regulate cell proliferation, survival, apoptosis, cell migration, and angiogenesis. Aberrant expression of STAT3 and mutant active forms of KRAS have been well-established in the induction and maintenance of multiple cancers. STAT3 and KRAS mutant proteins have been considered anti-cancer targets; however, they are also considered to be clinically “undruggable” intracellular molecules, except for KRAS(G12C). Here we report a first-in-class molecule, a novel, single domain camelid VHH antibody (15 kDa), SBT-100, that binds to both STAT3 and KRAS and can penetrate the tumor cell membrane, and significantly inhibit cancer cell growth. Additionally, SBT-100 inhibits KRAS GTPase activity and downstream phosphorylation of ERK in vitro. In addition, SBT-100 inhibits the growth of multiple human cancers in vitro and in vivo. These results demonstrate the feasibility of targeting hard-to-reach aberrant intracellular transcription factors and signaling proteins simultaneously with one VHH to improve cancer therapies.

Anti-histamine effects of dipotassium glycyrrhizinate on lung fibroblasts, implicating its therapeutic mechanism for pulmonary fibrosis.

To examine the possible anti-histamine effects of dipotassium glycyrrhizinate (DG), a dipotassium salt of glycyrrhizic acid, on histamine-mediated lung fibroblast activation, differentiation and proliferation; to investigate the potential and underlying mechanisms for pulmonary fibrosis (PF) treatment. Rat primary lung fibroblasts were extracted to establish cell models; histamine, DG and loratadine (LTD, a histamine receptor antagonist) were applied. Cell proliferation, migration and cell cycle were explored; intracellular signal proteins were detected; mitochondrial membrane potential was examined. The anti-histamine effects of DG were found in a similar pattern of LTD on lung fibroblasts. DG inhibited histamine-induced cell activation, proliferation and migration; DG altered histamine-mediated mitochondrial membrane potentials. DG reduced the histamine-induced PAR-2 (a tryptase receptor) expression to impair mast cell tryptase co-working. Histamine-induced expressions of MMP-2, FAK, TNF-α, P38, iNOS were decreased by DG, while Bax and caspase-3, P53 were increased by DG against histamine effects. Histamine drove cells from G0/G1 to S phases, whereas DG rested cells by inhibiting G0/G1 and G2/M phases. This study provided the evidences that DG can inhibit histamine-induced effects on lung fibroblasts and promote apoptosis of abnormally activated lung fibroblasts, implicating its potential therapeutic mechanisms against PF development, also for those histamine-related diseases.

Physiologic and pathophysiologic roles of AKAP12.

A kinase anchoring protein (AKAP) 12 is a scaffolding protein that improves the specificity and efficiency of spatiotemporal signal through assembling intracellular signal proteins into a specific complex. AKAP12 is a negative mitogenic regulator that plays an important role in controlling cytoskeletal architecture, maintaining endothelial integrity, regulating glial function and forming blood-brain barrier (BBB) and blood retinal barrier (BRB). Moreover, elevated or reduced AKAP12 contributes to a variety of diseases. Complex connections between AKAP12 and various diseases including chronic liver diseases (CLDs), inflammatory diseases and a series of cancers will be tried to delineate in this paper. We first describe the expression, distribution and physiological function of AKAP12. Then we summarize the current knowledge of different connections between AKAP12 expression and various diseases. Some research groups have found paradoxical roles of AKAP12 in different diseases and further confirmation is needed. This paper aims to assess the role of AKAP12 in physiology and diseases to help lay the foundation for the design of small molecules for specific AKAP12 to correct the pathological signal defects.

Abstract 853: The biological role and mechanisms of XPO1 R749Q mutation in colon cancer

Abstract Exportin-1 (XPO1) is known to be overexpressed or mutated in several cancer types. XPO1 is the target of selinexor, which has received approval in hematologic cancers but not in solid tumors. We recently performed a large-scale genomic analysis of 42,793 patients with cancer spanning 322 cancer types (Taylor et. al. Cancer Discovery 2019). We identified a highly statistically significant hotspot mutation in XPO1 R749Q occurring mainly in colorectal cancer patients. We have generated 2 different isogenic colorectal cancer cell lines bearing the XPO1 R749Q mutation (HCT116 and LS174T) using CRISPR-CAS9. We observed that in vitro and in vivo, these cell lines did not reveal a role for XPO1 R749Q on cell growth and proliferation. A chemical compound library screen of >200 FDA-approved cancer therapies revealed a strong therapeutic resistance of XPO1 R749Q cells relative to wildtype (XPO1 WT) cells, specifically to chemotherapies used in the treatment of colon cancer. However, XPO1 R749Q remained sensitive to inhibition with the selective XPO1 inhibitor selinexor. Mass spectrometry analysis of nuclear and cytoplasmic fractionated proteins revealed XPO1 R749Q mutant cells had increased export of proteins from the nucleus compared to XPO1 WT cells. Upon ionizing radiation or chemotherapy, XPO1 R749Q cells had less DNA damage as measured by comet assay and γ-H2Ax immunofluorescence. XPO1 R749Q cells are resistant to chemotherapy but respond to selinexor in vitro. Selinexor effectively blocked nuclear export of canonical cargoes of XPO1 in XPO1 R749Q and XPO1 WT cells. The combination of selinexor and irinotecan showed high levels of synergy in vitro. In vivo observations in mice xenografted with XPO1 R749Q mutant HCT116 cells showed moderate tumor response to selinexor or irinotecan monotherapies but prolonged tumor responses to combination therapy. Single-cell intracellular signaling protein analysis using the Isoplexis platform discovered increased mTOR signaling in the XPO1 R749Q vs. WT cells. Western blot validation confirmed increased mTOR signaling in XPO1 R749Q mutants. In conclusion, our current study sheds novel insights into therapeutic targeting of cancers where XPO1 mutations occur, such as colorectal cancer. Specifically, these mutations confer resistance to DNA-damaging therapies such as chemotherapy and radiation; however, they may predict for sensitivity to targeted agents such as XPO1 inhibition. We have generated unique genetic models for study of XPO1 that are currently lacking. Our study established the biological and mechanistic consequences of XPO1 R749Q mutations in cancer, thereby having a significant positive impact on the treatments of patients with cancer. Further work is ongoing for the identification of cargo proteins transported by mutated XPO1 that connect the effects of XPO1 R749Q mutation on nuclear export to response to DNA damage, mTOR signaling and XPO1 inhibition in tumors in vitro and in vivo. Citation Format: Tulasigeri M. Totiger, Monika Chojnacka, Jumana Afaghani, Sana Chaudhry, Skye Montoya, Maurizio Affer, Justin Taylor. The biological role and mechanisms of XPO1 R749Q mutation in colon cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 853.

Orphan Nuclear Receptor NR4A2 Is Constitutively Expressed in Cartilage and Upregulated in Inflamed Synovium From hTNF-Alpha Transgenic Mice.

Orphan nuclear receptor 4A2 (NR4A2/Nurr1) is a constitutively active transcription factor with potential roles in the onset and progression of inflammatory arthropathies. NR4A2 is overexpressed in synovium and cartilage from individuals with rheumatoid arthritis (RA), psoriatic arthritis, and osteoarthritis. This study documents the expression and tissue localization of NR4A2 and upstream regulator nuclear factor kappa B (NF-κB) in the human tumor necrosis factor-alpha (hTNF-α) transgenic mouse model of RA. Since TNF-α is a potent inducer of NR4A2 in vitro, we hypothesized that NR4A2 would also be upregulated and active during disease progression in this model. Expression levels of NR4A2, related receptors NR4A1 (Nur77) and 3 (NOR1), and NF-κB1 transcripts were quantified by RT-qPCR in hTNF-α and wild-type joints at three stages of disease. The protein distribution of NR4A2 and NF-κB subunit RelA (p65) was analyzed by quantitative immunohistochemistry. Global gene expression of 88 RA-related genes was also screened and compared between groups. Consistent with previous reports on the hTNF-α model, transgenic mice exhibited significant weight loss and severely swollen paws by 19 weeks of age compared to age-matched wild-type controls. NR4A1-3 and NF-κB1 were constitutively expressed at disease onset and in healthy joints. NF-κB1 transcript levels increased 2-fold in hTNF-α paws with established disease (12 weeks), followed by a 2-fold increase in NR4A2 at the late disease stage (19 weeks). NR4A2 and RelA proteins were overexpressed in inflamed synovium prior to symptoms of arthritis, suggesting that gene expression changes documented in whole paws were largely driven by elevated expression in diseased synovium. Broader screening of RA-related genes by RT-qPCR identified several differentially expressed genes in hTNF-α joints including those encoding inflammatory cytokines and chemokines, matrix-degrading enzymes and inhibitors, cell surface receptors, intracellular signaling proteins and transcription factors. Consensus binding sites for NR4A receptors and NF-κB1 were enriched in the promoters of differentially expressed genes suggesting central roles for these transcription factors in this model. This study is the first comprehensive analysis of NR4A2 in an animal model of RA and validates the hTNF-α model for testing of small molecules and genetic strategies targeting this transcription factor.

Overexpression of Tetraspanin31 contributes to malignant potential and poor outcomes in gastric cancer.

Tetraspanin has important functions in many cancers by aggregating with various proteins that interact with intracellular signaling proteins. The molecular function of Tetraspanin31 (TSPAN31), located in the 12q14 amplified region in various cancers, remains unclear in gastric cancer (GC). We tested whether TSPAN31 acts as a cancer‐promoting gene through its activation or overexpression in GC. We analyzed seven GC cell lines and 189 primary tumors, which were curatively resected in our hospital between 2011 and 2013. Overexpression of the TSPAN31 protein was frequently detected in three GC cell lines (42.9%) and 62 primary GC specimens (32.8%). Overexpression of TSPAN31 was significantly correlated with lymphatic invasion, venous invasion, more advanced pT and pN stages, and a higher recurrence rate. Moreover, TSPAN31 positivity was an independent factor predicting worse patient outcomes (p = 0.0283, hazard ratio 3.97). Ectopic overexpression of TSPAN31 facilitated cell proliferation of GC cells, and knockdown of TSPAN31 inhibited cell proliferation, migration, invasion, and epithelial–mesenchymal transition of GC cells through the PI3K‐Akt pathway and increased cell apoptosis in a TP53 mutation‐independent manner. In vivo analysis also revealed knockdown of TSPAN31 suppressed tumor progression. In addition, knockdown of TSPAN31 improved chemosensitivity to cisplatin through the suppression of ABCC2. These findings suggest that TSPAN31 plays a crucial role in tumor‐malignant potential through overexpression, highlighting its utility as a prognostic factor and a potential therapeutic target in GC.

An endothelial proinflammatory phenotype precedes the development of the engraftment syndrome after autologous Hct.

Engraftment syndrome (ES) is a common complication after autologous hematopoietic cell transplantation (auto-HCT) whose pathophysiological substrate remains unclear. We investigated whether endothelial damage could contribute to ES. Circulating ECs-damage biomarkers were measured in plasma from patients with (ES; n = 14) or without ES (non-ES; n = 20), collected at different time points: before HCT, 5 (S5) and 10 days (S10) after HCT, and at either the ES onset (SON) or the discharge day (SDIS). Also, cultured endothelial cells (ECs) were exposed to serum samples, obtained at the same points, to evaluate changes in ECs-activation (ICAM-1, VE-Cadherin) biomarkers, the reactivity of ECs towards leukocytes, and activation of intracellular signaling proteins related to inflammation (p38MAPK) and proliferation (Erk1/2). Results showed that circulating VWF, sTNFR1 and sVCAM-1 levels were higher in ES patients at all the points assessed, especially at SON. In vitro results showed an increased ICAM-1 expression on ECs exposed to ES samples vs. non-ES samples, especially to S5, with elevated leukocyte adhesion. Also, a lower VE-Cadherin expression and an increased phosphorylation of p38MAPK and Erk1/2 proteins were observed in ECs exposed to ES vs. non-ES samples. Our results indicate that endothelial activation precedes ES development and could be one of its pathophysiological substrates.

High Interferon Signature Leads to Increased STAT1/3/5 Phosphorylation in PBMCs From SLE Patients by Single Cell Mass Cytometry.

The establishment of an “interferon (IFN) signature” to subset SLE patients on disease severity has led to therapeutics targeting IFNα. Here, we investigate IFN signaling in SLE using multiplexed protein arrays and single cell cytometry by time of flight (CyTOF). First, the IFN signature for SLE patients (n=81) from the Stanford Lupus Registry is determined using fluidigm qPCR measuring 44 previously determined IFN-inducible transcripts. IFN-high (IFN-H) patients have increased SLE criteria and renal/CNS/immunologic involvement, and increased autoantibody reactivity against spliceosome-associated antigens. CyTOF analysis is performed on non-stimulated and stimulated (IFNα, IFNγ, IL-21) PBMCs from SLE patients (n=25) and HCs (n=9) in a panel identifying changes in phosphorylation of intracellular signaling proteins (pTOF). Another panel is utilized to detect changes in intracellular cytokine (ICTOF) production in non-stimulated and stimulated (PMA/ionomycin) PBMCs from SLE patients (n=31) and HCs (n=17). Bioinformatic analysis by MetaCyto and OMIQ reveal phenotypic changes in immune cell subsets between IFN-H and IFN-low (IFN-L) patients. Most notably, IFN-H patients exhibit increased STAT1/3/5 phosphorylation downstream of cytokine stimulation and increased phosphorylation of non-canonical STAT proteins. These results suggest that IFN signaling in SLE modulates STAT phosphorylation, potentially uncovering possible targets for future therapeutic approaches.

Rho GTPase gene expression and breast cancer risk: a Mendelian randomization analysis

The Rho GTPase family consists of 20 genes encoding intracellular signalling proteins that influence cytoskeletal dynamics, cell migration and cell cycle progression. They are implicated in breast cancer progression but their role in breast cancer aetiology is unknown. As aberrant Rho GTPase activity could be associated with breast cancer, we aimed to determine the potential for a causal role of Rho GTPase gene expression in breast cancer risk, using two-sample Mendelian randomization (MR). MR was undertaken in 122,977 breast cancer cases and 105,974 controls, including 69,501 estrogen receptor positive (ER+) cases and 105,974 controls, and 21,468 ER negative (ER−) cases and 105,974 controls. Single nucleotide polymorphisms (SNPs) underlying expression quantitative trait loci (eQTLs) obtained from normal breast tissue, breast cancer tissue and blood were used as genetic instruments for Rho GTPase expression. As a sensitivity analysis, we undertook co-localisation to examine whether findings reflected shared causal variants or genomic confounding. We identified genetic instruments for 14 of the 20 human Rho GTPases. Using eQTLs obtained from normal breast tissue and normal blood, we identified evidence of a causal role of RHOD in overall and ER+ breast cancers (overall breast cancer: odds ratio (OR) per standard deviation (SD) increase in expression level 1.06; (95% confidence interval (CI) 1.03, 1.09; P = 5.65 × 10–5) and OR 1.22 (95% CI 1.11, 1.35; P = 5.22 × 10–5) in normal breast tissue and blood respectively). There was a consistent direction of association for ER− breast cancer, although the effect-estimate was imprecisely estimated. Using eQTLs from breast cancer tissue and normal blood there was some evidence that CDC42 was negatively associated with overall and ER + breast cancer risk. The evidence from colocalization analyses strongly supported our MR results particularly for RHOD. Our study suggests a potential causal role of increased RHOD gene expression, and, although the evidence is weaker, a potential protective role for CDC42 gene expression, in overall and ER+ breast cancers. These finding warrant validation in independent samples and further biological investigation to assess whether they may be suitable targets for drug targeting.

Essential function of adaptor protein Nck1 in platelet-derived growth factor receptor signaling in human lens epithelial cells

Binding of platelet-derived growth factor-BB (PDGF-BB) to its cognate receptor (PDGFR) promotes lens epithelial cell (LEC) proliferation and migration. After cataract surgery, these LEC behaviors have been proposed as an influential cause of posterior capsule opacification (PCO). Stimulated PDFGR undergoes dimerization and tyrosine phosphorylation providing docking sites for a SH2-domain-containing noncatalytic region of tyrosine kinase (Nck). Nck is an adaptor protein acting as a linker of the proximal and downstream signaling events. However, the functions of Nck1 protein in LEC have not been investigated so far. We reported here a crucial role of Nck1 protein in regulating PDGFR-mediated LEC activation using LEC with a silenced expression of Nck1 protein. The knockdown of Nck1 suppressed PDGF-BB-stimulated LEC proliferation and migration and disrupted the cell cycle progression especially G1/S transition. LEC lacking Nck1 protein failed to exhibit actin polymerization and membrane protrusions. The downregulation of Nck1 protein in LEC impaired PDGFR‐induced phosphorylation of intracellular signaling proteins, including Erk1/2, Akt, CREB and ATF1, which resulted in inhibition of LEC responses. Therefore, these data suggest that the loss of Nck1 expression may disturb LEC activation and Nck1 may potentially be a drug target to prevent PCO and lens-related disease.

Dipotassium Glycyrrhizinate Inhibits Histamine-Induced Proliferation by Suppressing Cell Cycle and P38/FAK Pathway of Lung Cancer Cells In-Vitro

Histamine is released from activated mast cells which have been found around tumor tissue area. The aim of this study was to examine the function of histamine on human lung cancer cell line A549 cell; to investigate the anti-histamine effects of dipotassium glycyrrhizinate (DG) on histamine mediated proliferation and migration of A549 cells and to explore the potential inhibitory mechanism of DG on lung cancer cell metastasis. The in-vitro histamine/A549 cells model was established, the cell proliferation, migration and cell cycle were examined, and the intracellular glutathione (GSH) level and signal proteins were detected. Data showed that histamine promote cell viability, proliferation and migration, while DG inhibited the histamine-induced cell proliferation and migration. DG decreased histamine-induced expressions of FAK, matrix metalloproteinases-2/9 (MMP-2/9), P38, PCNA, and Bcl-2, and DG up-regulated Bax, P53 and Becline-1 expressions against histamine’s effects. DG altered the histamine-driven M-phase in cell cycle and decreased the histamine-elevated GSH content in A549 cells, leading to cell apoptosis. This study revealed that DG inhibited the histamine-induced cell proliferation and promoted cell apoptosis of A549 cells, implicating its potential therapeutic mechanisms on lung cancer cell proliferation, metastasis and development.

Signaling Pathways that Regulate Macropinocytosis in Mammalian Cells.

Macropinocytosis is an evolutionarily conserved endocytic pathway that mediates non-selective uptake of extracellular fluid in bulk. Macropinocytosis is initiated by localized polymerization of the actin cytoskeleton, which generates plasma membrane protrusions that enclose part of the environment into large endocytic vesicles. From amoebae to mammalian cells, the actin dynamics that drive macropinosome formation are regulated by a conserved set of intracellular signaling proteins including Ras superfamily GTPases and PI3-kinases. In mammalian cells, multiple upstream signaling pathways control activity of these core regulators in response to cell-extrinsic and cell-intrinsic stimuli. Growth factor signaling pathways play a central role in macropinocytosis induction. In addition, an increasing number of functionally diverse processes has been identified as macropinocytosis regulators, including several nutrient-sensing and developmental signaling pathways. Many of these signaling pathways have proto-oncogenic properties, and their dysregulation drives the high macropinocytic activity that is commonly observed in cancer cells. These regulatory principles illustrate how macropinocytosis is controlled by complex upstream inputs to exert diverse cellular functions in physiological and pathological contexts.

Interleukin-17 Cytokines and Receptors: Potential Amplifiers of Tendon Inflammation.

Interleukin (IL)-17A, a pro-inflammatory cytokine that is linked to the pathology of several inflammatory diseases, has been shown to be upregulated in early human tendinopathy and to mediate inflammatory and tissue remodelling events. However, it remains unclear which cells in tendons can respond to IL-17A, and how IL-17A, and its family members IL-17F and IL-17AF, can affect intracellular signalling activation and mRNA expression in healthy and diseased tendon-derived fibroblasts. Using well-phenotyped human tendon samples, we show that IL-17A and its receptors IL-17RA and IL-17RC are present in healthy hamstring, and tendinopathic and torn supraspinatus tendon tissue. Next, we investigated the effects of IL-17A, IL-17F, or IL-17AF on cultured patient-derived healthy and diseased tendon-derived fibroblasts. In these experiments, IL-17A treatment significantly upregulated IL6, MMP3, and PDPN mRNA expression in diseased tendon-derived fibroblasts. IL-17AF treatment induced moderate increases in these target genes, while little change was observed with IL-17F. These trends were reflected in the activation of intracellular signalling proteins p38 and NF-B p65, which were significantly increased by IL-17A, modestly increased by IL-17AF, and not increased by IL-17F. In combination with TNF-α, all three IL-17 cytokines induced IL6 and MMP3 mRNA expression to similar levels. Therefore, this study confirms that healthy and diseased tendon-derived fibroblasts are responsive to IL-17 cytokines and that IL-17A induces the most profound intracellular signalling activation and mRNA expression of inflammatory genes, followed by IL-17AF, and finally IL-17F. The ability of IL-17 cytokines to induce a direct response and activate diverse pro-inflammatory signalling pathways through synergy with other inflammatory mediators suggests a role for IL-17 family members as amplifiers of tendon inflammation and as potential therapeutic targets in tendinopathy.

Proteasome Inhibition Attenuates Self-Renewal in Human Acute Myeloid Leukemia By Targeting NF-Kappa B in Leukemia Stem Cells

In acute myeloid leukemia (AML), relapse following standard chemotherapy is common, leading to 2-year survival rates of less than 30%. Relapse is caused by leukemia stem cells (LSCs), a rare population of mostly quiescent cells that are chemo-refractory and can recapitulate the disease. Our overall goal is to define mechanisms of self-renewal that could be targeted to cure AML and prevent relapse. Functionally, self-renewal is defined by the ability to propagate leukemia in vivo.We use transcriptional and protein profiling to define the functional states of LSCs. We previously demonstrated that NRAS G12V facilitates self-renewal in a mouse model of AML (Mll-AF9/NRAS G12V). Using single-cell RNA sequencing and in vivo leukemia assays, we showed that the stem cell compartment in this model has two distinct subpopulations which differ in their self-renewal and proliferative abilities. The subset of LSCs marked by CD36 LowCD69 High (CD69 High) expression can self-renew and are poorly proliferative. The CD36 HighCD69 Low (CD36 High) subset is unable to self-renew and is highly proliferative. These data demonstrated that self-renewal and rapid proliferation are mutually exclusive functions among LSCs. We demonstrated that the gene expression profiles associated with these functionally distinct LSC subsets are also similarly differentially expressed in primary human LSCs, at the single-cell level (Sachs Cancer Research 2020). Given the functional differences between the CD36 LowCD69 High and CD36 HighCD69 Low subsets in murine AML, we tested whether CD69 and CD36 likewise discriminate self-renewal and proliferation in human AML. We sorted primary human AML samples according to CD69 and CD36 expression and found that Lin -CD69 High cells formed more colonies than Lin -CD36 High cells in every sample tested (n=6). Notably, in three of the six samples, the Lin -CD36 High population was unable to form any colonies (Panel A). These data suggest that CD69 may identify a self-renewing subset of human AML as it does in murine AML. Next, we asked whether Lin -CD69 High and Lin -CD36 High subsets harbor unique signaling protein activation profiles that could potentially be targeted therapeutically. We used CyTOF (mass cytometry) to compare the levels of 12 intracellular signaling proteins between these subpopulations in 14 human AML samples with intermediate and poor risk genetics. CyTOF quantitatively measures a panel of proteins at the single-cell level and allows us to profile activated signaling pathways within well-defined immunophenotypic subpopulations. We found upregulation of both total and phosphorylated (t- and p-) NF-kappa B, pERK, p4EBP1, pMAPKAPKII, and its upstream activator, pP38 in the Lin -CD69 High subset relative to the Lin -CD36 High. Notably, CD69 is a NF-kappa B target gene. Indeed, our single-cell transcriptional data showed that NF-kappa B target genes were upregulated in the self-renewing, CD69 High subset of murine and human LSCs (Sachs Cancer Research 2020). NF-kappa B is a well-known mediator of AML-self-renewal. Next, we asked whether targeting NF-kappa B with proteasome inhibitors might reduce self-renewal capacity in human AML. We plated six primary human samples in colony-forming assays with pan-proteasome inhibitor, carfilzomib, immunoproteasome inhibitor, PR957, or vehicle and found that both inhibitors reduced colony formation in every sample tested. We harvested colonies from each of the inhibitor and vehicle groups and plated them in secondary colony-forming assays. Colonies harvested from vehicle-treated samples formed colonies but those harvested from either carfilzomib or PR957-treated samples were unable to form any secondary colonies (Panel B). Using CyTOF, we validated that both inhibitors reduce NF-kappa B levels in these samples.These data demonstrate that CD69 marks a self-renewing subset of human AML, as it does in our murine AML model, and suggest that the transcriptional mechanisms of self-renewal that we defined in our murine model at the single-cell level may be shared with human AML. Additionally, these data demonstrate enhanced NF-kappa B levels in this self-renewing subset and that targeting the NF-kappa B pathway with proteasome inhibitors attenuates self-renewal in primary human AML samples (Panel C). These findings suggest that proteasome inhibition may be an effective approach to treating and preventing relapse in AML. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Photobiomodulation Improves the Inflammatory Response and Intracellular Signaling Proteins Linked to Vascular Function and Cell Survival in the Brain of Aged Rats.

Photobiomodulation is a non-pharmacological tool widely used to reduce inflammation in many tissues. However, little is known about its effects on the inflammatory response in the aged brain. We conducted the study to examine anti-inflammatory effects of photobiomodulation in aging brains. We used aged rats (20months old) with control (handled, laser off) or transcranial laser (660nm wavelength, 100 mW power) treatments for 10 consecutive days and evaluated the level of inflammatory cytokines and chemokines, and the expression and activation of intracellular signaling proteins in the cerebral cortex and the hippocampus. Inflammatory analysis showed that aged rats submitted to transcranial laser treatment had increased levels of IL-1alpha and decreased levels of IL-5 in the cerebral cortex. In the hippocampus, the laser treatment increased the levels of IL-1alpha and decreased levels of IL-5, IL-18, and fractalkine. Regarding the intracellular signaling proteins, a reduction in the ERK and p38 expression and an increase in the STAT3 and ERK activation were observed in the cerebral cortex of aged rats from the laser group. In addition, the laser treatment increased the hippocampal expression of p70S6K, STAT3, and p38 of aged rats. Taken together, our data indicate that transcranial photobiomodulation can improve the inflammatory response and the activation of intracellular signaling proteins linked to vascular function and cell survival in the aged brain.

Вплив цинку на метаболічні процеси в нормі та при патології

Zinc belongs to the independent human trace elements. Its biological role is taken into account with the activity of attracting endocrine secretion. As a component of some enzymes, zinc affects the processes of reproduction, live and carbohydrate metabolism. Zinc is an important trace element with a variety of biological functions, which is involved in cell growth, apoptosis and metabolism, as well as in the regulation of endocrine, immune and neuronal functions. At the cellular level, zinc stimulates the formation of polysomes, inhibits free radical oxidation, which is catalyzed by iron. The important role of zinc in nucleic metabolism, transcription processes, stabilization of nucleic acids, proteins and especially components of biological membranes. Zinc plays an important role in skeletal development. Zinc accelerates the synthesis of collagen in wounds, has a stabilizing effect on cytoplasmic membranes, and prevents the release of hydrolytic enzymes. In mitochondria, zinc inhibits the transport of electrons in the respiratory chain. This property is realized in sperm, where there is a lot of zinc and there is low respiratory activity of mitochondria. Zinc is necessary for the development of brain tissue. Zincergic neurons are found in the cerebral cortex. Zinc has been shown to play an important role in signal transduction by modulating excitatory and inhibitory cellular receptors and intracellular signaling proteins. Zinc has a special effect on the development and course of many diseases, such as Down syndrome, alcoholism, angina, dysmenorrhea, atherosclerosis and more.

Multiplexed Single-Cell Plasmonic Immunoassay of Intracellular Signaling Proteins Enables Non-Destructive Monitoring of Cell Fate.

It is of significant importance in cancer biology to identify signaling pathways that play key roles in cell fate determination. Dissecting cellular signaling pathways requires the measurement of a large number of signaling proteins. However, tools for simultaneously monitoring multiple signaling pathway components in single living cells remain limited at present. Herein, we describe an approach, termed multiplexed single-cell plasmonic immunosandwich assay (mxscPISA), for simultaneous detection of multiple signaling proteins in individual living cells. This approach enabled simultaneous non-destructive monitoring of multiple (up to five, currently the highest multiplexing capacity in living cells) cytoplasmic and nucleus signaling proteins in individual cells with ultrahigh detection sensitivity. As a proof of principle, the epidermal growth factor receptor (EGFR) pathway, which plays a central role in cell fate determination, was investigated using this approach in this study. We found that there were differential attenuation rate of pro-survival and accumulation rate of pro-death signaling protein of the EGFR pathway in response to EGFR inactivation. These findings implicate that, after EGFR inactivation, a transient imbalance between survival and apoptotic signaling outputs contributed to the final cell fate of death. The mxscPISA approach can be a promising tool to reveal a signaling dynamic pattern at the single-cell level and to identify key components of signaling pathways that contribute to the final cell fate using only a limited number of cells.

The Effect of LRP-1 Gene and Mir-205 on Cardiovascular Disease in Iraqi Patients with Familial Hypercholesterolemia

Familial hypercholesterolemia (FH) is a genetic disorder characterized by autosomal inheritance in genes related to LDL-C metabolism, with the major clinical features of hyper-LDL-cholesterolemia and premature coronary artery disease. (LRP-1) is a member of the LDLR family. It is a membrane receptor with scavenging and signaling properties. LRP-1 interacts with a wide range of extracellular ligands as well as intracellular scaffolding and signaling proteins, which makes it important in crucial clinical circumstances like cardiovascular disease, cancer, and neurological illnesses. Mir-205 uses these molecules as biomarker for cardiovascular diseases. This study aims to measure gene expression for the LPR-1 gene and its relationship to the development of cardiovascular disease in familial hypercholesterolemia and non-familial hypercholesterolemia. Also, it studies the indication whether mir-205 regulates the action of the LRP-1 gene in terms of increasing or decreasing gene expression. However, the available methods for measuring LRP1 levels are direct and quantitative using Poly Chain Reaction (RT-PCR) in real time, not at its ends. In the present study, blood was isolated from 150 individuals distributed into three groups: Group 1 included: 50 samples from a healthy group; Group 2: 50 samples from non-Familial hypercholesterolemia patients; Group 3:50 samples Familial hyperchol-esterolemia patients. The results showed that LRP1 protein expression was significantly reduced in patients with F.H compared with normal control in a small cohort from an Iraqi population. This pilot study suggests that the reduced LRP1 protein expression may be associated with cardiovascular disease progression.

Effects of particulate matter and nicotine for the MPP+-induced SH-SY5Y cells: Implication for Parkinson’s disease

Exposure to particulate matter (PM) has been considered a potential risk factor for various neurodegenerative diseases, whereas nicotine has protective effects on Parkinson’s disease (PD). However, it is still unclear whether or how PM alone and in combination with nicotine affects the pathogenesis of PD. We investigated the potential neurotoxicity of PM and the protective properties of nicotine in an in vitro PD model. A 1-methyl-4-phenylpyridimium (MPP+)-induced neurotoxicity model was established with SH-SY5Y cells. Cell viability and apoptosis were measured using MTT and TUNEL assays, respectively. Intracellular reactive oxygen species (ROS) levels were analyzed using the cell-permeant fluorescent probe DCFH-DA. We investigated mitochondrial apoptotic markers such as Bax, Bcl2, cytochrome C, and cleaved caspase-3 and analyzed their levels by Western blotting. SH-SY5Y cells exposed to PM and MPP+ exhibited significantly increased intracellular ROS and decreased cell viability with those exposed to PM alone. PM strikingly exacerbated MPP+-induced mitochondrial dysfunction, including an increase in the Bax/Bcl2 ratio and the release of cytochrome C and cleaved caspase-3. On the other hand, pretreatment of SH-SY5Y cells with nicotine reduced the MPP+-induced loss of cell viability and levels of intracellular ROS and mitochondrial apoptotic signaling proteins. However, pretreatment with nicotine did not prevent PM-induced toxicity in MPP+-treated SHSY5Y cells. PM and MPP+ synergistically increased ROS levels and mitochondrial apoptosis, which led to SH-SY5Y cell death. The protective effect of nicotine cannot rescue PM-induced synergistic neurotoxicity in the MPP+-induced PD model. Our findings verified the opposing roles of PM and nicotine in a model of PD pathogenesis. A large number of in vivo and in vitro studies would verify the roles of PM and nicotine in the future.