Upregulation Of Protein Kinase Research Articles

Anlotinib destabilizes PAX3-FOXO1 to induce rhabdomyosarcoma cell death via upregulating NEK2

BackgroundRhabdomyosarcoma (RMS) is one of the most common soft tissue sarcomas in children and adolescents, in which PAX3-FOXO1 fusion gene positive patients have very poor prognosis. PAX3-FOXO1 has been identified as an independent prognostic predictor in RMS, with no currently available targeted therapeutic intervention. The novel tyrosine kinase inhibitor anlotinib exhibits a wide range of anticancer effects in multiple types of cancers; however, there have been no relevant studies regarding its application in RMS. Materials and methodsWe investigated the effects of PAX3-FOXO1 on the therapeutic efficacy of anlotinib using the CCK-8 assay, flow cytometry, invasion assay, wound healing assay, western blotting, quantitative polymerase chain reaction(qPCR), and xenograft experiments. RNA-seq and co-immunoprecipitation assays were conducted to determine the specific mechanism by which anlotinib regulates PAX3-FOXO1 expression. ResultsAnlotinib effectively inhibited RMS cell proliferation and promoted apoptosis and G2/M phase arrest while impeding tumor growth in vivo. Downregulation of PAX3-FOXO1 enhances the antitumor effects of anlotinib. Anlotinib upregulates protein kinase NEK2 and increases the degradation of PAX3-FOXO1 via the ubiquitin-proteasome pathway, leading to a reduction in PAX3-FOXO1 protein levels. ConclusionAnlotinib effectively inhibited the malignant progression of RMS and promoted degradation of the fusion protein PAX3-FOXO1. Anlotinib could be a targeted therapeutic approach to treat PAX3-FOXO1 fusion-positive RMS.

Essential role of glycoprotein Ibα in platelet activation

AbstractGlycoprotein Ibα (GPIbα), the ligand-binding subunit of platelet GPIb-IX complex, interacts with von Willebrand factor (VWF) exposed at the injured vessel wall, initiating platelet adhesion, activation, hemostasis, and thrombus formation. The cytoplasmic tail of GPIbα interacts with 14-3-3ζ, regulating the VWF-GPIbα–elicited signal transduction and VWF binding function of GPIbα. However, we unexpectedly found that the GPIbα–14-3-3ζ association, beyond VWF-dependent function, is essential for general platelet activation. We found that the myristoylated peptide of GPIbα C-terminus MPαC, a potential GPIbα inhibitor, by itself induced platelet aggregation, integrin αIIbβ3 activation, granule secretion, and phosphatidylserine (PS) exposure. Conversely, the deletion of the cytoplasmic tail of GPIbα in mouse platelets (10aa−/−) decreased platelet aggregation, integrin αIIbβ3 activation, granule secretion, and PS exposure induced by various physiological agonists. Phosphoproteome-based kinase activity profiling revealed significantly upregulated protein kinase C (PKC) activity in MPαC-treated platelets. MPαC-induced platelet activation was abolished by the pan-PKC inhibitor and PKCα deletion. Decreased PKC activity was observed in both resting and agonist-stimulated 10aa−/− platelets. GPIbα regulates PKCα activity by sequestering 14-3-3ζ from PKCα. In vivo, the deletion of the GPIbα cytoplasmic tail impaired mouse hemostasis and thrombus formation and protected against platelet-dependent pulmonary thromboembolism. Therefore, our findings demonstrate an essential role for the GPIbα cytoplasmic tail in regulating platelet general activation and thrombus formation beyond the VWF–GPIbα axis.

Multi-modal mechanisms of the metastasis suppressor, NDRG1: Inhibition of WNT/β-catenin signaling by stabilization of protein kinase Cα

The metastasis suppressor, N-myc downstream regulated gene-1 (NDRG1), inhibits pro-oncogenic signaling in pancreatic cancer (PC). This investigation dissected a novel mechanism induced by NDRG1 on WNT/β-catenin signaling in multiple PC cell-types. NDRG1 overexpression decreased β-catenin and down-regulated glycogen synthase kinase-3β (GSK-3β) protein levels and its activation. However, β-catenin phosphorylation at Ser33, Ser37, and Thr41 that are classically induced by GSK-3β were significantly increased after NDRG1 overexpression, suggesting a GSK-3β-independent mechanism. Intriguingly, NDRG1 overexpression up-regulated protein kinase Cα (PKCα), with PKCα silencing preventing β-catenin phosphorylation at Ser33, Ser37, and Thr41, and decreasing β-catenin expression. Further, NDRG1 and PKCα were demonstrated to associate, with PKCα stabilization occurring after NDRG1 overexpression. In fact, PKCα half-life increased from 1.5 ± 0.8 h (3) in control cells to 11.0 ± 2.5 h (3) after NDRG1 overexpression. Thus, NDRG1 overexpression leads to the association of NDRG1 with PKCα and PKCα stabilization, resulting in β-catenin phosphorylation at Ser33, Ser37, and Thr41. In fact, the association between PKCα, NDRG1, and β-catenin was identified, with the formation of a potential metabolon that promotes the latter β-catenin phosphorylation. This anti-oncogenic activity of NDRG1 was multi-modal, with the above mechanism accompanied by the down-regulation of the nucleo-cytoplasmic shuttling protein, p21-activated kinase 4 (PAK4), that is involved in β-catenin nuclear translocation, inhibition of AKT phosphorylation (Ser473), and decreased β-catenin phosphorylation at Ser552 that suppresses its transcriptional activity. These mechanisms of NDRG1 activity are important to dissect to understand the marked anti-cancer efficacy of NDRG1-inducing thiosemicarbazones that up-regulate PKCα and inhibit WNT signaling.

Abstract 5431: Identification of serine/threonine kinase 39 (STK39) as a driver for multidrug resistance in hepatocellular carcinoma

Abstract Hepatocellular carcinoma (HCC) is a common type of cancer globally and is associated with a poor prognosis due to its high recurrence rate and resistance to treatment. Solid evidence showed the existence of cancer stem cells (CSCs) that are crucial for therapeutic resistance. However, there are only a few kinase inhibitors that have been found to specifically target liver T-ICs for the HCC treatment. To understand the regulatory mechanism of liver CSCs, we employed RNA-Seq analysis to compare the gene expression profiles, with a particular focus on protein kinases, between (1) sorted CD133+ liver CSCs and CD133- differentiated cells extracted from two HCC cell lines (Huh7 and PLC8024), as well as cultured hepatospheres and adherent differentiated cells of HCC cell line PLC/PRF/5. Our analysis identified Serine/Threonine kinase 39 (STK39) as a commonly up-regulated protein kinase in both CD133+ liver CSCs and CSC-enriched hepatospheres. Based on our analysis of the TCGA data, a positive correlation has consistently been observed between STK39 mRNA expression and the expression of liver CSC markers, including CD24, CD133, and CD47. Utilizing publicly available transcriptome datasets from the NCBI Gene Expression Omnibus (GEO), including GSE14520 and GSE25097, we found that overexpression of STK39 is detected in HCC tumor tissues (p<0.001) and is significantly associated with poorer overall survival and disease-free survival. Through the use of lentiviral based overexpression and knockdown approaches, we have demonstrated the role of STK39 in regulating liver CSC properties, including self-renewal, tumorigenicity, cell invasiveness, and the expression of liver CSC markers. STK39 expression was found to be significantly elevated in in HCC cells that were resistant to both sorafenib and lenvatinib, and its inhibition resulted in increased sensitivity of HCC cells to these treatments. To identify the downstream target of STK39 for regulation of cancer stemness, tandem affinity purification coupled with mass spectrometry was employed. The analysis led to the identification of PARP1 as a novel protein binding partner of STK39. Upon suppression of STK39 in HCC cells, it was observed that the phosphorylation of PARP1 at Threonine 368 (T368) was significantly inhibited, indicating its role as a downstream effector of STK39-mediated liver CSC functions. Interestingly, STK39-mediated PARP1 phosphorylation led to development of resistance to PARP1 inhibitor, rucaparib. Additionally, STK39 plays a critical role in regulating PARP1-mediated chromatin decondensation, which enables it to open chromatin structure at the promoter regions of SOX2/OCT4. In summary, treatment of molecular targeted drugs including rucaparib and lenvatinib with STK39 inhibitor may be a potential therapeutic strategy for treatment of this deadly disease. Citation Format: Carmen Oi Ning Leung, Terence Kin-Wah Lee. Identification of serine/threonine kinase 39 (STK39) as a driver for multidrug resistance in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5431.

Just a spoonful of metformin helps the medicine go down.

Diffuse intrinsic pontine glioma (DIPG) is a devastating brain tumor with a need for novel therapies. So far, monotherapies have failed to prolong survival for these patients, and combinatorial strategies have often shown severe, dose-limiting toxicities. In this issue of the JCI, Duchatel, Jackson, and colleagues address this challenge by introducing a drug combination that mitigates side effects and overcomes resistance. After identifying the PI3K/mTOR pathway as a therapeutic vulnerability, they treated DIPG-bearing mice with paxalisib and saw responses but also observed hyperglycemia as a severe side effect. Combining paxalisib with metformin mitigated this toxicity, but also upregulated protein kinase C (PKC) signaling. To tackle this mechanism of resistance, the authors added the PKC inhibitor enzastaurin to their drug combination and showed that this triple therapy led to improved survival. This approach paves the way for improved outcomes for patients with DIPG and other brain tumors.

Prefrontal cortical synaptoproteome profile combined with machine learning predicts resilience towards chronic social isolation in rats

Chronic social isolation (CSIS) of rats serves as an animal model of depression and generates CSIS-resilient and CSIS-susceptible phenotypes. We aimed to investigate the prefrontal cortical synaptoproteome profile of CSIS-resilient, CSIS-susceptible, and control rats to delineate biochemical pathways and predictive biomarker proteins characteristic for the resilient phenotype. A sucrose preference test was performed to distinguish rat phenotypes. Class separation and machine learning (ML) algorithms support vector machine with greedy forward search and random forest were then used for discriminating CSIS-resilient from CSIS-susceptible and control rats. CSIS-resilient compared to CSIS-susceptible rat proteome analysis revealed, among other proteins, downregulated glycolysis intermediate fructose-bisphosphate aldolase C (Aldoc), and upregulated clathrin heavy chain 1 (Cltc), calcium/calmodulin-dependent protein kinase type II (Cam2a), synaptophysin (Syp) and fatty acid synthase (Fasn) that are involved in neuronal transmission, synaptic vesicular trafficking, and fatty acid synthesis. Comparison of CSIS-resilient and control rats identified downregulated mitochondrial proteins ATP synthase subunit beta (Atp5f1b) and citrate synthase (Cs), and upregulated protein kinase C gamma type (Prkcg), vesicular glutamate transporter 1 (Slc17a7), and synaptic vesicle glycoprotein 2 A (Sv2a) involved in signal transduction and synaptic trafficking. The combined protein differences make the rat groups linearly separable, and 100% validation accuracy is achieved by standard ML models. ML algorithms resulted in four panels of discriminative proteins. Proteomics-data-driven class separation and ML algorithms can provide a platform for accessing predictive features and insight into the molecular mechanisms underlying synaptic neurotransmission involved in stress resilience.

Abstract 19102: E-Cigarettes Induce Persistent Cardiac Autonomic Dysfunction and Molecular Signatures of Dilated and Arrhythmogenic Cardiomyopathy

Introduction: Evidence is mounting that use of e-cigarettes (e-cigs) promotes cardiopulmonary injury and dysfunction, but the long-term consequences remain unclear. Consumption of menthol e-cigs has surged amid imminent bans on combustible menthol cigarettes and cigars. Recently, we found e-cig aerosols with menthol (but not tobacco) flavor acutely induced spontaneous ventricular arrhythmias in mice. Hypothesis: Menthol e-cigs induces persistent cardiac autonomic imbalance. Methods: Adult C57BL/6J male mice with electrocardiogram (ECG) and blood pressure (BP) telemeters were exposed for 4 weeks to menthol e-cig aerosols (JUUL, 180 puffs/day, 5 days/week; n=8) or filtered air (Air; n=7) and challenged with restraint stress to determine cardiovascular reactivity to stress. Hearts (n=5/group) were collected immediately after final exposure for phosphoproteomics. Results: Menthol e-cig exposures increased HR, decreased HR variability, and evoked ventricular premature beats only on exposure day 1, but consistently increased BP throughout the 20-day exposure (+16 mmHg systolic BP vs. Air). Additional exposures in females (n=4) recapitulated these acute effects. Pre-treatment with atenolol (n=4 males) abolished e-cig-induced arrhythmias, suggesting β 1 -adrenergic mediation of e-cig-induced arrhythmias. After 4-week JUUL Menthol exposure, mice had basal sinus bradycardia and impaired stress reactivity, characterized by reduced maximal HR and rapid recovery to baseline. All three effects persisted up to 3 weeks following cessation and were accompanied by prolonged ventricular repolarization (QT c ). Phosphoproteomic analysis revealed upregulation of protein kinase A signaling, and mitochondrial dysfunction in e-cig-exposed mice. Phosphoproteins associated with dilated & arrhythmogenic cardiomyopathy (ATP2A2, FN1, CACNB2, PFKM, ADCY6, and PRKAR1A) were differentially regulated in e-cig-exposed mice. Conclusions: We demonstrate that menthol e-cigs induce persistent cardiac autonomic imbalance associated with molecular signatures of dilated & arrhythmogenic cardiomyopathy. Autonomic imbalance and cardiac electrical dysfunction may contribute to the long-term health effects of repeated use of e-cigarettes.

Abstract 2443: Serine/threonine kinase 39 (STK39) regulates cancer stemness through regulating PARP-mediated SOX2/OCT4 expression in hepatocellular carcinoma

Abstract Hepatocellular carcinoma (HCC) is one of the common malignancies worldwide. Poor prognosis of HCC patients is attributed to high frequency of tumor relapse and therapeutic resistance. Increasing evidence showed the existence of cancer stem cells (CSCs) which is crucial for tumor initiation and therapeutic resistance. However, there are limited kinase inhibitors available that show specific targeting against liver T-ICs for HCC treatment. To understand the mechanism for regulation of liver CSCs, we have employed RNA-Seq profiling to compare the gene expression profiles with focuses on protein kinases between (1) sorted CD133+ liver CSCs and CD133- differentiated counterparts isolated from two HCC cell lines (Huh7 and PLC8024) and cultured hepatospheres and adherent differentiated cells of HCC cell line PLC/PRF/5. This analysis revealed Serine/Threonine kinase 39 (STK39), also known as SPAK, to be the commonly up-regulated protein kinase common in both CD133+ liver CSCs and CSC enriched hepatospheres. Consistently, based on the TCGA data analysis, STK39 mRNA expression is positively correlated with the expression of liver CSC markers including CD24, CD133, and CD47. Using publicly available transcriptome datasets deposited in the NCBI Gene Expression Omnibus (GEO) including GSE14520 and GSE25097, we found that overexpression of STK39 was also observed in HCC tumor tissues (p<0.001) and correlated with poorer overall survival and disease-free survival. By lentiviral based overexpression and knockdown approaches, we demonstrated the role of STK39 in regulation of liver CSC properties, including self-renewal, tumorigenicity, cell invasiveness and expression of liver CSC markers. Specially, STK39 expression was highly elevated in sorafenib- and lenvatinib-resistant HCC cells, and its inhibition led to sensitization of HCC cells towards these treatments. In order to identify downstream target of STK39 for regulation of cancer stemness, tandem affinity purification coupled with mass spectrometry was employed. Upon analysis, PARP1, was identified to be the novel protein binding partner of STK39. Upon suppression of STK39 in HCC cells, phosphorylation of PARP1 at Threonine 368 (T368) was found to be highly inhibited, suggesting its role as downstream effector of STK39-mediated liver CSC functions. Furthermore, STK39 regulates PARP1-mediated chromatin decondensation, and thus opens chromatin structure at the promoter regions of SOX2/OCT4. In summary, STK39 regulates cancer stemness and drug resistance by directly modulating PARP1-mediated SOX2/OCT4 expression via chromatin remodelling. Targeting STK39/PARP1 signaling cascade alone or in combination with molecular targeted drugs including rucaparib and lenvatnib can be the potential therapeutic strategy for treatment of this deadly disease. Citation Format: Carmen Oi Ning Leung, Terence Kin-Wah Lee. Serine/threonine kinase 39 (STK39) regulates cancer stemness through regulating PARP-mediated SOX2/OCT4 expression in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2443.

ANP promotes HTR-8/SVneo cell invasion by upregulating protein kinase N 3 via autophagy inhibition.

Preeclampsia is a gestational disease characterized by two major pathological changes-shallow trophoblast invasion and impaired spiral artery remodeling. Atrial natriuretic peptide (ANP) is a kind of peptide hormone that regulates blood pressure, while the lack of active ANP participates in preeclampsia pathogenesis. However, the underlying mechanism of how ANP modulates trophoblasts function remains unclarified. Here, we performed isobaric tags for relative and absolute quantification (iTRAQ) in ANP-treated HTR-8/SVneo cells and identified Protein Kinase 3 (PKN3) as the downstream factor of ANP, which was downregulated in preeclamptic placenta. Chromatin immunoprecipitation analysis and luciferase assays showed that NFYA was one of the transcription factors for the PKN3 promoter, which was also regulated by ANP treatment in HTR-8/SVneo cells. Transmission electron microscopy and Western Blotting in HTR-8/SVneo cells indicated that ANP inhibited autophagy via AMPK-mTORC1 signaling, while excess autophagy was observed in preeclamptic placenta. The increased expression of PKN3 and enhanced cell invasion ability in HTR-8/SVneo cells induced by ANP could be abolished by autophagy activation or transfection with PKN3 shRNA or NFYA shRNA or NPR-A shRNA via regulating the invasion-related genes and the epithelial mesenchymal transition molecules. Our results demonstrated that ANP could enhance trophoblast invasion by upregulating PKN3 via NFYA promotion through autophagy inhibition in an AMPK/mTORC1 signaling-dependent manner.

Hypoxia-induced downregulation of microRNA-186-5p in endothelial cells promotes non-small cell lung cancer angiogenesis by upregulating protein kinase C alpha

The tumor microenvironment stimulates the angiogenic activity of endothelial cells (ECs) to facilitate tumor vascularization, growth, and metastasis. The involvement of microRNA-186-5p (miR-186) in regulating the aberrant activity of tumor-associated ECs has so far not been clarified. In the present study, we demonstrated that miR-186 is significantly downregulated in ECs microdissected from human non-small cell lung cancer (NSCLC) tissues compared with matched non-malignant lung tissues. Invitro analyses of primary human dermal microvascular ECs (HDMECs) exposed to different stimuli indicated that this miR-186 downregulation is triggered by hypoxia viaactivation of hypoxia-inducible factor 1 alpha (HIF1α). Transfection of HDMECs with miR-186 mimic (miR-186m) significantly inhibited their proliferation, migration, tube formation, and spheroid sprouting. In contrast, miR-186 inhibitor (miR-186i) exerted pro-angiogenic effects. Invivo, endothelial miR-186 overexpression inhibited the vascularization of Matrigel plugs and the initial growth of tumors composed of NSCLC cells (NCI-H460) and HDMECs. Mechanistic analyses revealed that the gene encoding for protein kinase C alpha (PKCα) is a bona fide target of miR-186. Activation of this kinase significantly reversed the miR-186m-repressed angiogenic activity of HDMECs. These findings indicate that downregulation of miR-186 in ECs mediates hypoxia-stimulated NSCLC angiogenesis by upregulating PKCα.

The WNT/Ca2+ pathway promotes atrial natriuretic peptide secretion by activating protein kinase C/transforming growth factor-β activated kinase 1/activating transcription factor 2 signaling in isolated beating rat atria.

WNT signaling plays an important role in cardiac development, but abnormal activity is often associated with cardiac hypertrophy, myocardial infarction, remodeling, and heart failure. The effect of WNT signaling on regulation of atrial natriuretic peptide (ANP) secretion is unclear. Therefore, the purpose of this study was to investigate the effect of Wnt agonist 1 (Wnta1) on ANP secretion and mechanical dynamics in beating rat atria. Wnta1 treatment significantly increased atrial ANP secretion and pulse pressure; these effects were blocked by U73122, an antagonist of phospholipase C. U73122 also abolished the effects of Wnta1-mediated upregulation of protein kinase C (PKC) β and γ expression, and the PKC antagonist Go 6983 eliminated Wnta1-induced secretion of ANP. In addition, Wnta1 upregulated levels of phospho-transforming growth factor-β activated kinase 1 (p-TAK1), TAK1 banding 1 (TAB1) and phospho-activating transcription factor 2 (p-ATF2); these effects were blocked by both U73122 and Go 6983. Wnta1-induced ATF2 was abrogated by inhibition of TAK1. Furthermore, Wnta1 upregulated the expression of T cell factor (TCF) 3, TCF4, and lymphoid enhancer factor 1 (LEF1), and these effects were blocked by U73122 and Go 6983. Tak1 inhibition abolished the Wnta1-induced expression of TCF3, TCF4, and LEF1 and Wnta1-mediated ANP secretion and changes in mechanical dynamics. These results suggest that Wnta1 increased the secretion of ANP and mechanical dynamics in beating rat atria by activation of PKC-TAK1-ATF2-TCF3/LEF1 and TCF4/LEF1 signaling mainly via the WNT/Ca2+ pathway. It is also suggested that WNT-ANP signaling is implicated in cardiac physiology and pathophysiology.

Thermoneutral Housing and a Western Diet Combination Exacerbates Dysferlin-Deficient Muscular Dystrophy.

Most mouse models of muscular dystrophy (MD) show mild phenotypes, which limits the translatability of experimental therapies to patients. A growing body of evidence suggests that MD is accompanied by metabolic abnormalities that could potentially exacerbate the primary muscle wasting process. Since thermoneutral (TN) housing of mice (~30°C) has been shown to affect many metabolic parameters, particularly when combined with a Western diet (WD), our aim was to determine whether the combination of TN and WD exacerbates muscle wasting in dysferlin-deficient BLAJ mice, a common model of limb-girdle MD type 2b (LGMD2b). The 2-mo-old wild-type (WT) and BLAJ mice were housed at TN or room temperature (RT) and fed a WD or regular chow for 9 mo. Ambulatory function, muscle histology, and protein immunoblots of skeletal muscle were assessed. BLAJ mice at RT and fed a chow diet showed normal ambulation function similar to WT mice, whereas 90% of BLAJ mice under WD and TN combination showed ambulatory dysfunction (p < 0.001), and an up to 4.1-fold increase in quadriceps and gastrocnemius fat infiltration. Western blotting revealed decreased autophagy marker microtubules-associated protein 1 light chain 3-B (LC3BII/LC3BI) ratio and up-regulation of protein kinase B/AKT and ribosomal protein S6 phosphorylation, suggesting inefficient cellular debris and protein clearance in TN BLAJ mice fed a WD. Male and female BLAJ mice under TN and WD combination showed heterogenous fibro-fatty infiltrate composition. TN and WD combination exacerbates rodent LGMD2b without affecting WT mice. This improves rodent modeling of human MD and helps elucidate how metabolic abnormalities may play a causal role in muscle wasting.

Modulating Microglia/Macrophage Activation by CDNF Promotes Transplantation of Fetal Ventral Mesencephalic Graft Survival and Function in a Hemiparkinsonian Rat Model.

Parkinson’s disease (PD) is characterized by the loss of dopaminergic neurons in substantia nigra pars compacta, which leads to the motor control deficits. Recently, cell transplantation is a cutting-edge technique for the therapy of PD. Nevertheless, one key bottleneck to realizing such potential is allogenic immune reaction of tissue grafts by recipients. Cerebral dopamine neurotrophic factor (CDNF) was shown to possess immune-modulatory properties that benefit neurodegenerative diseases. We hypothesized that co-administration of CDNF with fetal ventral mesencephalic (VM) tissue can improve the success of VM replacement therapies by attenuating immune responses. Hemiparkinsonian rats were generated by injecting 6-hydroxydopamine (6-OHDA) into the right medial forebrain bundle of Sprague Dawley (SD) rats. The rats were then intrastriatally transplanted with VM tissue from rats, with/without CDNF administration. Recovery of dopaminergic function and survival of the grafts were evaluated using the apomorphine-induced rotation test and small-animal positron emission tomography (PET) coupled with [18F] DOPA or [18F] FE-PE2I, respectively. In addition, transplantation-related inflammatory response was determined by uptake of [18F] FEPPA in the grafted side of striatum. Immunohistochemistry (IHC) examination was used to determine the survival of the grated dopaminergic neurons in the striatum and to investigate immune-modulatory effects of CDNF. The modulation of inflammatory responses caused by CDNF might involve enhancing M2 subset polarization and increasing fractal dimensions of 6-OHDA-treated BV2 microglial cell line. Analysis of CDNF-induced changes to gene expressions of 6-OHDA-stimulated BV2 cells implies that these alternations of the biomarkers and microglial morphology are implicated in the upregulation of protein kinase B signaling as well as regulation of catalytic, transferase, and protein serine/threonine kinase activity. The effects of CDNF on 6-OHDA-induced alternation of the canonical pathway in BV2 microglial cells is highly associated with PI3K-mediated phagosome formation. Our results are the first to show that CDNF administration enhances the survival of the grafted dopaminergic neurons and improves functional recovery in PD animal model. Modulation of the polarization, morphological characteristics, and transcriptional profiles of 6-OHDA-stimualted microglia by CDNF may possess these properties in transplantation-based regenerative therapies.

Ginsenoside Rg2 Attenuates Doxorubicin-induced Cardiomyocyte Apoptosis via the PI3K/Akt Pathway

Doxorubicin, sold under the brand name Adriamycin among others, is an important drug for cancer therapy; however, its use is limited by its cardiotoxicity. Ginsenoside Rg2 is extracted from Panax ginseng C.A.Mey., Araliaceae, which is believed to have cardioprotective properties. However, to date, there have been no reports on whether ginsenoside Rg2 could protect cardiomyocytes against doxorubicin. In this study, we investigated the action and the underlying mechanisms of cardioprotection of ginsenoside Rg2 upon doxorubicin treatment. Cell counting kit-8 was used to determine cell viability; in addition, terminal deoxynucleotidyl transferase–mediated dUTP nick-end labeling staining was used to detect apoptotic cells. Western blotting was used to investigate the relevant pathways. LY294002, a phosphatidylinositol 3-kinase inhibitor, was also used in this study. Ginsenoside Rg2 significantly (p < 0.01) neutralized cardiomyocyte apoptosis induced by doxorubicin in a dose-dependent manner, but this effect was blocked by LY294002. Furthermore, ginsenoside Rg2 upregulated protein kinase B phosphorylation through the phosphatidylinositol 3-kinase/protein kinase B pathway and inhibited p53 expression. These results suggest that ginsenoside Rg2 could attenuate doxorubicin-induced cardiomyocyte apoptosis via the phosphatidylinositol 3-kinase/protein kinase B pathway.Graphical abstract

Aerobic exercise training-induced follistatin-like 1 secretion in the skeletal muscle is related to arterial stiffness via arterial NO production in obese rats.

Follistatin‐like 1 (FSTL1), which is mainly secreted from skeletal muscle and myocardium, upregulates protein kinase B (Akt) and endothelial nitric oxide synthase (eNOS) phosphorylation in vascular endothelial cells. It is unclear whether skeletal muscle‐ and myocardium‐derived FSTL1 secretion induced by aerobic exercise training is involved in the reduction of arterial stiffness via arterial NO production in obese rats. This study aimed to clarify whether aerobic exercise training‐induced FSTL1 secretion in myocardium and skeletal muscle is associated with a reduction in arterial stiffness via arterial Akt‐eNOS signaling pathway in obese rats. Sixteen Otsuka Long‐Evans Tokushima Fatty (OLETF) obese rats were randomly divided into two groups: sedentary control (OLETF‐CON) and eight‐week aerobic exercise training (treadmill for 60min at 25m/min, 5days/week, OLETF‐AT). Eight Long‐Evans Tokushima Otsuka (LETO) rats were used as a healthy sedentary control group. In OLETF‐CON, serum FSTL1, arterial Akt and eNOS phosphorylation, and arterial nitrite/nitrate (NOx) levels were significantly lower, and carotid‐femoral pulse wave velocity (cfPWV) was significantly greater than those in LETO. These parameters were improved in the OLETF‐AT compared to the OLETF‐CON. In the OLETF‐AT, FSTL1 levels in slow‐twitch fiber‐rich soleus muscle were significantly greater than those in the OLETF‐CON, but not in myocardium, fast‐twitch fiber‐rich tibialis anterior muscle, and adipose tissue. Serum FSTL1 levels were positively correlated with soleus FSTL1, arterial eNOS phosphorylation, and NOx levels and negatively correlated with cfPWV. Thus, aerobic exercise training‐induced FSTL1 secretion in slow‐twitch fiber‐rich muscles may be associated with a reduction in arterial stiffness via arterial NO production in obese rats.

Bioactive Components of Houttuynia cordata Thunb and Their Potential Mechanisms Against COVID-19 Using Network Pharmacology and Molecular Docking Approaches.

We investigated the molecular mechanism by which Houttuynia cordata Thunb (HCT) may intervene in coronavirus disease 2019 (COVID-19) and COVID-19-induced cytokine storms using network pharmacology and molecular docking approaches. Using the Traditional Chinese medicine Systems Pharmacology Database and Analysis Platform (TCMSP), a "component-target-pathway" topology map of HCT for COVID-19 treatment was constructed using Cytoscape. Core target genes were analyzed using the STRING database, and the signal pathway map and biological mechanism of COVID-19 therapy were obtained using cluster profilers. Active components of HCT were docked with severe respiratory syndrome coronavirus 2 (SARS-CoV-2) 3C-like protease (3CLpro) and RNA-dependent RNA polymerase (RdRp) using AutoDockTools. Data visualization and statistical analysis were conducted using the R program. A molecular dynamic simulation was carried out with the Groningen Machine for Chemical Simulation program. HCT had six active anti-COVID-19 ingredients and 45 molecular targets. Their crucial target proteins for COVID-19 treatment were the RELA (nuclear factor kappa B [NF-κB] p65 subunit), interleukin 6, and mitogen-activated protein kinase 1. In functional enrichment analysis, the potential molecular targets of active components of HCT for COVID-19 treatment belonged to 18 signaling pathways (adjusted P = 2.12E-11). Gene ontology obtained by Kyoto Encyclopedia of Genes and Genome enrichment screening showed that the primary mechanism of COVID-19 treatment was upregulation of protein kinase C followed by downregulations of T cell differentiation and proliferation and NF-κB signaling. Molecular docking showed that the active components of HCT (quercetin and kaempferol) had similar binding affinities for SARS-CoV-2 3CLpro and SARS-CoV-2 RdRp, primary COVID-19 target proteins as did clinically used drugs. These results were confirmed with molecular dynamics simulation. In conclusion, multiple components of HCT, especially quercetin and kaempferol, have the potential to treat COVID-19 infection and COVID-19-induced cytokine storm by targeting multiple proteins.

Glut5 Knockdown in the Nucleus Tractus Solitarii Alleviates Fructose-Induced Hypertension in Rats

BackgroundSeveral studies have suggested mechanisms whereby excessive fructose intake increases blood pressure (BP). Glucose transporter 5 (GLUT5) is a fructose transporter expressed on enterocytes, and its involvement in the nucleus tractus solitarius (NTS)–modulated increase in BP following fructose intake remains unclear. ObjectivesHerein, we investigated whether NTS Glut5 knockdown (KD) can alleviate fructose-induced hypertension in rat models. MethodsMale Wistar-Kyoto rats (6–8 weeks old; average weight: 230 g) were randomly assigned into 4 groups [control (Con), fructose (Fru), fructose + scrambled (Fru + S), and Fru + KD]. The Con group rats had ad libitum access to regular water, and the other 3 groups were provided 10% fructose water ad libitum for 4 weeks (2 weeks before lentiviral transfection in the Fru + S and Fru + KD groups). Glut5 short hairpin RNA was delivered into the NTS of rats using a lentivirus system. Fructose-induced hypertension was assessed via the tail-cuff technique, a noninvasive blood pressure measurement approach. GLUT5-associated and other insulin signaling pathways in the NTS of rats were assessed using immunofluorescence and immunoblotting analyses. We evaluated between-group differences using the Mann-Whitney U test or Kruskal-Wallis 1-way ANOVA. ResultsCompared with the Fru + S group, the Fru + KD group had reduced sympathetic nerve hyperactivity (48.8 ± 3.2 bursts/min; P < 0.05), improved central insulin signaling, upregulated protein kinase B (AKT; 3.0-fold) and neuronal NO synthase (nNOS; 2.78-fold) expression, and lowered BP (17 ± 1 mmHg, P < 0.05). Moreover, Glut5 KD restored signaling dependent on adenosine 5′-monophosphate–activated protein kinase and reduced fructose-induced oxidative stress 2.0-fold, and thus decreased NAD(P)H oxidase in p67-phox 1.9-fold within the NTS. ConclusionsFructose-induced reactive oxygen species generates in the NTS of rats through GLUT5 and receptor for advanced glycation end products signaling, thus impairing the AKT–nNOS–NO signaling pathway and ultimately causing hypertension.

UBR5 promotes tumor immune evasion through enhancing IFN-γ-induced PDL1 transcription in triple negative breast cancer.

Background: The up-regulation of PD-L1 is recognized as an adaption of cancer cells to evade immune surveillance and attack. However, the intrinsic mechanisms of the induction of PD-L1 by interferon-γ (IFN-γ) in tumor microenvironment remain incompletely characterized. Ubiquitin ligase E3 component N-recognition protein 5 (UBR5) has a critical role in tumorigenesis of triple negative breast cancer (TNBC) by triggering specific immune responses to the tumor. Dual targeting of UBR5 and PD-L1 exhibited superior therapeutic benefits in a preclinical TNBC model in short term.Methods: The regulation of UBR5 to PD-L1 upon IFN-γ stimulation was evaluated through in UBR5 deficiency, reconstitution or overexpression cell line models by quantitative PCR, immunohistochemistry and RNA-seq. The effects of PD-L1 regulation by UBR5 and double blockade of both genes were evaluated in mouse TNBC model. Luciferase reporter assay, chromatin immunoprecipitation-qPCR and bioinformatics analysis were performed to explore the transcription factors involved in the regulation of UBR5 to PD-L1.Results: E3 ubiquitin ligase UBR5 plays a key role in IFN-γ-induced PDL1 transcription in TNBC in an E3 ubiquitination activity-independent manner. RNA-seq-based transcriptomic analyses reveal that UBR5 globally affects the genes in the IFN-γ-induced signaling pathway. Through its poly adenylate binding (PABC) domain, UBR5 enhances the transactivation of PDL1 by upregulating protein kinase RNA-activated (PKR), and PKR's downstream factors including signal transducers and activators of transcription 1 (STAT1) and interferon regulatory factor 1 (IRF1). Restoration of PD-L1 expression in UBR5-deficient tumor cells recoups their malignancy in vivo, whereas CRISPR/Cas9-mediated simultaneous abrogation of UBR5 and PD-L1 expression yields synergistic therapeutic benefits than either blockade alone, with a strong impact on the tumor microenvironment.Conclusions: This study identifies a novel regulator of PDL1 transcription, elucidates the underlying molecular mechanisms and provides a strong rationale for combination cancer immunotherapies targeting UBR5 and PD-L1.

Zingiber officinale attenuates 6-hydroxydopamine induced oxidative stress and apoptosis through AKT, Nrf2, MAPK, NF-κB signaling pathway in PC12 cells

To explore the protective impact of the ginger root ethanol extract (GRE) against the oxidative stress induced by 6-hydroxydopamine (6-OHDA) and apoptotic and its mechanism. Parkinson's disease (PD) model was established using 6-OHDA in the cells of rat adrenal pheochromocytoma (PC12). The GRE pretreatment increased PC12 cell viability of injury induced by 6-OHDA. The GRE effectively suppressed 6-OHDA-induced death, apoptosis through decreased Bax and cleaved-caspase 3 expression, and up-regulated expression of B-cell lymphoma 2 (Bcl-2). GRE also inhibited 6-OHDA-inducel oxidative stress, decreased reactive oxygen species (ROS) and up-regulates the heme oxygenase (HO-1), antioxidant enzymes, catalase and, glutathione (GSH), superoxide dismutase (SOD), 8-oxoguanine glycosylase1 (OGG1) and NAD(P)H quinone oxidoreductase1 (NQO1). Besides, GRE up-regulating Protein kinase B (Akt), nuclear erythroid 2-related factor 2 (Nrf2), down-regulating nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) translocation, together with the mitogen-activated protein kinase (MAPK) phosphorylation. In conclusions, the GRE prevented apoptosis and oxidative stress in 6-OHDA-induced PC12 cells. The ginger ethanol extract could be treatment and prevention of PD.

The Clinical Significance and Potential Molecular Mechanism of Upregulated CDC28 Protein Kinase Regulatory Subunit 1B in Osteosarcoma.

Background CDC28 Protein Kinase Regulatory Subunit 1B (CKS1B) is a member of cyclin-dependent kinase subfamily and the relationship between CKS1B and osteosarcoma (OS) remains to be explored. Methods 80 OS and 41 nontumor tissue samples were arranged to conduct immunohistochemistry (IHC) to evaluate CKS1B expression between OS and nontumor samples. The standard mean deviation (SMD) was calculated based on in-house IHC and tissue microarrays and exterior high-throughput datasets for further verification of CKS1B expression in OS. The effect of CKS1B expression on clinicopathological and overall survival of OS patients was measured through public high-throughput datasets, and analysis of immune infiltration and single-cell RNA-seq was applied to ascertain molecular mechanism of CKS1B in OS. Results A total of 197 OS samples and 83 nontumor samples (including tissue and cell line) were obtained from in-house IHC, microarrays, and exterior high-throughput datasets. The analysis of integrated expression status demonstrated upregulation of CKS1B in OS (SMD = 1.38, 95% CI [0.52–2.25]) and the significant power of CKS1B expression in distinguishing OS samples from nontumor samples (Area under the Curve (AUC) = 0.89, 95% CI [0.86–0.91]). Clinicopathological and prognosis analysis indicated no remarkable significance but inference of immune infiltration and single-cell RNA-seq prompted that OS patients with overexpressed CKS1B were more likely to suffer OS metastasis while MYC Protooncogene may be the upstream regulon of CKS1B in proliferating osteoblastic OS cells. Conclusions In this study, sufficient evidence was provided for upregulation of CKS1B in OS. The advanced effect of CKS1B on OS progression indicates a foreground of CKS1B as a biomarker for OS.