Conformational dynamics of α-1 acid glycoprotein (AGP) in cancer: A comparative study of glycosylated and unglycosylated AGP.

α-1 acid glycoprotein (AGP) is one of the most abundant plasma proteins. It fulfills two important functions: immunomodulation, and binding to various drugs and receptors. These different functions are closely associated and modulated via changes in glycosylation and cancer missense mutations. From a structural point of view, glycans alter the local biophysical properties of the protein leading to a diverse ligand-binding spectrum. However, glycans can typically not be observed in the resolved X-ray crystallography structure of AGP due to their high flexibility and microheterogeneity, so limiting our understanding of AGP's conformational dynamics 70 years after its discovery. We here investigate how mutations and glycosylation interfere with AGP's conformational dynamics changing its biophysical behavior, by using molecular dynamics (MD) simulations and sequence-based dynamics predictions. The MD trajectories show that glycosylation decreases the local backbone flexibility of AGP and increases the flexibility of distant regions through allosteric effects. We observe that mutations near the glycosylation site affect glycan's conformational preferences. Thus, we conclude that mutations control glycan dynamics which modulates the protein's backbone flexibility directly affecting its accessibility. These findings may assist in the drug design targeting AGP's glycosylation and mutations in cancer.

Multiallelic Copy Number Variation in ORM1 is Associated with Plasma Cell-Free DNA Levels as an Intermediate Phenotype for Venous Thromboembolism.

Venous thromboembolism (VTE) is a common disease with high heritability. However, only a small portion of the genetic variance of VTE can be explained by known genetic risk factors. Neutrophil extracellular traps (NETs) have been associated with prothrombotic activity. Therefore, the genetic basis of NETs could reveal novel risk factors for VTE. A recent genome-wide association study of plasma cell-free DNA (cfDNA) levels in the Genetic Analysis of Idiopathic Thrombophilia 2 (GAIT-2) Project showed a significant associated locus near ORM1. We aimed to further explore this candidate region by next-generation sequencing, copy number variation (CNV) quantification, and expression analysis using an extreme phenotype sampling design involving 80 individuals from the GAIT-2 Project. The RETROVE study with 400 VTE cases and 400 controls was used to replicate the results. A total of 105 genetic variants and a multiallelic CNV (mCNV) spanning ORM1 were identified in GAIT-2. Of these, 17 independent common variants, a region of 22 rare variants, and the mCNV were significantly associated with cfDNA levels. In addition, eight of these common variants and the mCNV influenced ORM1 expression. The association of the mCNV and cfDNA levels was replicated in RETROVE (p-value = 1.19 × 10-6). Additional associations between the mCNV and thrombin generation parameters were identified. Our results reveal that increased mCNV dosages in ORM1 decreased gene expression and upregulated cfDNA levels. Therefore, the mCNV in ORM1 appears to be a novel marker for cfDNA levels, which could contribute to VTE risk.

ORM 1 as a biomarker of increased vascular invasion and decreased sorafenib sensitivity in hepatocellular carcinoma.

This study aimed to clarify the role of Orosomucoid 1 (ORM1) in the development and therapy resistance in hepatocellular carcinoma (HCC). The mRNA expression level of ORM1 was analyzed via integrative analysis of Gene Express Omnibus (GEO) and The Cancer Genome Atlas (TCGA) datasets. The protein expression level of ORM1 in our cohort was determined using immunohistochemistry. Correlation analysis was used to investigate the relationship between ORM1 expression and clinical parameters. The Cell Counting Kit-8 assay was used to clarify the role of ORM1 in HCC malignant behaviors, including cell growth and sorafenib sensitivity, in vitro. The results indicated that ORM1 was significantly downregulated in the hepatic cancer cells compared to that in the non-cancerous cells. However, it was upregulated in microvascular invasion samples, especially in the cancer embolus compared to that in the surrounding tumor cells. Though Kaplan-Meier analysis did not show an association of ORM1 expression with the overall survival rates of HCC patients, univariate analysis indicated that ORM1 expression was highly correlated with tumor grade and stage. An in vitro assay also revealed that downregulation of ORM1 led to the suppression of tumor growth and enhancement of sorafenib sensitivity without epithelial-to-mesenchymal transition (EMT) alteration, which was consistent with our bioinformatic analysis. Hence, ORM1 played a key role in HCC tumorigenesis and may serve as a potential target for the development of therapeutics against HCC in the future.

Integrated GWAS and Gene Expression Suggest ORM1 as a Potential Regulator of Plasma Levels of Cell-Free DNA and Thrombosis Risk.

Plasma cell-free DNA (cfDNA) is a surrogate marker of neutrophil extracellular traps (NETs) that contribute to immunothrombosis. There is growing interest about the mechanisms underlying NET formation and elevated cfDNA, but little is known about the factors involved. We aimed to identify genes involved in the regulation of cfDNA levels using data from the Genetic Analysis of Idiopathic Thrombophilia (GAIT-2) Project.Imputed genotypes, whole blood RNA-Seq data, and plasma cfDNA quantification were available for 935 of the GAIT-2 participants from 35 families with idiopathic thrombophilia. We performed heritability and GWAS analysis for cfDNA. The heritability of cfDNA was 0.26 (p = 3.7 × 10-6), while the GWAS identified a significant association (rs1687391, p = 3.55 × 10-10) near the ORM1 gene, on chromosome 9. An eQTL (expression quantitative trait loci) analysis revealed a significant association between the lead GWAS variant and the expression of ORM1 in whole blood (p = 6.14 × 10-9). Additionally, ORM1 expression correlated with levels of cfDNA (p = 4.38 × 10-4). Finally, genetic correlation analysis between cfDNA and thrombosis identified a suggestive association (ρ g = 0.43, p = 0.089).All in all, we show evidence of the role of ORM1 in regulating cfDNA levels in plasma, which might contribute to the susceptibility to thrombosis through mechanisms of immunothrombosis.

Hydrolase Activity of the Genetic Variants of Human Alpha-1-Acid Glycoprotein.

In human plasma, the main agent of hydrolysis of the ester-type prodrug of levodopa, designated ONO-2160, is alpha-1-acid glycoprotein (AGP), which is a mixture of the F1*S and A variants at molar ratios of 3:1 to 2:1. In this study, the mechanism of AGP esterase-like activity was investigated by evaluating the contribution of the F1*S and A variants to ONO-2160 hydrolysis and identifying the AGP hydrolase active site. We found that although both variants hydrolyzed ONO-2160, their hydrolase activities were different. The intrinsic plasma clearance of the F1*S variant (0.441 mL/h/mg protein) was approximately 30 times higher than that of the A variant (0.0148 mL/h/mg protein), indicating that the F1*S variant contributed the most to AGP esterase-like activity. To identify the hydrolase active site of AGP, we performed inhibition studies of ONO-2160 hydrolysis using 12 AGP-binding drugs with various ligand-binding constants and binding selectivities to the two AGP variants. Inhibition of activity was positively correlated with the constant of ligand binding to the F1*S variant. In addition, compounds with high affinity to the F1*S variant inhibited ONO-2160 hydrolysis the most. Together, our data indicate that ONO-2160 is predominantly hydrolyzed by the F1*S variant at its ligand-binding site.

Orosomucoid 1 promotes epirubicin resistance in breast cancer by upregulating the expression of matrix metalloproteinases 2 and 9.

Orosomucoid 1 (ORM1) has been shown to be upregulated in the serum of breast cancer patients; however, the expression and function of ORM1 in breast cancer remains unknown. We measured the expression of ORM1 in breast cancer tissues and cell lines using qRT-PCR. A colony formation assay was done to assess cell proliferation and Transwell and wound healing assays were performed to determine the migration and invasion capacity of the cells, respectively. In addition, a CCK-8 assay was used to measure epirubicin cytotoxicity and western blot assays were done to analyze the putative mechanisms of epirubicin sensitivity. We found that the expression of ORM1 was upregulated in breast cancer tissues and cell lines. The expression of ORM1 enhanced the proliferation and migration of the cell lines. In contrast, down-regulation of ORM1 inhibited the expression of MMP-2 and MMP-9 and activation of the AKT/ERK signaling pathway. Therefore, ORM1 may represent a potential therapeutic target for breast cancer and promote epirubicin resistance by regulating the expression of MMP-2 and MMP-9, as well as activating the AKT/ERK signaling pathway.

α1-Acid Glycoprotein Enhances the Immunosuppressive and Protumor Functions of Tumor-Associated Macrophages.

Blood levels of acute-phase protein α1-acid glycoprotein (AGP, orosmucoid) increase in patients with cancer. Although AGP is produced from hepatocytes following stimulation by immune cell-derived cytokines under conditions of inflammation and tumorigenesis, the functions of AGP in tumorigenesis and tumor progression remain unknown. In the present study, we revealed that AGP contributes directly to tumor development by induction of programmed death ligand 1 (PD-L1) expression and IL6 production in macrophages. Stimulation of AGP induced PD-L1 expression in both human monocyte-derived macrophages through STAT1 activation, whereas AGP had no direct effect on PD-L1 expression in tumor cells. AGP also induced IL6 production from macrophages, which stimulated proliferation in tumor cells by IL6R-mediated activation of STAT3. Furthermore, administration of AGP to AGP KO mice phenocopied effects of tumor-associated macrophages (TAM) on tumor progression. AGP decreased IFNγ secretion from T cells and enhanced STAT3 activation in subcutaneous tumor tissues. In addition, AGP regulated PD-L1 expression and IL6 production in macrophages by binding with CD14, a coreceptor for Toll-like receptor 4 (TLR4), and inducing TLR4 signaling. These results provide the first evidence that AGP is directly involved in tumorigenesis by interacting with TAMs and that AGP might be a target molecule for anticancer therapy. SIGNIFICANCE: AGP-mediated suppression of antitumor immunity contributes to tumor progression by inducing PD-L1 expression and IL6 production in TAMs.

Impact of Interindividual Differences in Plasma Fraction Unbound on the Pharmacokinetics of a Novel Syk Kinase Inhibitor in Beagle Dogs.

Inconsistencies in pharmacokinetic parameters between individual animals in preclinical studies are a common occurrence. Often such differences between animals are simply accepted as experimental variability rather than as indications of specific differences in animal phenotype that could lead to a different interpretation of the data. The fraction unbound in plasma is one factor influencing pharmacokinetic parameters and is typically determined using pooled plasma from multiple animals, making the assumption that there is limited population variance. However, this assumption is not often tested and may not hold true if there are polymorphisms affecting binding or variation in the concentrations of individual plasma proteins that could give rise to different fraction unbound phenotypes in individual animals. During profiling of a novel Syk inhibitor, AZ8399, striking interindividual differences in total plasma clearance and volume of distribution were observed between dogs consistent with differences in fraction unbound between animals. Determination of the fraction unbound showed a ∼5-fold difference in fraction unbound between the animals in the study. Broader analysis of individual dogs across a colony demonstrated a correlation between individual animal fraction unbound with total plasma clearance and volume of distribution. The concentrations of the common drug-binding proteins albumin and α1-acid glycoprotein in plasma were determined, and α1-acid glycoprotein levels were found to correlate with fraction unbound. Finally, single-nucleotide polymorphisms were identified at c.502 and c.522 of exon 5 of the dog α1-acid glycoprotein gene that may be correlated to the α1-acid glycoprotein concentration phenotype observed. SIGNIFICANCE STATEMENT: The current work demonstrates the potential for significant interindividual differences in plasma fraction unbound in beagle dogs and goes on to examine the underlying cause for the compound described. The findings suggest that the application of a population mean value of fraction unbound generated from a pooled sample may not always be appropriate and could introduce significant errors in scaling of in vitro clearance values, PBPK understanding, and interpretation of PKPD or toxicokinetic data in the context of unbound concentrations.

Species Difference in Hydrolysis of an Ester-type Prodrug of Levodopa in Human and Animal Plasma: Different Contributions of Alpha-1 Acid Glycoprotein.

Previously, we found that ONO-2160, an ester-type prodrug of levodopa (3-hydroxy-l-tyrosine), was mainly hydrolyzed in human plasma by α1-acid glycoprotein (AGP) with a partial contribution of albumin. In this study, we investigated whether ONO-2160 was hydrolyzed in the plasma of preclinical species (dog, rabbit, rat, and mouse) and humans and whether AGP and albumin are involved in its hydrolysis. ONO-2160 was hydrolyzed to some extent in the plasma of all tested species with the order of magnitude mouse > human > rabbit > rat > dog. Except for dogs, ONO-2160 was partially hydrolyzed by animal AGP and albumin. This indicated that, similar to albumin, AGP possesses esterase-like activity in mice, rats, and rabbits, as well as humans. A comparison of the values of intrinsic clearance per milliliter of plasma demonstrated that AGP was the major contributor to the hydrolysis of ONO-2160 in rabbit plasma, whereas albumin was primarily responsible for the hydrolysis of ONO-2160 in mouse plasma. This was confirmed by experiments using AGP-knockout mouse plasma. This study reports the first evidence for the existence of species differences in the hydrolysis of ONO-2160 in plasma related to the different contributions of AGP and albumin.

Thapsigargin suppresses alpha 1-acid glycoprotein secretion independently of N-glycosylation and ER stress.

Alpha-1 acid glycoprotein (AGP) is a major acute-phase protein that is involved in drug/ligand binding and regulation of immune response. In response to inflammation, AGP secretion from the liver increases, resulting in elevated concentration of plasma AGP. AGP exhibits multiple N-glycosylation sites, and thus, is highly glycosylated. Although AGP glycosylation is considered to affect its functions, the significance of AGP glycosylation for its secretion is unclear. In this study, we investigated the effects of AGP glycosylation using glycosylation-deficient mouse AGP mutants lacking one, four, or all five N-glycosylation sites. Furthermore, we examined the effects of endoplasmic reticulum (ER) stress-inducing reagents, including tunicamycin and thapsigargin, which induce ER stress in an N-glycosylation-dependent and -independent manner, respectively. Here, we found that glycosylation deficiency and ER stress induce a little or no effect on AGP secretion. Conversely, thapsigargin significantly suppressed AGP secretion in glycosylation-independent manner. These findings indicate that AGP secretion is regulated via thapsigargin-sensitive pathway that might be further controlled by the intracellular calcium concentrations.

Proteomic Profile of Urinary Extracellular Vesicles Identifies AGP1 as a Potential Biomarker of Primary Aldosteronism.

CONTEXT: Primary aldosteronism (PA) represents 6% to 10% of all essential hypertension patients and is diagnosed using the aldosterone-to-renin ratio (ARR) and confirmatory studies. The complexity of PA diagnosis encourages the identification of novel PA biomarkers. Urinary extracellular vesicles (uEVs) are a potential source of biomarkers, considering that their cargo reflects the content of the parent cell. OBJECTIVE: We aimed to evaluate the proteome of uEVs from PA patients and identify potential biomarker candidates for PA. METHODS: Second morning spot urine was collected from healthy controls (n = 8) and PA patients (n = 7). The uEVs were isolated by ultracentrifugation and characterized. Proteomic analysis on uEVs was performed using LC-MS Orbitrap. RESULTS: Isolated uEVs carried extracellular vesicle markers, showed a round shape and sizes between 50 and 150 nm. The concentration of uEVs showed a direct correlation with urinary creatinine (r = 0.6357; P = 0.0128). The uEV size mean (167 ±â€…6 vs 183 ±â€…4nm) and mode (137 ±â€…7 vs 171 ±â€…11nm) was significantly smaller in PA patients than in control subjects, but similar in concentration. Proteomic analysis of uEVs from PA patients identified an upregulation of alpha-1-acid glycoprotein 1 (AGP1) in PA uEVs, which was confirmed using immunoblot. A receiver operating characteristic curve analysis showed an area under the curve of 0.92 (0.82 to 1; P = 0.0055). CONCLUSION: Proteomic and further immunoblot analyses of uEVs highlights AGP1 as potential biomarker for PA.

Dissecting the regulatory roles of ORM proteins in the sphingolipid pathway of plants.

Sphingolipids are a vital component of plant cellular endomembranes and carry out multiple functional and regulatory roles. Different sphingolipid species confer rigidity to the membrane structure, facilitate trafficking of secretory proteins, and initiate programmed cell death. Although the regulation of the sphingolipid pathway is yet to be uncovered, increasing evidence has pointed to orosomucoid proteins (ORMs) playing a major regulatory role and potentially interacting with a number of components in the pathway, including both enzymes and sphingolipids. However, experimental exploration of new regulatory interactions is time consuming and often infeasible. In this work, a computational approach was taken to address this challenge. A metabolic network of the sphingolipid pathway in plants was reconstructed. The steady-state rates of reactions in the network were then determined through measurements of growth and cellular composition of the different sphingolipids in Arabidopsis seedlings. The Ensemble modeling framework was modified to accurately account for activation mechanisms and subsequently used to generate sets of kinetic parameters that converge to the measured steady-state fluxes in a thermodynamically consistent manner. In addition, the framework was appended with an additional module to automate screening the parameters and to output models consistent with previously reported network responses to different perturbations. By analyzing the network's response in the presence of different combinations of regulatory mechanisms, the model captured the experimentally observed repressive effect of ORMs on serine palmitoyltransferase (SPT). Furthermore, predictions point to a second regulatory role of ORM proteins, namely as an activator of class II (or LOH1 and LOH3) ceramide synthases. This activating role was found to be modulated by the concentration of free ceramides, where an accumulation of these sphingolipid species dampened the activating effect of ORMs on ceramide synthase. The predictions pave the way for future guided experiments and have implications in engineering crops with higher biotic stress tolerance.

miR-362-3p Targets Orosomucoid 1 to Promote Cell Proliferation, Restrain Cell Apoptosis and Thereby Mitigate Hypoxia/Reoxygenation-Induced Cardiomyocytes Injury.

This study aimed to investigate the mechanism of how miR-362-3p/orosomucoid 1 (ORM1) involved in hypoxia/reoxygenation (H/R)-induced cardiomyocytes injury. Based on data obtained from Gene Expression Omnibus (GEO) database, we revealed that ORM1 was highly expressed and positively correlated with the expression of inflammatory factors (MAPK1, MAPK3, IL1B and CASP9). miR-362-3p was identified as an upstream regulatory miRNA of ORM1 and negatively modulated the mRNA and protein expression levels of ORM1 in H/R-injured cardiomyocytes. Moreover, we found that miR-362-3p was downregulated in cardiomyocytes injured by H/R. The promoting influence of miR-362-3p mimic on the proliferation and the inhibitory effect of miR-362-3p mimic on the apoptosis of H/R-stimulated cardiomyocytes were eliminated by overexpression of ORM1. Furthermore, miR-362-3p affected the expression of MAPK1, MAPK3, IL1B and CASP9 in H/R-injured cardiomyocytes through targeting ORM1. Our outcomes illustrated that miR-362-3p exhibited a protective influence on H/R-induced cardiomyocytes through targeting ORM1.

Canine orosomucoid (alpha-1 acid glycoprotein) variants and their influence on drug plasma protein binding.

Orosomucoid polymorphisms influence plasma drug binding in humans; however, canine variants and their effect on drug plasma protein binding have not yet been reported. In this study, the orosomucoid gene (ORM1) was sequenced in 100 dogs to identify the most common variant and its allele frequency determined in 1,464 dogs (from 64 breeds and mixed-breed dogs). Plasma protein binding extent of amitriptyline, indinavir, verapamil, and lidocaine were evaluated by equilibrium dialysis using plasma from ORM1 genotyped dogs (n = 12). Free and total drug plasma concentrations were quantified by liquid chromatography-mass spectrometry. From the five polymorphisms identified in canine ORM1, two were nonsynonymous. The most common was c.70G>A (p.Ala24Thr) with an allele frequency of 11.2% (n = 1464). Variant allele frequencies varied by breed, reaching 74% in Shetland Sheepdogs (n = 21). Free drug fractions did not differ significantly (p > .05; Mann-Whitney U) between plasma collected from dogs with c.70AA (n = 4) and those with c.70GG (n = 8) genotypes. While c.70G>A did not affect the extent of plasma protein binding in our study, the potential biological and pharmacological implication of this newly discovered ORM1 variant in dogs should be further investigated.

Erythromycin has therapeutic efficacy on muscle fatigue acting specifically on orosomucoid to increase muscle bioenergetics and physiological parameters of endurance.

At present, there are still no official or semi-official recommendations for the treatment of muscle fatigue. We previously reported that acute phase protein orosomucoid (ORM) can enhance muscle endurance and exert anti-fatigue effect. In attempting to seek anti-fatigue drugs that target ORM, we found macrolide antibiotics, particularly erythromycin, were effective. Erythromycin can significantly prolong the time of mice forced-swimming and treadmill running, increase muscle fatigue index, alleviate fatigue-induced tissue damage, and elevate glycogen content, mitochondria function and ATP level in the muscle. Also, erythromycin increases ORM protein expression in a dose- and time- dependent manner both in vitro and in vivo. Further studies found that erythromycin could increase the activity of ORM promoter and the stability of ORM mRNA, which might both be responsible for the ORM up-regulation. ORM knockdown or knockout could abolish the promoting effect of erythromycin in mice forced-swimming time, muscle fatigue index and glycogen level. Furthermore, those effects were also abolished in mice with C-C motif chemokine receptor 5 (CCR5) antagonist administration or AMPKα2 deficiency. Therefore, erythromycin could enhance muscle glycogen and endurance via up-regulating the level of ORM and activating CCR5-AMPK pathway, indicating it might act as a potential drug to treat muscle fatigue.

Dramatically changed immune-related molecules as early diagnostic biomarkers of non-small cell lung cancer.

Non-small cell lung cancer (NSCLC) is the main type of lung cancer, with a low 5-year survival rate because of the absence of effective clinical biomarkers for early diagnosis. Based on the immunosurveillance theory, we proposed that changes in the immune system are more pronounced than tumour-associated antigens during the early stage of cancer. Therefore, a new strategy was designed to screen early diagnostic biomarkers from peripheral leukocytes in early-stage NSCLCs with transcriptome sequencing. A total of 358 immune-related differentially expressed genes were identified between early-NSCLC patients and healthy individuals. Orosomucoid-1 (ORM1, a acute phase protein), the total ORM and chitotriosidase-1 (involved in degradation of chitobiose) were selected for further verification in 210 serum samples by western blotting, ELISA and nephelometry immunoassay (based on immuno-scatter turbidmetry). Receiver operating characteristic curve analysis show that ORM1 and total ORM have excellent diagnostic efficacies, with area under the curve of 0.862 and 0.920, respectively, which significantly distinguished very early-NSCLC (IA) from healthy samples. Flow cytometry results showed that CD15+ neutrophils made up 73% of ORM1+ peripheral leukocytes. In mouse lung cancer model, serum ORM1, but not liver ORM1, changed significantly in the early stage of NSCLC. ORM1 expression in peripheral leukocytes was regulated by TGF-ß and mediated by the TGF-ß/Smad signalling pathway. Our results indicated that combined ORM and TGF-ß could be a promising clinical biomarker in the diagnosis of early NSCLC.

Fucosylated α1-acid glycoprotein as a biomarker to predict prognosis following tumor immunotherapy of patients with lung cancer.

Immunotherapy targeting immune checkpoint molecules has provided remarkable clinical benefits in cancer patients but no clinically relevant biomarker for predicting treatment outcomes exists. Recently, we demonstrated that glycan structures of serum α1-acid glycoprotein (AGP) changed dramatically in cancer patients and that α1,3fucosylated AGP (fAGP) levels increased along with disease progression and decreased responding to chemotherapy treatments. Here, the fAGP was analyzed in sera prospectively obtained from 39 patients with advanced lung cancer who underwent immunotherapy with anti-PD-1 antibody, nivolumab. Twenty-three patients had significantly high fAGP levels above the cut-off value (H-fAGP) at one month after starting the treatment and 20 patients in this group, whose tumor sizes did not decrease, maintained high fAGP levels continuously and subsequently died. However, the other 16 patients, whose fAGP levels decreased or maintained below the cut-off value (L-fAGP), survived during a 2-year observation even though 5 patients in this group had no tumor shrinkage. Accordingly, the overall survival rate was found to significantly correlate with the fAGP level. Multivariate analyses revealed that the H-fAGP was an independent risk factor for cancer progression. Therefore, the fAGP level appeared to be a reliable biomarker for predicting clinical efficacy of immunotherapy with nivolumab.

Alpha-1-acid glycoprotein 1 is upregulated in pancreatic ductal adenocarcinoma and confers a poor prognosis.

Pancreatic cancer is an aggressive malignancy that carries a high mortality rate. A major contributor to the poor outcome is the lack of effective molecular markers. The purpose of this study was to develop protein markers for improved prognostication and noninvasive diagnosis. A mass spectrometry (MS)-based discovery approach was applied to pancreatic cancer tissues and healthy pancreas. In the verification phase, extracellular proteins with differential expression were further quantified in targeted mode using parallel reaction monitoring (PRM). Next, a tissue microarray (TMA) cohort including 140 pancreatic cancer resection specimens was constructed, in order to validate protein expression status and investigate potential prognostic implications. The levels of protein candidates were finally assessed in a prospective series of 110 serum samples in an accredited clinical laboratory using the automated Cobas system. Protein sequencing with nanoliquid chromatography tandem MS (nano-LC-MS/MS) and targeted PRM identified alpha-1-acid glycoprotein 1 (AGP1) as an upregulated protein in pancreatic cancer tissue. Using TMA and immunohistochemistry, AGP1 expression was significantly associated with shorter overall survival (HR = 2.22; 95% CI 1.30-3.79, P = 0.004). Multivariable analysis confirmed the results (HR = 1.87; 95% CI 1.08-3.24, P = 0.026). Circulating levels of AGP1 yielded an area under the curve (AUC) of 0.837 for the discrimination of resectable pancreatic cancer from healthy controls. Combining AGP1 with CA 19-9 enhanced the diagnostic performance, with an AUC of 0.963. This study suggests that AGP1 is a novel prognostic biomarker in pancreatic cancer tissue. Serum AGP1 levels may be useful as part of a biomarker panel for early detection of pancreatic cancer but further studies are needed.

Role of acute-phase protein ORM in a mice model of ischemic stroke.

The only Food and Drug Administration-approved treatment for acute ischemic stroke is tissue plasminogen activator, and the discovery of novel therapeutic targets is critical. Here, we found orosomucoid (ORM), an acute-phase protein mainly produced by the liver, might act as a treatment candidate for an ischemic stroke. The results showed that ORM2 is the dominant subtype in mice normal brain tissue. After middle cerebral artery occlusion (MCAO), the level of ORM2 is significantly increased in the ischemic penumbra compared with the contralateral normal brain tissue, whereas ORM1 knockout did not affect the infarct size. Exogenous ORM could significantly decrease infarct size and neurological deficit score. Inspiringly, the best administration time point was at 4.5 and 6 hr after MCAO. ORM could markedly decrease the Evans blue extravasation, and improve blood-brain barrier-associated proteins expression in the ischemic penumbra of MACO mice and oxygen-glucose deprivation (OGD)-treated bEnd3 cells. Meanwhile, ORM could significantly alleviate inflammation by inhibiting the production of interleukin 1ß (IL-1ß), IL-6, and tumor necrosis factor α (TNF-α), reduce oxidative stress by improving the balance of malondialdehyde (MDA) and superoxide dismutase (SOD), inhibit apoptosis by decreasing caspase-3 activity in ischemic penumbra of MCAO mice and OGD-treated bEnd.3 cells. Because of its protective role at multiple levels, ORM might be a promising therapeutic target for ischemic stroke.

A downstream molecule of 1,25-dihydroxyvitamin D3, alpha-1-acid glycoprotein, protects against mouse model of renal fibrosis.

Renal fibrosis, the characteristic feature of progressive chronic kidney disease, is associated with unremitting renal inflammation. Although it is reported that 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), the active form of vitamin D, elicits an anti-renal fibrotic effect, its molecular mechanism is still unknown. In this study, renal fibrosis and inflammation observed in the kidney of unilateral ureteral obstruction (UUO) mice were reduced by the treatment of 1,25(OH)2D3. The plasma protein level of alpha-1-acid glycoprotein (AGP), a downstream molecule of 1,25(OH)2D3, was increased following administration of 1,25(OH)2D3. Additionally, increased mRNA expression of ORM1, an AGP gene, was observed in HepG2 cells and THP-1-derived macrophages that treated with 1,25(OH)2D3. To investigate the involvement of AGP, exogenous AGP was administered to UUO mice, resulting in attenuated renal fibrosis and inflammation. We also found the mRNA expression of CD163, a monocyte/macrophage marker with anti-inflammatory potential, was increased in THP-1-derived macrophages under stimulus from 1,25(OH)2D3 or AGP. Moreover, AGP prevented lipopolysaccharide-induced macrophage activation. Thus, AGP could be a key molecule in the protective effect of 1,25(OH)2D3 against renal fibrosis. Taken together, AGP may replace vitamin D to function as an important immune regulator, offering a novel therapeutic strategy for renal inflammation and fibrosis.