Identification of kynurenine and quinolinic acid as promising serum biomarkers for drug-induced interstitial lung diseases.

BACKGROUND: Drug-induced interstitial lung disease (DILD) is a lung injury caused by various types of drugs and is a serious problem in both clinical practice and drug development. Clinical management of the condition would be improved if there were DILD-specific biomarkers available; this study aimed to meet that need. METHODS: Biomarker candidates were identified by non-targeted metabolomics focusing on hydrophilic molecules, and further validated by targeted approaches using the serum of acute DILD patients, DILD recovery patients, DILD-tolerant patients, patients with other related lung diseases, and healthy controls. RESULTS: Serum levels of kynurenine and quinolinic acid (and kynurenine/tryptophan ratio) were elevated significantly and specifically in acute DILD patients. The diagnostic potentials of these biomarkers were superior to those of conventional lung injury biomarkers, Krebs von den Lungen-6 and surfactant protein-D, in discriminating between acute DILD patients and patients with other lung diseases, including idiopathic interstitial pneumonia and lung diseases associated with connective tissue diseases. In addition to identifying and evaluating the biomarkers, our data showed that kynurenine/tryptophan ratios (an indicator of kynurenine pathway activation) were positively correlated with serum C-reactive protein concentrations in patients with DILD, suggesting the potential association between the generation of these biomarkers and inflammation. Our in vitro experiments demonstrated that macrophage differentiation and inflammatory stimulations typified by interferon gamma could activate the kynurenine pathway, resulting in enhanced kynurenine levels in the extracellular space in macrophage-like cell lines or lung endothelial cells. Extracellular quinolinic acid levels were elevated only in macrophage-like cells but not endothelial cells owing to the lower expression levels of metabolic enzymes converting kynurenine to quinolinic acid. These findings provide clues about the molecular mechanisms behind their specific elevation in the serum of acute DILD patients. CONCLUSIONS: The serum concentrations of kynurenine and quinolinic acid as well as kynurenine/tryptophan ratios are promising and specific biomarkers for detecting and monitoring DILD and its recovery, which could facilitate accurate decisions for appropriate clinical management of patients with DILD.

Significant association between HLA-B*35:01 and onset of drug-induced liver injury caused by Kampo medicines in Japanese patients.

AIM: Drug-induced liver injury (DILI) is a severe and life-threatening immune-mediated adverse effect, occurring rarely among treated patients. We examined genomic biomarkers in the Japanese population that predict the onset of DILI after using a certain class of drugs, such as Kampo products (Japanese traditional medicines). METHODS: A total of 287 patients diagnosed as DILI by hepatology specialists were recruited after written informed consent was obtained. A genome-wide association analysis and human leukocyte antigen (HLA) typing in four digits were performed. RESULTS: We found a significant association (p = 9.41 × 10-10 ) of rs146644517 (G > A) with Kampo product-related DILI. As this polymorphism is located in the HLA region, we evaluated the association of HLA types and found that 12 (63.2%) of 19 Kampo-DILI patients contained HLA-B*35:01, whereas only 15.2% were positive for this HLA among healthy volunteers. The odds ratio was 9.56 (95% confidence interval 3.75-24.46; p = 2.98 × 10-6 , corrected p = 4.17 × 10-5 ), and it increased to 13.55 compared with the DILI patients not exposed to Kampo products. The individual crude drug components in the Kampo products, including Scutellaria root (ougon in Japanese), rhubarb (daiou), Gardenia fruit (sanshishi), and Glycyrrhiza (kanzou), were significantly associated with HLA-B*35:01. CONCLUSIONS: HLA-B*35:01 is a genetic risk factor and a potential predictive biomarker for Kampo-induced DILI in the Japanese population.

Identification and characterization of lysophosphatidylcholine 14:0 as a biomarker for drug-induced lung disease.

Drug-induced interstitial lung disease (DILD) occurs when drug exposure causes inflammation of the lung interstitium. DILD can be caused by different types of drugs, and some DILD patterns results in a high mortality rate; hence, DILD poses a serious problem in clinical practice as well as drug development, and strategies to diagnose and distinguish DILD from other lung diseases are necessary. We aimed to identify novel biomarkers for DILD by performing lipidomics analysis on plasma samples from patients with acute and recovery phase DILD. Having identified lysophosphatidylcholines (LPCs) as candidate biomarkers for DILD, we determined their concentrations using validated liquid chromatography/mass spectrometry biomarker assays. In addition, we evaluated the ability of LPCs to discriminate patients with acute phase DILD from those with recovery phase DILD, DILD-tolerant, or other lung diseases, and characterized their association with clinical characteristics. Lipidomics analysis revealed a clear decrease in LPC concentrations in the plasma of patients with acute phase DILD. In particular, LPC(14:0) had the highest discriminative index against recovery phase and DILD-tolerant patients. LPC(14:0) displayed no clear association with causal drugs, or subjects' backgrounds, but was associated with disease severity. Furthermore, LPC(14:0) was able to discriminate between patients with DILD and other lung diseases, including idiopathic interstitial pneumonia and lung disease associated with connective tissue disease. LPC(14:0) is a promising biomarker for DILD that could improve the diagnosis of DILD and help to differentiate DILD from other lung diseases, such as idiopathic interstitial pneumonia and connective tissue disease.

Stratifin as a novel diagnostic biomarker in serum for diffuse alveolar damage.

Among the various histopathological patterns of drug-induced interstitial lung disease (DILD), diffuse alveolar damage (DAD) is associated with poor prognosis. However, there is no reliable biomarker for its accurate diagnosis. Here, we show stratifin/14-3-3σ (SFN) as a biomarker candidate found in a proteomic analysis. The study includes two independent cohorts (including totally 26 patients with DAD) and controls (total 432 samples). SFN is specifically elevated in DILD patients with DAD, and is superior to the known biomarkers, KL-6 and SP-D, in discrimination of DILD patients with DAD from patients with other DILD patterns or other lung diseases. SFN is also increased in serum from patients with idiopathic DAD, and in lung tissues and bronchoalveolar lavage fluid of patients with DAD. In vitro analysis using cultured lung epithelial cells suggests that extracellular release of SFN occurs via p53-dependent apoptosis. We conclude that serum SFN is a promising biomarker for DAD diagnosis.

Characterization of serotonin as a candidate biomarker of severity and prognosis of COVID-19 using LC/MS analysis.

The coronavirus disease 2019 (COVID-19) pandemic has been associated with high mortality worldwide. Owing to its complicated pathophysiology, diagnostic and prognostic biomarkers for effective patient management remain scarce. We analyzed kynurenine, tryptophan, and serotonin levels in the serum of patients with COVID-19 via liquid chromatography/mass spectrometry analysis. Serum serotonin levels were decreased in patients with more severe COVID-19, along with increased kynurenine and decreased tryptophan concentrations. Patients with moderate disease who subsequently worsened showed significantly lower serotonin concentrations compared with those who did not experience severe disease. Serum serotonin levels may represent a valuable biomarker for COVID-19 severity and prognosis.

Serum stratifin and presepsin as candidate biomarkers for early detection of COVID-19 disease progression.

The prognosis of patients with severe cases of COVID-19 is poor; thus, biomarkers for earlier prediction of COVID-19 progression are vital. We measured levels of five lung injury-related biomarkers, SP-D, KL-6, presepsin, kallistatin and stratifin, in serum samples collected serially during hospitalization from 31 patients with mild/moderate or severe/critical COVID-19 pneumonia, and their predictive performances were compared. Like the previously reported presepsin, a new biomarker candidate, stratifin, was significantly elevated with the onset of severe or critical symptoms in COVID-19 patients and decreased with symptom improvement. Notably, changes in stratifin and presepsin levels were distinctly earlier than those in SP-D, KL-6 and even SpO2/FiO2 values. Furthermore, serum levels of these biomarkers were significantly higher at the pre-severe stage (before the start of oxygen support) of patients who eventually advanced to severe/critical stages than in the patients who remained at the mild/moderate stage. These results were confirmed in an independent cohort, including 71 mild/moderate and 14 severe/critical patients, for whom the performance of stratifin and presepsin in discriminating between mild/moderate and pre-severe conditions of COVID-19 patients was superior to that of the SpO2/FiO2 ratio. Therefore, we concluded that stratifin and presepsin could be used as prognostic biomarkers for severe COVID-19 progression.

Frequency of null genotypes of glutathione S-transferase M1 and T1 in Japanese patients with drug-induced liver injury.

AIM: Previous reports suggest that the null genotype (*0/*0) of glutathione S-transferase (GST) M1 and/or GSTT1 could be risk factors for drug-induced liver injury (DILI). However, multi-institutional pharmacogenetic research with various suspected drugs has rarely been performed in Japan. Therefore, the aim of this study was to investigate the role of GSTM1 and GSTT1 null genotype in the occurrence of DILI in Japanese patients. METHODS: Blood samples of 270 DILI patients from 23 hospitals throughout Japan collected between 2010 and 2018 were subjected to genotyping of null genotypes of GSTM1 and GSTT1 using the SmartAmp-2 method. We also collected information on DILI types, time to onset of DILI, pharmacological classification of suspected drugs and Digestive Disease Week-Japan score, as well as genotypes of GSTM1 and GSTT1 in each patient with DILI. RESULTS: The distribution of a combination of null genotypes of GSTM1 and GSTT1 in Japanese patients with DILI was significantly different from that reported in the general Japanese population. Notably, the incidence of the GSTM1 null genotype in patients with DILI was significantly higher than that of the control population. A significant relationship between the frequency of GSTM1 and GSTT1 null genotypes and pharmacological classification of suspected drugs, clinical laboratory data for liver function, time to onset of DILI, and Digestive Disease Week-Japan scores was not observed. CONCLUSIONS: The GSTM1 null genotype was associated with an increased incidence of DILI in Japanese patients.

Cross-reactive humoral immune responses against seasonal human coronaviruses in COVID-19 patients with different disease severities.

BACKGROUND: The cross-reactive antibody response against seasonal human coronaviruses (HCoVs) was evaluated according to disease severity in patients with COVID-19 in Japan. METHODS: In total, 194 paired serum samples collected from 97 patients with COVID-19 (mild, 35; severe, 62) were analyzed on admission and during convalescence. IgG antibodies against the nucleocapsid (N) and spike (S) proteins of SARS-CoV-2 and four seasonal HCoVs (HCoV-NL63, -229E, -OC43, and -HKU1) were detected by enzyme-linked immunosorbent assays. RESULTS: There was no difference in optical density (OD) values for seasonal HCoVs on admission between the severe and mild cases. In addition, a specific pattern of disease severity-associated OD values for HCoVs was not identified. Significant increases in OD values from admission to convalescence for HCoV-HKU1and -OC43 IgG-S, and for HCoV-NL63 and -229E IgG-N were observed in the severe cases. Significant differences were observed between the mild and severe cases for HCoV-HKU1 and -OC43 IgG-S OD values during convalescence. Correlations were found between the fold changes for HCoV-OC43 IgG-S OD values, and for SARS-CoV-2 IgG-S OD values, and C-reactive protein, lactate dehydrogenase, and lymphocyte levels. CONCLUSION: There was no association between the antibody titer for seasonal HCoVs in the early phase of COVID-19 and disease severity.

Phosphorylation of Ser1452 on BRG1 inhibits the function of the SWI/SNF complex in chromatin activation.

BRG1, one of core subunits of the SWI/SNF chromatin remodeling complex, is frequently mutated in cancers. Previously, we reported significant downregulation of the phosphorylation level of BRG1 on Ser1452 (<10%) in cell lines derived from ovarian clear cell carcinoma with frequent recurrence and acquired drug resistance. In this study, we tried to elucidate the roles of BRG1 phosphorylation, using cell lines expressing wild-type, phosphorylation-mimic (brg1-S1452D), or non-phosphorylatable (brg1-S1452A) BRG1. Quantitative proteomic analyses revealed upregulation of proteins and phosphoproteins related to linker histone H1s, histone methylation, and protein ubiquitylation in brg1-S1452D cells, which may coordinately promote the chromatin inactivation and ubiquitin-dependent degradation of target proteins. Consistent with these results, brg1-S1452D cells exhibited an increase in condensed chromatin and polyubiquitylated proteins. In brg1-S1452D cells, we also detected downregulation of various cancer-related proteins (e.g., EGFR and MET) as well as decreased migration, proliferation, and sensitivity to taxanes and oxaliplatin. Together, our results reveal that BRG1 phosphorylation drives tumor malignancy by inhibiting the functions of SWI/SNF complex in chromatin activation, thereby promoting expression of various cancer-related proteins. SIGNIFICANCE: For the first time we demonstrated that the mutation on Ser1452 phosphorylation site of BRG1, a component of SWI/SNF chromatin remodeling complex, changed protein and phosphoprotein levels of linker histone H1s, binding competitor of histone H1s, and histone methylase/demethylase involved in the heterochromatic histone modifications to promote the chromatin inactivation. In phosphorylation-mimic mutant, significant decrease of various cancer-related proteins as well as migration, proliferation, and sensitivity to specific antitumor agents were detected. Our results reveal that BRG1 phosphorylation drives tumor malignancy by inhibiting the functions of SWI/SNF complex in chromatin activation, thereby promoting expression of various cancer-related proteins.

Validation of tissue factor pathway inhibitor 2 as a specific biomarker for preoperative prediction of clear cell carcinoma of the ovary.

BACKGROUND: Tissue factor pathway inhibitor 2 (TFPI2) is a novel serum biomarker that discriminates ovarian clear cell carcinoma (CCC) from borderline ovarian tumors (BOTs) and non-clear cell epithelial ovarian cancers (EOCs). Here, we examined the performance of TFPI2 for preoperative diagnosis of CCC. METHODS: Serum samples were obtained preoperatively from patients with ovarian masses, who needed surgical treatment at five hospitals in Japan. The diagnostic powers of TFPI2 and cancer antigen 125 (CA125) serum levels to discriminate CCC from BOTs, other EOCs, and benign lesions were compared. RESULTS: A total of 351 patients including 69 CCCs were analyzed. Serum TFPI2 levels were significantly higher in CCC patients (mean ± SD, 508.2 ± 812.0 pg/mL) than in patients with benign lesions (154.7 ± 46.5), BOTs (181 ± 95.5) and other EOCs (265.4 ± 289.1). TFPI2 had a high diagnostic specificity for CCC (79.5%). In patients with benign ovarian endometriosis, no patient was positive for TFPI2, but 71.4% (15/21) were CA125 positive. TFPI2 showed good performance in discriminating stage II-IV CCC from BOTs and other EOCs (AUC 0.815 for TFPI2 versus 0.505 for CA125) or endometriosis (AUC 0.957 for TFPI2 versus 0.748 for CA125). The diagnostic sensitivity of TFPI2 to discriminate CCC from BOTs and other EOCs was improved from 43.5 to 71.0% when combined with CA125. CONCLUSIONS: High specificity of TFPI2 for preoperative detection of CCC was verified with the defined cutoff level of TFPI2 in clinical practice. TFPI2 and CA125 may contribute substantially to precise prediction of intractable CCC.

Plasma Lipid Profiling of Three Types of Drug-Induced Liver Injury in Japanese Patients: A Preliminary Study.

Drug-induced liver injury (DILI) is a major adverse event caused by drug treatment, which can be categorized into three types: hepatocellular, mixed, and cholestatic. Although nearly every class of drugs can cause DILI, an overall understanding of lipid profiles in DILI patients is lacking. We used lipidomics to analyze the plasma lipid profiles of patients to understand their hepatic pathophysiology and identify DILI biomarkers. We identified 463 lipids and compared their levels between the acute and recovery phases of the three types of DILI patients. Mixed and cholestatic types demonstrated specific plasma lipid alterations between the phases, but the hepatocellular type did not. Moreover, as specific indicators of mixed-type DILI, levels of several ceramides increased in the acute phase, while those of arachidonic acid-containing ether-linked phosphoglycerolipids decreased. In contrast, as specific indicators of cholestatic-type DILI, levels of palmitic acid-containing saturated or monounsaturated phosphatidylcholines increased in the acute phase, while those of arachidonic acid- or docosahexaenoic acid-containing ether-linked phosphoglycerolipids and phosphatidylinositols decreased. We also identified lipids with a relatively high capacity to discriminate the acute phase from the recovery phase and healthy subjects. These findings may help with understanding the pathophysiology of different DILI types and identify candidate biomarkers.

Hepatic Adaptation to Therapeutic Doses of Acetaminophen: An Exploratory Study in Healthy Individuals.

PURPOSE: Acetaminophen (APAP) has hepatotoxic potential when overdosed. Recent studies have reported serum alanine aminotransferase (ALT) elevations that resolve spontaneously with continued use of the drug, referred to as adaptation, in several individuals receiving therapeutic doses of APAP. However, the clinical significance of these ALT elevations remains unclear. This study was performed to investigate the incidence and characteristics of hepatic adaptation to therapeutic doses of APAP in healthy individuals. METHODS: In a randomized, single-blind, placebo-controlled study, 242 healthy Japanese individuals were enrolled. Each person received 3 g/d of APAP (n = 202) or placebo (n = 40) for 28 days. All study participants underwent analysis of genetic polymorphisms of CYP2E1 and UGT1A1; measurements of plasma APAP concentration and urine metabolites (glucuronide, sulfate, cysteine, and mercapturate); liver function monitoring, including ALT, microRNA-122, and high-mobility group box 1. Individuals with ALT levels remaining below the upper limit of normal (ULN; 40 U/L) during the study period were defined as tolerant and those with ALT elevations above the ULN as susceptible. Susceptible individuals who developed ALT elevations exceeding 2 × ULN discontinued use of the study drug for tolerability consideration. Susceptible individuals who had ALT elevations that decreased toward the ULN spontaneously with continued use of the study drug were classified as adaptation. FINDINGS: In the APAP group, 129 individuals (66%) were classified as tolerant and 65 (34%) as susceptible. Among 65 susceptible individuals, 12 (18%) discontinued use of APAP because of ALT elevations (>2 × ULN), whereas 53 (82%) completed 28-day APAP dosing. Thirty of 65 susceptible individuals (46%) had adaptation within 28 days. In the placebo group, no individuals was withdrawn from the study because of elevated ALT levels, 33 individuals (89%) were classified as tolerant, and 4 (11%) were classified as susceptible. None had clinical signs of liver injury. ALT level correlated significantly with microRNA-122 but not with high-mobility group box 1. No association was found between plasma APAP concentrations and ALT levels. Urinary excretion of APAP mercapturate was higher in susceptible than in tolerant individuals (P = 0.018, Wilcoxon or Kruskal-Wallis test). The frequency of homozygotes and compound heterozygotes for UGT1A1∗28 and UGT1A1∗6 (∗28/∗28, ∗6/∗6, and ∗6/∗28) was higher in susceptible than in tolerant individuals (13.9% vs 3.9%; P = 0.011, χ2 test). IMPLICATIONS: These findings indicate that in healthy individuals, APAP at a therapeutic dose can cause transient and self-limiting ALT elevation, reflecting subclinical hepatocellular damage, and these ALT elevations may be associated with the disposition of APAP metabolites and genetic factors. UMIN-CTR identifier: UMIN000019607.

Proteomic analysis of aortic smooth muscle cell secretions reveals an association of myosin heavy chain 11 with abdominal aortic aneurysm.

Abdominal aortic aneurysm (AAA) is a life-threatening disease, and no disease-specific circulating biomarkers for AAA screening are currently available. We have identified a smooth muscle cell (SMC)-specific biomarker for AAA. We cultured aneurysmal tunica media that were collected from eight patients undergoing elective open-repair surgeries. Secreted proteins in culture medium were subjected to liquid chromatography/tandem mass spectrometry. Myosin heavy chain 11 (myosin-11) was identified as a SMC-specific protein in the tunica media-derived secretions of all patients. We then examined myosin-11 protein concentrations by ELISA in plasma samples from patients with AAA ( n = 35) and age-matched healthy control subjects ( n = 34). Circulating myosin-11 levels were significantly higher in patients with AAA than control subjects. The area under the receiver-operating characteristic curve (AUC) of myosin-11 was 0.77, with a specificity of 65% at a sensitivity of 91%. Multivariate logistic regression analysis showed a significant association between the myosin-11 level and presence of AAA. When the myosin-11 level was combined with hypertension, it improved the prediction of AAA (AUC 0.88) more than hypertension per se. We then investigated the correlation between aortic diameter and circulating myosin-11 levels using AAA serum samples from patients undergoing endovascular aneurysm repair ( n = 20). Circulating myosin-11 levels were significantly correlated with maximum aortic diameter. Furthermore, changes in myosin-11 concentrations from the baseline 12 mo after endovascular aneurysm repair were associated with those in aortic diameter. These data suggest that circulating levels of myosin-11, which is a SMC-specific myosin isoform, may be useful as a biomarker for AAA. NEW & NOTEWORTHY Extensive studies have revealed that inflammation- or proteolysis-related proteins are proposed as biomarkers for abdominal aortic aneurysm (AAA). Changes in these protein concentrations are not specific for smooth muscle, which is a major part of AAA pathologies. Hence, no disease-specific circulating markers for AAA are currently available. We found, using secretome-based proteomic analysis on human AAA tunica media, that myosin heavy chain 11 was associated with AAA. Circulating myosin heavy chain 11 may be a new tissue-specific AAA marker.

In vitro mouse spermatogenesis with an organ culture method in chemically defined medium.

We previously reported the successful induction and completion of mouse spermatogenesis by culturing neonatal testis tissues. The culture medium consisted of α-minimum essential medium (α-MEM), supplemented with Knockout serum replacement (KSR) or AlbuMAX, neither of which were defined chemically. In this study, we formulated a chemically defined medium (CDM) that can induce mouse spermatogenesis under organ culture conditions. It was found that bovine serum albumin (BSA) purified through three different procedures had different effects on spermatogenesis. We also confirmed that retinoic acid (RA) played crucial roles in the onset of spermatogonial differentiation and meiotic initiation. The added lipids exhibited weak promoting effects on spermatogenesis. Lastly, luteinizing hormone (LH), follicle stimulating hormone (FSH), triiodothyronine (T3), and testosterone (T) combined together promoted spermatogenesis until round spermatid production. The CDM, however, was not able to produce elongated spermatids. It was also unable to induce spermatogenesis from the very early neonatal period, before 2 days postpartum, leaving certain factors necessary for spermatogenic induction in mice unidentified. Nonetheless, the present study provided important basic information on testis organ culture and spermatogenesis in vitro.

Depressive-Like Behaviors Are Regulated by NOX1/NADPH Oxidase by Redox Modification of NMDA Receptor 1.

Involvement of reactive oxygen species (ROS) has been suggested in the development of psychiatric disorders. NOX1 is a nonphagocytic form of NADPH oxidase whose expression in the nervous system is negligible compared with other NOX isoforms. However, NOX1-derived ROS increase inflammatory pain and tolerance to opioid analgesia. To clarify the role of NOX1 in the brain, we examined depressive-like behaviors in mice deficient in Nox1 (Nox1-/Y). Depressive-like behaviors induced by chronic social defeat stress or administration of corticosterone (CORT) were significantly ameliorated in Nox1-/Y Generation of ROS was significantly elevated in the prefrontal cortex (PFC) of mice administrated with CORT, while NOX1 mRNA was upregulated only in the ventral tegmental area (VTA) among brain areas responsible for emotional behaviors. Delivery of miRNA against NOX1 to VTA restored CORT-induced depressive-like behaviors in wild-type (WT) littermates. Administration of CORT to WT, but not to Nox1-/Y, significantly reduced transcript levels of brain-derived neurotrophic factor (bdnf), with a concomitant increase in DNA methylation of the promoter regions in bdnf Delivery of miRNA against NOX1 to VTA restored the level of BDNF mRNA in WT PFC. Redox proteome analyses demonstrated that NMDA receptor 1 (NR1) was among the molecules redox regulated by NOX1. In cultured cortical neurons, hydrogen peroxide significantly suppressed NMDA-induced upregulation of BDNF transcripts in NR1-expressing cells but not in cells harboring mutant NR1 (C744A). Together, these findings suggest a key role of NOX1 in depressive-like behaviors through NR1-mediated epigenetic modification of bdnf in the mesoprefrontal projection.SIGNIFICANCE STATEMENT NADPH oxidase is a source of reactive oxygen species (ROS) that have been implicated in the pathogenesis of various neurological disorders. We presently showed the involvement of a nonphagocytic type of NADPH oxidase, NOX1, in major depressive disorders, including behavioral, biochemical, and anatomical changes in mice. The oxidation of NR1 by NOX1-derived ROS was demonstrated in prefrontal cortex (PFC), which may be causally linked to the downregulation of BDNF, promoting depressive-like behaviors. Given that NOX1 is upregulated only in VTA but not in PFC, mesocortical projections appear to play a crucial role in NOX1-dependent depressive-like behaviors. Our study is the first to present the potential molecular mechanism underlying the development of major depression through the NOX1-induced oxidation of NR1 and epigenetic modification of bdnf.

Clinical Significance of Tissue Factor Pathway Inhibitor 2, a Serum Biomarker Candidate for Ovarian Clear Cell Carcinoma.

BACKGROUND: There is currently no reliable serum biomarker for ovarian clear cell carcinoma (CCC), a highly lethal histological subtype of epithelial ovarian cancer (EOC). Previously, using a proteome-based approach, we identified tissue factor pathway inhibitor 2 (TFPI2) as a candidate serum biomarker for CCC. In this study, we sought to evaluate the clinical diagnostic performance of TFPI2 in preoperative prediction of CCC. METHODS: Serum TFPI2 levels were measured in serum samples from a retrospective training set consisting of patients with benign and borderline ovarian tumors, EOC subtypes, and uterine diseases. Via receiver operating characteristic (ROC) analyses, we compared the diagnostic performance of TFPI2 with that of CA125 in discrimination of patients with ovarian CCC from other patient groups. The observed diagnostic performances were examined in a prospective validation set. RESULTS: The 268-patient training set included 29 patients with ovarian CCC. Unlike CA125, which was also elevated in patients with endometriosis and several EOC subtypes, serum TFPI2 levels were specifically elevated only in ovarian CCC patients, consistent with the mRNA expression pattern in tumor tissues. The area under the ROC curve (AUC) of serum TFPI2 was obviously higher than that of CA125 for discrimination of CCC from other ovarian diseases (AUC = 0.891 versus 0.595). Applying a cut-off value of 280 pg/mL, TFPI2 could distinguish early-stage (FIGO I and II) CCC from endometriosis with 72.2% sensitivity, 93.3% specificity, and 88.8% accuracy. Similar results were confirmed in an independent 156-patient prospective validation set. CONCLUSIONS: TFPI2 is a useful serum biomarker for preoperative clinical diagnosis of CCC.

Data for identification of GPI-anchored peptides and ω-sites in cancer cell lines.

We present data obtained using a focused proteomics approach to identify the glycosylphosphatidylinositol (GPI)-anchored peptides in 19 human cancer cell lines. GPI-anchored proteins (GPI-APs), which localize to the outer leaflet of the membrane microdomains commonly referred to as lipid rafts play important roles in diverse biological processes. Due to the complex structure of the GPI-anchor moiety, it has been difficult to identify GPI-anchored peptide sequences on the proteomic scale by database searches using tools such as MASCOT. Here we provide data from 73 ω-sites derived from 49 GPI-APs in 19 human cancer cell lines. This article contains data related to the research article entitled "Identification of glycosylphosphatidylinositol-anchored proteins and ω-sites using TiO2-based affinity purification followed by hydrogen fluoride treatment" (Masuishi et al., 2016) [1].

Identification of glycosylphosphatidylinositol-anchored proteins and ω-sites using TiO2-based affinity purification followed by hydrogen fluoride treatment.

Glycosylphosphatidylinositol anchored proteins (GPI-APs) in the outer leaflet of the membrane microdomains, commonly referred to as lipid rafts, play important roles in many biological processes such as signal transduction, cell adhesion, protein trafficking, and antigen presentation. From a topological viewpoint, elucidating the presence and localization of GPI-anchor modification sites (ω-sites) is important for the study of the biophysical properties and anchoring mechanisms of these proteins. However, very few reports have actually identified ω-sites of GPI-APs. To enable large-scale site-specific analysis of GPI anchoring, we developed a method for identification of ω-sites by mass spectrometry by combining titanium dioxide-based affinity purification and hydrogen fluoride treatment. This method was able to identify ~3-fold more GPI-APs than our previous method: the new technique identified a total of 73 ω-sites derived from 49 GPI-APs. In 13 of the 49 GPI-APs identified, the GPI-anchor attached to multiple amino acids in the C-terminal site, yielding a variety of different protein species. This method allows us to simultaneously identify many GPI-AP protein species with different ω-sites. We also demonstrated the C-terminal GPI anchor attachment signal peptide, based on information about the GPI anchor binding sites of 49 GPI-APs. Thus, our results provide evidence for new insight into the GPI-anchored proteome and the role of GPI anchoring. BIOLOGICAL SIGNIFICANCE: GPI-anchored proteins (GPI-APs) are localized to the outer leaflet of the plasma membranes. Because the GPI anchor is a complex structure, the identification of GPI-anchored peptides by mass spectrometry has always been considered difficult. To improve the feasibility of large-scale site-specific analysis of GPI anchoring, we developed a method for identification of GPI-anchored peptides by combining titanium dioxide-based affinity purification with hydrogen fluoride treatment. Using this novel technique, we identified a total of 73 ω-sites derived from 49 GPI-APs. These data may help us to develop a comprehensive understanding of the GPI-anchored proteome and the role of GPI anchoring. Moreover, this method could be used to discover GPI-APs as candidate biomarkers.

Phosphoproteome analysis demonstrates the potential role of THRAP3 phosphorylation in androgen-independent prostate cancer cell growth.

Elucidating the androgen-independent growth mechanism is critical for developing effective treatment strategies to combat androgen-independent prostate cancer. We performed a comparative phosphoproteome analysis using a prostate cancer cell line, LNCaP, and an LNCaP-derived androgen-independent cell line, LNCaP-AI, to identify phosphoproteins involved in this mechanism. We performed quantitative comparisons of the phosphopeptide levels in tryptic digests of protein extracts from these cell lines using MS. We found that the levels of 69 phosphopeptides in 66 proteins significantly differed between LNCaP and LNCaP-AI. In particular, we focused on thyroid hormone receptor associated protein 3 (THRAP3), which is a known transcriptional coactivator of the androgen receptor. The phosphorylation level of THRAP3 was significantly lower at S248 and S253 in LNCaP-AI cells. Furthermore, pull-down assays showed that 32 proteins uniquely bound to the nonphosphorylatable mutant form of THRAP3, whereas 31 other proteins uniquely bound to the phosphorylation-mimic form. Many of the differentially interacting proteins were identified as being involved with RNA splicing and processing. These results suggest that the phosphorylation state of THRAP3 at S248 and S253 might be involved in the mechanism of androgen-independent prostate cancer cell growth by changing the interaction partners.

Mass spectrometric analysis of the phosphorylation levels of the SWI/SNF chromatin remodeling/tumor suppressor proteins ARID1A and Brg1 in ovarian clear cell adenocarcinoma cell lines.

Protein phosphorylation is one of the major factors involved in tumor progression and malignancy. We performed exploratory studies aimed at identifying phosphoproteins characteristic to cell lines derived from ovarian clear cell adenocarcinoma (CCA), a highly malignant type of ovarian cancer. Comparative phosphoproteome analysis revealed that the phosphopeptides of five SWI/SNF chromatin remodeling/tumor suppressor components, including ARID1A and BRG1, were significantly down-regulated in CCA cells. We then quantitatively determined the phosphorylation levels of ARID1A and BRG1 by immunoprecipitation-multiple reaction monitoring (IP-MRM) that we used for analysis of the cognate phospho- and nonphosphopeptides of low-abundance proteins. The phosphorylation level of Brg1 at Ser1452 was down-regulated in CCA cells, whereas the phosphorylation level of ARID1A at Ser696 did not significantly differ between CCA and non-CCA cells. These results were consistent with the results of immunoblotting showing that Brg1 levels were comparable, but ARID1A levels were lower, in CCA cells relative to non-CCA cells. This is the first report to demonstrate reduced phosphorylation of Brg1 in CCA-derived cells. Our data also indicated that the IP-MRM/MS method we used is a powerful tool for validation of the phosphoproteins detected by shotgun analysis of phosphopeptides.