Advancements in protein glycosylation biomarkers for ovarian cancer through mass spectrometry-based approaches.

INTRODUCTION: Ovarian cancer, characterized by metastasis and reduced 5-year survival rates, stands as a substantial factor in the mortality of gynecological malignancies worldwide. The challenge of delayed diagnosis originates from vague early symptoms and the absence of efficient screening and diagnostic biomarkers for early cancer detection. Recent studies have explored the intricate interplay between ovarian cancer and protein glycosylation, unveiling the potential significance of glycosylation-oriented biomarkers. AREAS COVERED: This review examines the progress in glycosylation biomarker research, with particular emphasis on advances driven by mass spectrometry-based technologies. We document milestones achieved, discuss encountered limitations, and also highlight potential areas for future research and development of protein glycosylation biomarkers for ovarian cancer. EXPERT OPINION: The association of glycosylation in ovarian cancer is well known, but current research lacks desired sensitivity and specificity for early detection. Notably, investigations into protein-specific and site-specific glycoproteomics have the potential to significantly enhance our understanding of ovarian cancer and facilitate the identification of glycosylation-based biomarkers. Furthermore, the integration of advanced mass spectrometry techniques with AI-driven analysis and glycome databases holds the promise for revolutionizing biomarker discovery for ovarian cancer, ultimately transforming diagnosis and improving patient outcomes.

In-depth investigation of altered glycosylation in human haptoglobin associated cancer by mass spectrometry.

Serum haptoglobin (Hp), a highly sialylated biomolecule with four N-glycosylation sites, is a positive acute-phase response glycoprotein that acts as an immunomodulator. Hp has gained considerable attention due to its potential as a signature molecule that exhibits aberrant glycosylation in inflammatory disorders and malignancies. Its glycosylation can be analyzed qualitatively and quantitatively by various methods using mass spectrometry. In this review, we have provided a brief overview of Hp structure and biological function and described mass spectrometry-based techniques for analyzing glycosylation ranging from macroheterogeneity to microheterogeneity of Hp in diseases and cancer. The sugars on haptoglobin can be a sweet bridge to link the potential of cancer-specific biomarkers to clinically relevant applications.

Host tp53 mutation induces gut dysbiosis eliciting inflammation through disturbed sialic acid metabolism.

BACKGROUND: Host tp53 mutations are frequently found during the early stages of colitis-associated colorectal cancer (CAC), but whether such mutations induce gut microbiota dysbiosis and chronic intestinal inflammation that contributes to the development of CAC, remains unknown. RESULTS: We found that zebrafish tp53 mutant larvae exhibited elevated intestinal inflammation, by monitoring the NFκB activity in the mid-distal intestines of zebrafish larvae using an NFκB:EGFP transgenic reporter line in vivo as well as neutrophil infiltration into the intestine. This inflammation was due to dysbiotic gut microbiota with reduced diversity, revealed using both 16S rRNA amplicon sequencing and a germfree larva model. In this dysbiosis, Aeromonas spp. were aberrantly enriched as major pathobionts and exhibited the capacity for aggressive colonization in tp53 mutants. Importantly, the ex-germfree experiments supported the causality of the host tp53 mutation for inducing the inflammation. Transcriptome and high-performance liquid chromatography analyses of the host gastrointestinal tracts identified dysregulated sialic acid (SA) metabolism concomitant with increased host Neu5Gc levels as the key determinant of aberrant inflammation, which was reversed by the sialidase inhibitors oseltamivir and Philippin A. CONCLUSIONS: These results demonstrate a crucial role for host tp53 in maintaining symbiosis and immune homeostasis via SA metabolism. Disturbed SA metabolism via a tp53 mutation may be exploited by specific elements of the gut microbiome, eliciting both dysbiosis and inflammation. Manipulating sialometabolism may therefore provide an efficacious therapeutic strategy for tp53 mutation-induced dysbiosis, inflammation, and ultimately, related cancers. Video Abstract.

Isomer-Specific Monitoring of Sialylated N-Glycans Reveals Association of α2,3-Linked Sialic Acid Epitope With Behcet's Disease.

Behcet's disease (BD) is an immune disease characterized by chronic and relapsing systemic vasculitis of unknown etiology, which can lead to blindness and even death. Despite continuous efforts to discover biomarkers for accurate and rapid diagnosis and optimal treatment of BD, there is still no signature marker with high sensitivity and high specificity. As the link between glycosylation and the immune system has been revealed, research on the immunological function of glycans is being actively conducted. In particular, sialic acids at the terminus of glycoconjugates are directly implicated in immune responses, cell-cell/pathogen interactions, and tumor progression. Therefore, changes in sialic acid epitope in the human body are spotlighted as a new indicator to monitor the onset and progression of immune diseases. Here, we performed global profiling of N-glycan compositions derived from the sera of 47 healthy donors and 47 BD patients using matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) to preferentially determine BD target glycans. Then, three sialylated biantennary N-glycans were further subjected to the separation of linkage isomers and quantification using porous graphitized carbon-liquid chromatography (PGC-LC)/multiple reaction monitoring (MRM)-MS. We were able to successfully identify 11 isomers with sialic acid epitopes from the three glycan compositions consisting of Hex5HexNAc4NeuAc1, Hex5HexNAc4Fuc1NeuAc1, and Hex5HexNAc4NeuAc2. Among them, three isomers almost completely distinguished BD from control with high sensitivity and specificity with an area under the curve (AUC) of 0.945, suggesting the potential as novel BD biomarkers. In particular, it was confirmed that α2,3-sialic acid at the terminus of biantennary N-glycan was the epitope associated with BD. In this study, we present a novel approach to elucidating the association between BD and glycosylation by tracing isomeric structures containing sialic acid epitopes. Isomer-specific glycan profiling is suitable for analysis of large clinical cohorts and may facilitate the introduction of diagnostic assays for other immune diseases.

Novel analysis procedure for red ginseng polysaccharides by matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry.

BACKGROUND: Red ginseng polysaccharides (RGPs) have been acknowledged for their outstanding immunomodulation and anti-tumor activities. However, their studies are still limited by the complexity of their structural features, the absence of purification and enrichment methods, and the rarity of the analytical instruments that apply to the analysis of such macromolecules. Thus, this study is an attempt to establish a new mass spectrometry (MS)-based analysis procedure for RGPs. METHODS: Saponin pre-excluded powder of RG (RG-SPEP, 10 mg) was treated with 200 µL of distilled water and centrifuged for 5 h at 1000 rpm and 85 °C. Ethanol-based precipitation and centrifugation were applied to obtain RGPs from the heated extracts. Further, endo-carbohydrase treatments were performed to produce specific saccharide fragments. Solid-phase extraction (SPE) processes were implemented to purify and enrich the enzyme-treated RGPs, while matrix-assisted laser desorption/ionization time-of-flight/time-of-flight (MALDI-TOF/TOF) MS was employed for the partial structural analysis of the obtained RGPs. RESULTS: Utilizing cellulase, porous graphitized carbon (PGC), hydrophilic interaction chromatography (HILIC), and MALDI-TOF/TOF MS, the neutral and acidic RGPs were qualitatively analyzed. Hexn and Hexn -18 (cellulose analogs) were determined to be novel neutral RGPs. Additionally, the [Unknown + Hexn] species were also determined as new acidic RGPs. Furthermore, HexAn (H) was determined as another form of the acidic RGPs. CONCLUSION: Compared to the previous methods of analysis, these unprecedented applications of HILIC-SPE and MALDI-TOF/TOF MS to analyze RGPs proved to be fairly effective for fractionating and detecting neutral and acidic components. This new procedure exhibits great potential as a specific tool for searching and determining various polysaccharides in many herbal medicines.

GALNT3 suppresses lung cancer by inhibiting myeloid-derived suppressor cell infiltration and angiogenesis in a TNFR and c-MET pathway-dependent manner.

The deregulation of polypeptide N-acetyl-galactosaminyltransferases (GALNTs) contributes to several cancers, but their roles in lung cancer remain unclear. In this study, we have identified a tumor-suppressing role of GALNT3 in lung cancer. We found that GALNT3 suppressed lung cancer development and progression in both xenograft and syngeneic mouse models. Specifically, GALNT3 suppressed lung cancer initiation by inhibiting the self-renewal of lung cancer cells. More importantly, GALNT3 attenuated lung cancer growth by preventing the creation of a favorable tumor microenvironment (TME), which was attributed to GALNT3's ability to inhibit myeloid-derived suppressor cell (MDSC) infiltration into tumor sites and subsequent angiogenesis. We also identified a GALNT3-regulated gene (GRG) signature and found that lung cancer patients whose tumors exhibit the GRG signature showed more favorable prognoses. Further investigation revealed that GALNT3 suppressed lung cancer cell self-renewal by reducing ß-catenin levels, which led to reduced expression of the downstream targets of the WNT pathway. In addition, GALNT3 inhibited MDSC infiltration into tumor sites by suppressing both the TNFR1-NFκB and cMET-pAKT pathways. Specifically, GALNT3 inhibited the nuclear localization of NFκB and the c-MET-induced phosphorylation of AKT. This then led to reduced production of CXCL1, a chemokine required for MDSC recruitment. Finally, we confirmed that the GALNT3-induced inhibition of the TNFR1-NFκB and cMET-pAKT pathways involved the O-GalNAcylation of the TNFR1 and cMET receptors. In summary, we have identified GALNT3 as the first GALNT member capable of suppressing lung cancer and uncovered a novel mechanism by which GALNT3 regulates the TME.

In-depth characterization of non-human sialic acid (Neu5Gc) in human serum using label-free ZIC-HILIC/MRM-MS.

Sialic acid Neu5Gc, a non-human glycan, is recognized as a new harmful substance that can cause vascular disease and cancer. Humans are unable to synthesize Neu5Gc due to a genetic defect that converts Neu5Ac to Neu5Gc, but Neu5Gc is often observed in human biological samples. Therefore, the demand for accurately measuring the amount of Neu5Gc present in human blood or tissues is rapidly increasing, but there is still no method to reliably quantify trace amounts of a non-human sugar. In particular, selective isolation and detection of Neu5Gc from human serum is analytically challenging due to the presence of excess sialic acid Neu5Ac, which has physicochemical properties very similar to Neu5Gc. Herein, we developed the label-free approach based on ZIC-HILIC/MRM-MS that can enrich sialic acids released from human serum and simultaneously monitor Neu5Ac and Neu5Gc. The combination of complete separation of Neu5Gc from abundant Neu5Ac by hydrophilic and electrostatic interactions with selective monitoring of structure-specific cross-ring cleavage ions generated by negative CID-MS/MS was remarkably effective for quantification of Neu5Ac and Neu5Gc at the femtomole level. Indeed, we were able to successfully determine the absolute quantitation of Neu5Gc from 30 healthy donors in the range of 3.336 ± 1.252 pg/µL (mean ± SD), 10,000 times lower than Neu5Ac. In particular, analysis of sialic acids in protein-free serum revealed that both Neu5Ac and Neu5G are mostly bound to proteins and/or lipids, but not in free form. In addition, the correlation between expression level of Neu5Gc and biological factors such as BMI, age, and sex was investigated. This method can be widely used in studies requiring sialic acid-related measurements such as disease diagnosis or prediction of immunogenicity in biopharmaceuticals as it is both fast and highly sensitive.

Discovery of N-glycan Biomarkers for the Canine Osteoarthritis.

Protein glycosylation is a post-translational modification that impacts on protein activity, stability, and interactions. It was sensitively altered by the cellular state and, therefore, is now used for a diagnostic or prognostic indicator of various human diseases such as cancer. To evaluate the clinical feasibility in the veterinary area, the N-glycan biomarkers were discovered from canine serum for the diagnosis of osteoarthritis (OA), which is one of the most common diseases of dogs. N-glycome was obtained from 20 µL of canine serum by the enzymatic cleavage followed by the purification and enrichment using solid-phase extraction. Independent compositions of 163 and 463 N-glycans were found from healthy control (n = 41) and osteoarthritis patients (n = 92), respectively. Initially, 31 of the potential biomarkers were screened by the p-values below 1.0 × 10-10 from ANOVA. Then, the area under the curve (AUC) and the intensity ratio between OA patient and healthy control (P/C ratio) were calculated. Considering the diagnostic efficacy, the AUC bigger than 0.9 and the P/C ratio larger than 3.0 were used to discover 16 N-glycans as diagnostic biomarkers. Particularly, five of the diagnostic biomarkers were AUC above 0.99 and three of N-glycans had AUC 1.0. The results suggest a clear possibility for N-glycan biomarkers to be used as a clinical tool in the veterinary medical area enabling to provide objective and non-invasive diagnostic information.

Computational classification of core and outer fucosylation of N-glycoproteins in human plasma using collision-induced dissociation in mass spectrometry.

RATIONALE: Glycoprotein fucosylation, one of the major posttranslational modifications, is known to be highly involved in proteins related to various cancers. Fucosylation occurs in the core and/or outer sites of N-glycopeptides. Elucidation of the fucosylation type of N-glycoproteins is therefore important. However, it has remained a challenge to classify the fucosylation types of N-glycopeptides using collision-induced dissociation (CID) tandem mass (MS/MS) spectra. METHODS: The relative intensities of the Y1 F, Y2 F, Y3 F, and Y4 F product ions in the CID-MS/MS spectra of the IgG N-glycopeptides were measured for core fucosylation. The Core Fucose Index (CFI) was then calculated by multiplication of the relative intensities with a weight factor from logistic regression to differentiate between the core and none fucosylation. From the relative intensities of the B2 F and B3 SF ions of the MS/MS spectra of the AGP N-glycopeptides for outer fucosylation, the Outer Fucose Index (OFI) was calculated to differentiate between the outer and none fucosylation. RESULTS: In order to classify core and/or outer fucosylation of N-glycoproteins, we defined the fucosylation score (F-score) by a sigmoidal equation using a combination of the CFI and the OFI. For application, we classified the fucosylation types of N-glycoproteins in human plasma with 99.7% accuracy from the F-score. Human plasma samples showed 54.4%, 33.3%, 10.3%, and 1.6% for none, core, outer, and dual fucosylated N-glycopeptides, respectively. Core fucosylation was abundant at mono- and bi-antennary N-glycopeptides. Outer fucosylation was abundant at tri- and tetra-antennary N-glycopeptides. In total, 113 N-glycopeptides of 29 glycoproteins from 3365 glycopeptide spectral matches (GPSMs) were classified for different types of fucosylation. CONCLUSIONS: We established an F-score to classify three different fucosylation types: core, outer, and dual types of N-glycopeptides. The fucosylation types of 20 new N-glycopeptides from 11 glycoproteins in human plasma were classified using the F-score. Therefore, the F-score can be useful for the automatic classification of different types of fucosylation in N-glycoproteins of biological fluids including plasma, serum, and urine.

Family-based exome sequencing combined with linkage analyses identifies rare susceptibility variants of MUC4 for gastric cancer.

Genome-wide association studies of gastric cancer (GC) cases have revealed common gastric cancer susceptibility loci with low effect size. We investigated rare variants with high effect size via whole-exome sequencing (WES) of subjects with familial clustering of gastric cancer. WES of DNAs from the blood of 19 gastric cancer patients and 36 unaffected family members from 14 families with two or more gastric cancer patients were tested. Linkage analysis combined with association tests were performed using Pedigree Variant Annotation, Analysis, and Search Tool (pVAAST) software. Based on the logarithm of odds (LOD) and permutation-based composite likelihood ratio test (CLRT) from pVAAST, MUC4 was identified as a predisposing gene (LOD P-value = 1.9×10-5; permutation-based P-value of CLRT ≤ 9.9×10-9). In a larger cohort consisting of 597 GC patients and 9,759 healthy controls genotyped with SNP array, we discovered common variants in MUC4 regions (rs148735556, rs11717039, and rs547775645) significantly associated with GC supporting the association of MUC4 with gastric cancer. And the MUC4 variants were found in higher frequency in The Cancer Genome Atlas Study (TCGA) germline samples of patients with multiple cancer types. Immunohistochemistry indicated that MUC4 was downregulated in the noncancerous gastric mucosa of subjects with MUC4 germline missense variants, suggesting that loss of the protective function of MUC4 predisposes an individual to gastric cancer. Rare variants in MUC4 can be novel gastric cancer susceptibility loci in Koreans possessing the familial clustering of gastric cancer.

Glycosylation of serum haptoglobin as a marker of gastric cancer: an overview for clinicians.

Introduction: Gastric cancer (GC) is one of the leading causes of cancer-related death worldwide because of difficulties in early diagnosis. Aberrant glycosylation in serum proteins has been associated with many human diseases. Serum haptoglobin, a highly sialylated glycoprotein with four N-glycosylation sites, has gained considerable attention due to its potential as a signature molecule to display aberrant glycosylation in inflammatory disorders and various types of cancer. In particular, the relevance of haptoglobin glycosylation in GC has been investigated in a multifaceted way.Areas covered: The screening of haptoglobin glycosylation could offer an alternative approach toward GC diagnosis and detection. In this report, various assay platforms such as glycan profiling, site-specific glycopeptide profiling, and intact protein profiling are introduced for the detection of abnormal glycosylation of serum haptoglobin.Expert opinion: Although aberrant glycosylation of serum haptoglobin is associated with gastric cancer patients and might be a promising marker of GC screening, the development of a diagnosis platform to increase specificity and sensitivity for clinical use is still an analytical challenge. However, the continuous advancement of analytical technologies and methods will spur the paradigm shift from traditional serum markers, enabling the effective mining of human glycoproteome for GC diagnostic markers.

Comprehensive Profiling of Surface Gangliosides Extracted from Various Cell Lines by LC-MS/MS.

Gangliosides act as a surface marker at the outer cellular membrane and play key roles in cancer cell invasion and metastasis. Despite the biological importance of gangliosides, they have been still poorly characterized due to the lack of effective analytical tools. Herein, we performed molecular profiling and structural elucidation of intact gangliosides in various cell lines including CFPAC1, A549, NCI-H358, MCF7, and Caski. We identified and quantified a total of 76 gangliosides on cell membrane using C18 LC-MS/MS. Gangliosides found in each cell line exhibited high complexity and diversity both qualitatively and quantitatively. The most abundant species was GM3(d34:1) in CFPAC1, NCI-H358, and MCF7, while GM2(d34:1) and GM1(d34:1) were major components in A549 and Caski, respectively. Notably, glycan moieties showed more diversity between cancer cell lines than ceramide moieties. In addition, noncancerous pancreatic cell line (hTERT/HPNE) could be distinguished by gangliosides containing different levels of sialic acid compared with cancerous pancreatic cell line (CFPAC1). These results clearly demonstrated the feasibility of our analytical platform to comprehensive profile of cell surface gangliosides for identifying cell types and subgrouping cancer cell types.

An optimized mixed-mode stationary phase based on silica monolith particles for the separation of peptides and proteins in high-performance liquid chromatography.

A phase with both hydrophobic and hydrophilic functionalities has been synthesized by modification of ground silica monolith particles with C18 and 1-[3-(trimethoxysilyl)propyl] urea ligands. A series of phases was prepared by changing the ratio of the two ligands to determine the optimal ratio in view of separation efficiency. The resultant optimized stationary phase was packed in narrow-bore glass-lined stainless-steel columns (1 × 300 mm and 2.1 × 100 mm) and used for the separation of synthetic peptides and proteins. The average numbers of theoretical plates (N) of 52 100/column (174 000/m, 5.75 µm plate height) and 35 500/column (118 000/m, 8.47 µm plate height) were achieved with the 300 mm column at a flow rate of 25 µL/min (0.86 mm/s) in 60:40 v/v acetonitrile/30 mM aqueous ammonium formate for the mixture of peptides (Thr-Tyr-Ser, Val-Ala-Pro-Gly, angiotensin I, isotocin, and bradykinin) and for the mixture of proteins (myoglobin, human serum albumin, and insulin), respectively. Fast analysis of the peptides and proteins was also carried out at a flow rate of 0.9 mL/min (6.88 mm/s) with the 100 mm column and all the analytes were eluted within 2 min with good separation efficiency.

Parallel reaction monitoring with multiplex immunoprecipitation of N-glycoproteins in human serum for detection of hepatocellular carcinoma.

The N-glycosylation of proteins is one of the most important post-translational modifications relevant to various biological functions. The identification and quantification of N-glycoproteins in liquid chromatography-mass spectrometry (LC-MS) is challenging because of their low analytical sensitivity and selectivity. This is due to their microheterogeneity and the difficulty of synthesizing N-glycopeptides as an internal standard. Parallel reaction monitoring (PRM) is widely used in targeted LC-MS. The key advantage of LC-PRM is that it can identify N-glycopeptides using tandem mass spectrometry (MS/MS) fragmentation, even without an internal standard. We investigated the feasibility of analyzing N-glycoproteins using multiplex immunoprecipitation to improve sensitivity and selectivity. We targeted N-glycoproteins [α-fetoprotein (AFP), vitronectin (VTN), and α-1-antichymotrypsin (AACT)] that are abnormally glycosylated in hepatocellular carcinoma (HCC). Their tryptic N-glycopeptides were selected to determine the percentages of fucosylated N-glycopeptides using Y ions, which include glycopeptide fragments with amino acid sequences. Finally, we confirmed that the area under the receiver operating characteristic curve (AUC = 0.944) for the combination of AFP and VTN increased more so than for a single glycopeptide (AUC = 0.889 for AFP and 0.792 for VTN) with respect to discriminating between HCC and cirrhosis serum. This study shows that an LC-PRM method using multiplex N-glycoproteins immunoprecipitated from serum could be applied to develop and verify cancer biomarkers. Graphical abstract.

Identification of Missing Proteins in Human Olfactory Epithelial Tissue by Liquid Chromatography-Tandem Mass Spectrometry.

We performed proteomic analyses of human olfactory epithelial tissue to identify missing proteins using liquid chromatography-tandem mass spectrometry. Using a next-generation proteomic pipeline with a < 1.0% false discovery rate at the peptide and protein levels, we identified 3731 proteins, among which five were missing proteins (P0C7M7, P46721, P59826, Q658L1, and Q8N434). We validated the identified missing proteins using the corresponding synthetic peptides. No olfactory receptor (OR) proteins were detected in olfactory tissue, suggesting that detection of ORs would be very difficult. We also identified 49 and 50 alternative splicing variants mapped at the neXtProt and GENCODE databases, respectively, and 2000 additional single amino acid variants. This data set is available at the ProteomeXchange consortium via PRIDE repository (PXD010025).

Direct Monitoring of Fucosylated Glycopeptides of Alpha-Fetoprotein in Human Serum for Early Hepatocellular Carcinoma by Liquid Chromatography-Tandem Mass Spectrometry with Immunoprecipitation.

PURPOSE: Alpha-fetoprotein (AFP) is a widely used serological marker that is associated with hepatocellular carcinoma (HCC). Although the level of AFP is increased in HCC, its sensitivity for diagnosis is poor because AFP levels are also increased in liver diseases. Changes in glycoform, especially fucosylation, have been reported to be associated with the development of HCC. EXPERIMENTAL DESIGN: The authors introduce the monitoring of fucosylated glycopeptides by liquid chromatography (LC)-mass spectrometry (MS) combined with immunoprecipitation, where glycan-cleaved fragments with an amino acid sequence are used as transitions. Furthermore, neuraminidase for desialylation is useful to improve the MS detection limit (limit of detection [LOD] <2 ng mL-1 ) in 0.1 µL of serum. RESULTS: The performance of the relative percentage of fucosylated AFP (AFP-fuc%) for differentiating between early HCC and cirrhosis is better than that of serum AFP levels as indicated by a greater area under the receiver operator characteristic curve (area under the curve = 0.962 vs. 0.628) and sensitivity (92.3% vs. 53.9%), respectively. Furthermore, the inter- and intraday repeatability of AFP-fuc% in serum is less than 2.1%. CONCLUSIONS AND CLINICAL RELEVANCE: These findings suggest that glycopeptide-based LC-MS/MS is a promising method and that AFP-fuc% is a clinically useful parameter for differentiating between early HCC and liver cirrhosis.

Restoring Effects of Natural Anti-Oxidant Quercetin on Cellular Senescent Human Dermal Fibroblasts.

The oxidative damage initiated by reactive oxygen species (ROS) is a major contributor to the functional decline and disability that characterizes aging. The anti-oxidant flavonoid, quercetin, is a plant polyphenol that may be beneficial for retarding the aging process. We examined the restoring properties of quercetin on human dermal fibroblasts (HDFs). Quercetin directly reduced either intracellular or extracellular ROS levels in aged HDFs. To find the aging-related target genes by quercetin, microarray analysis was performed and two up-regulated genes LPL and KCNE2 were identified. Silencing LPL increased the expression levels of senescence proteins such as p16INK4A and p53 and silencing KCNE2 reversed gene expressions of EGR1 and p-ERK in quercetin-treated aged HDFs. Silencing of LPL and KCNE2 decreased the expression levels of anti-oxidant enzymes such as superoxide dismutase and catalase. Also, the mitochondrial dysfunction in aged HDFs was ameliorated by quercetin treatment. Taken together, these results suggest that quercetin has restoring effect on the cellular senescence by down-regulation of senescence activities and up-regulation of the gene expressions of anti-oxidant enzymes in aged HDFs.

Multi-paratopic VEGF decoy receptor have superior anti-tumor effects through anti-EGFRs and targeted anti-angiogenic activities.

Limitation of current anti-Vascular Endothelial Growth Factor (VEGF) cancer therapy is transitory responses, inevitable relapses and its insufficient tumor-targeting. Thus, multifaceted approaches, including the development of bispecific antibodies and combination strategies targeting different pathways have been proposed as an alternative. Here, we developed a novel multi-paratopic VEGF decoy receptor, Cetuximab-VEGF-Grab and Trastuzumab-VEGF-Grab, by genetically fusing VEGF decoy receptor (VEGF-Grab) to a single chain Fv of anti-Epidermal Growth Factor Receptor (EGFR) antibody (Cetuximab and Trastuzumab). These multi-paratopic VEGF decoy receptor, which recognize VEGF and EGFR family (EGFR or HER2), effectively suppressed both VEGF and EGFR pathways in vitro, to levels similar to those of the parental VEGF-Grab and anti-EGFR antibodies. In addition, the concurrent binding of multi-paratopic VEGF decoy receptor to VEGF and EGFR family enabled their specific localization to EGFR + tumor in vitro and in vivo. Furthermore, Cetuximab-VEGF-Grab and Trastuzumab-VEGF-Grab exhibited the enhanced anti-tumor activities compared to VEGF-Grab in EGFR + tumor xenograft mouse model via anti-EGFR and the targeted anti-angiogenic activities. These results indicate that multi-paratopic VEGF decoy receptor can be a promising agent, combining tumor-targeted anti-angiogenic therapy with efficient blockade of proliferative signals mediated by EGFR family.

Designation of fingerprint glycopeptides for targeted glycoproteomic analysis of serum haptoglobin: insights into gastric cancer biomarker discovery.

Gastric cancer (GC) is one of the leading causes of cancer-related death worldwide, largely because of difficulties in early diagnosis. Despite accumulating evidence indicating that aberrant glycosylation is associated with GC, site-specific localization of the glycosylation to increase specificity and sensitivity for clinical use is still an analytical challenge. Here, we created an analytical platform with a targeted glycoproteomic approach for GC biomarker discovery. Unlike the conventional glycomic approach with untargeted mass spectrometric profiling of released glycan, our platform is characterized by three key features: it is a target-protein-specific, glycosylation-site-specific, and structure-specific platform with a one-shot enzyme reaction. Serum haptoglobin enriched by immunoaffinity chromatography was subjected to multispecific proteolysis to generate site-specific glycopeptides and to investigate the macroheterogeneity and microheterogeneity. Glycopeptides were identified and quantified by nano liquid chromatography-mass spectrometry and nano liquid chromatography-tandem mass spectrometry. Ninety-six glycopeptides, each corresponding to a unique glycan/glycosite pairing, were tracked across all cancer and control samples. Differences in abundance between the two groups were marked by particularly high magnitudes. Three glycopeptides exhibited exceptionally high control-to-cancer fold changes along with receiver operating characteristic curve areas of 1.0, indicating perfect discrimination between the two groups. From the results taken together, our platform, which provides biological information as well as high sensitivity and reproducibility, may be useful for GC biomarker discovery. Graphical abstract ᅟ.

Lectin from Sambucus sieboldiana abrogates the anoikis resistance of colon cancer cells conferred by N-acetylglucosaminyltransferase V during hematogenous metastasis.

Anoikis is a form of anchorage-dependent apoptosis, and cancer cells adopt anokis-resistance molecular machinery to conduct metastasis. Here, we report that N-acetylglucosaminyltransferase V gene expression confers anoikis resistance during cancer progression. Overexpression of N-acetylglucosaminyltransferase V protected detached cancer cells from apoptotic death, and suppression or knockout of the gene sensitized cancer cells to the apoptotic death. The gene expression also stimulated anchorage-dependent as well as anchorage-independent colony formation of cancer cells following anoikis stress treatments. Importantly, treatment with the lectin from Sambucus sieboldiana significantly sensitized anoikis-induced cancer cell deaths in vitro as well as in vivo. We propose that the lectin alone or an engineered form could offer a new therapeutic treatment option for cancer patients with advanced tumors.