Comparative Membrane N-Glycomics of Different Breast Cancer Cell Lines To Understand Breast Cancer Brain Metastasis.

The mechanism of brain metastatic breast cancer has gained attention because of its increased incidence rate and its low survival rate. Aberrant protein glycosylation is thought to be a contributing factor in this metastatic mechanism, in which metastatic cancer cells can pass through the blood-brain barrier (BBB). The cell membrane is the outermost layer of a cell and in direct contact with the environment and with other cells, making membrane glycans especially important in many biological processes that include mediating cell-cell adhesion, cell signaling, and interactions. Thus, membrane glycomics has attracted more interest for a variety of disease studies in recent years. To reveal the role that membrane N-glycans play in breast cancer brain metastasis, in this study, membrane enrichment was achieved by ultracentrifugation. Liquid chromatography-tandem mass spectrometry was employed to analyze enriched membrane N-glycomes from five breast cancer cell lines and one brain cancer cell line. Relative quantitative glycomic data from each cell line were compared to MDA-MB-231BR, which is the brain-seeking cell line. The higher sialylation level observed in MDA-MB-231BR suggested the importance of sialylation as it might assist with cell invasion and the penetration of the BBB. Some highly sialylated N-glycans, such as HexNAc5Hex6DeoxyHex1NeuAc3 and HexNAc6Hex7DeoxyHex1NeuAc3, exhibited higher abundances in 231BR, indicating their possible contributions to breast cancer brain metastasis as well as their potential to be indicators for the breast cancer brain metastasis.

Revealing the Biological Attributes of N-Glycan Isomers in Breast Cancer Brain Metastasis Using Porous Graphitic Carbon (PGC) Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS).

Breast cancer is a leading cancer in women and is considered to be the second-most common metastatic cancer following lung cancer. An estimated 10-16% of breast cancer patients are suffering from brain metastasis, and the diagnostic cases of breast cancer brain metastasis are increasing. Nevertheless, the mechanisms behind this process are still unclear. Aberrant glycosylation has been proved to be related to many diseases and cancer metastasis. However, studies of N-glycan isomer function in breast cancer brain metastasis are limited. In this study, the expressions of N-glycan isomers derived from five breast cancer cell lines and one brain cancer cell line were investigated and compared to a brain-seeking cell line, 231BR, to acquire a better understanding of the role glycan isomers play in breast cancer brain metastasis. The high temperature nanoPGC-LC-MS/MS achieved an efficient isomeric separation and permitted the identification and quantitation of 144 isomers from 50 N-glycan compositions. There were significant expression alterations of these glycan isomers among the different breast cancer cell lines. The increase of total glycan abundance and sialylation level were observed to be associated with breast cancer invasion. With regard to individual isomers, the greatest number of sialylated isomers was observed along with significant expression alterations in 231BR, suggesting a relationship between glycan sialylation and breast cancer brain metastasis. Furthermore, the increase of the α2,6-sialylation level in 231BR likely contributes to the passage of breast cancer cells through the blood-brain barrier, thus facilitating breast cancer brain metastasis. Meanwhile, the upregulation of highly sialylated glycan isomers with α2,6-linked sialic acids were found to be associated with breast cancer metastasis. This investigation of glycan isomer expressions, especially the unique isomeric expression in brain-seeking cell line 231BR, provides new information toward understanding the potential roles glycan isomers play during breast cancer metastasis and more clues for a deeper insight of this bioprocess.

Enhanced Quantitative LC-MS/MS Analysis of N-linked Glycans Derived from Glycoproteins Using Sodium Deoxycholate Detergent.

Protein glycosylation is a common protein post-translational modification (PTM) in living organisms and has been shown to associate with multiple diseases, and thus may potentially be a biomarker of such diseases. Efficient protein/glycoprotein extraction is a crucial step in the preparation of N-glycans derived from glycoproteins prior to LC-MS analysis. Convenient, efficient and unbiased sample preparation protocols are needed. Herein, we evaluated the use of sodium deoxycholate (SDC) acidic labile detergent to release N-glycans of glycoproteins derived from biological samples such as cancer cell lines. Compared to the filter-aided sample preparation approach, the sodium deoxycholate (SDC) assisted approach was determined to be more efficient and unbiased. SDC removal was determined to be more efficient when using acidic precipitation rather than ethyl acetate phase transfer. Efficient extraction of proteins/glycoproteins from biological samples was achieved by combining SDC lysis buffer and beads beating cell disruption. This was suggested by a significant overall increase in the intensities of N-glycans released from cancer cell lines. Additionally, the use of SDC approach was also shown to be more reproducible than those methods that do not use SDC.

HILIC and ERLIC Enrichment of Glycopeptides Derived from Breast and Brain Cancer Cells.

Aberrant glycosylation has been linked to many different cancer types. The blood-brain barrier (BBB) is a region of the brain that regulates the entrance of ions, diseases, toxins, and so on. However, in breast cancer metastasis, the BBB fails to prevent the crossing of the cancer cells into the brain. Here we present a study of identifying and quantifying the glycosylation of six breast and brain cancer cell lines using hydrophilic interaction liquid chromatography (HILIC) and electrostatic repulsion liquid chromatography (ERLIC) enrichments and LC-MS/MS analysis. Qualitative and quantitative analyses of N-linked glycosylation were performed by both enrichment techniques for individual and complementary comparison. Potential cancer glycopeptide biomarkers were identified and confirmed by chemometric and statistical evaluations. A total of 497 glycopeptides were characterized, of which 401 were common glycopeptides (80.6% overlap) identified from both enrichment techniques. HILIC enrichment yielded 320 statistically significant glycopeptides in 231BR relative to the other cell lines out of 494 unique glycopeptides, and sequential HILIC-ERLIC enrichment yielded 214 statistically significant glycopeptides in 231BR compared with the other cell lines out of 404 unique glycopeptides. The results provide the first comprehensive glycopeptide listing for these six cell lines.

Integrated Transcriptomic and Glycomic Profiling of Glioma Stem Cell Xenografts.

Bone marrow-derived human mesenchymal stem cells (BM-hMSCs) have the innate ability to migrate or home toward and engraft in tumors such as glioblastoma (GBM). Because of this unique property of BM-hMSCs, we have explored their use for cell-mediated therapeutic delivery for the advancement of GBM treatment. Extravasation, the process by which blood-borne cells­such as BM-hMSCs­enter the tissue, is a highly complex process but is heavily dependent upon glycosylation for glycan-glycan and glycan-protein adhesion between the cell and endothelium. However, in a translationally significant preclinical glioma stem cell xenograft (GSCX) model of GBM, BM-hMSCs demonstrate unequal tropism toward these tumors. We hypothesized that there may be differences in the glycan compositions between the GSCXs that elicit homing ("attractors") and those that do not ("non-attractors") that facilitate or impede the engraftment of BM-hMSCs in the tumor. In this study, glycotranscriptomic analysis revealed significant heterogeneity within the attractor phenotype and the enrichment of high mannose type N-glycan biosynthesis in the non-attractor phenotype. Orthogonal validation with topical PNGase F deglycosylation on the tumor regions of xenograft tissue, followed by nLC-ESI-MS, confirmed the presence of increased high mannose type N-glycans in the non-attractors. Additional evidence provided by our glycomic study revealed the prevalence of terminal sialic acid-containing N-glycans in non-attractors and terminal galactose and N-acetyl-glucosamine N-glycans in attractors. Our results provide the first evidence for differential glycomic profiles in attractor and non-attractor GSCXs and extend the scope of molecular determinates in BM-hMSC homing to glioma.

Dietary supplementation of gingerols- and shogaols-enriched ginger root extract attenuate pain-associated behaviors while modulating gut microbiota and metabolites in rats with spinal nerve ligation.

Neuroinflammation is a central factor in neuropathic pain (NP). Ginger is a promising bioactive compound in NP management due to its anti-inflammatory property. Emerging evidence suggests that gut microbiome and gut-derived metabolites play a key role in NP. We evaluated the effects of two ginger root extracts rich in gingerols (GEG) and shogaols (SEG) on pain sensitivity, anxiety-like behaviors, circulating cell-free mitochondrial DNA (ccf-mtDNA), gut microbiome composition, and fecal metabolites in rats with NP. Sixteen male rats were divided into four groups: sham, spinal nerve ligation (SNL), SNL+0.75%GEG in diet, and SNL+0.75%SEG in diet groups for 30 days. Compared to SNL group, both SNL+GEG and SNL+SEG groups showed a significant reduction in pain- and anxiety-like behaviors, and ccf-mtDNA level. Relative to the SNL group, both SNL+GEG and SNL+SEG groups increased the relative abundance of Lactococcus, Sellimonas, Blautia, Erysipelatoclostridiaceae, and Anaerovoracaceae, but decreased that of Prevotellaceae UCG-001, Rikenellaceae RC9 gut group, Mucispirillum and Desulfovibrio, Desulfovibrio, Anaerofilum, Eubacterium siraeum group, RF39, UCG-005, Lachnospiraceae NK4A136 group, Acetatifactor, Eubacterium ruminantium group, Clostridia UCG-014, and an uncultured Anaerovoracaceae. GEG and SEG had differential effects on gut-derived metabolites. Compared to SNL group, SNL+GEG group had higher level of 1'-acetoxychavicol acetate, (4E)-1,7-Bis(4-hydroxyphenyl)-4-hepten-3-one, NP-000629, 7,8-Dimethoxy-3-(2-methyl-3-buten-2-yl)-2H-chromen-2-one, 3-{[4-(2-Pyrimidinyl)piperazino]carbonyl}-2-pyrazinecarboxylic acid, 920863, and (1R,3R,7R,13S)-13-Methyl-6-methylene-4,14,16-trioxatetracyclo[11.2.1.0∼1,10∼.0∼3,7∼]hexadec-9-en-5-one, while SNL+SEG group had higher level for (±)-5-[(tert-Butylamino)-2'-hydroxypropoxy]-1_2_3_4-tetrahydro-1-naphthol and dehydroepiandrosteronesulfate. In conclusion, ginger is a promising functional food in the management of NP, and further investigations are necessary to assess the role of ginger on gut-brain axis in pain management.

Analysis of the Neuroproteome Associated With Cell Therapy After Intranigral Grafting in a Mouse Model of Parkinson Disease.

Advances in large-scale proteomics analysis have been very useful in understanding pathogenesis of diseases and elaborating therapeutic strategies. Proteomics has been employed to study Parkinson disease (PD); however, sparse studies reported proteome investigation after cell therapy approaches. In this study, we used liquid chromatography-tandem mass spectrometry and systems biology to identify differentially expressed proteins in a translational mouse model of PD after cell therapy. Proteins were extracted from five nigrostriatal-related brain regions of mice previously lesioned with 6-hydroxydopamine in the substantia nigra. Protein expression was compared in non-grafted brain to 1 and 7 days after intranigral grafting of E12.5 embryonic ventral mesencephalon (VM). We found a total of 277 deregulated proteins after transplantation, which are enriched for lipid metabolism, oxidative phosphorylation and PD, thus confirming that our animal model is similar to human PD and that the presence of grafted cells modulates the expression of these proteins. Notably, seven proteins (Acta1, Atp6v1e1, Eci3, Lypla2, Pip4k2a, Sccpdh, and Sh3gl2) were commonly down-regulated after engraftment in all studied brain regions. These proteins are known to be involved in the formation of lipids and recycling of dopamine (DA) vesicle at the synapse. Moreover, intranigral transplantation of VM cells decreased the expression of proteins related to oxidative stress, especially in the nigrostriatal pathway containing the DA grafted neurons. In the same regions, an up-regulation of several proteins including α-synuclein and tyrosine hydroxylase was observed, whereas expression of tetraspanin 7 was shut down. Overall, these results suggest that intranigral transplantation of VM tissue in an animal model of PD may induce a decrease of oxidative stress in the nigrostriatal pathway and a restoration of the machinery of neurotransmitters, particularly DA release to promote DA transmission through a decrease of D2 DA receptors endocytosis. Identification of new mechanistic elements involved in the nigrostriatal reconstruction process, using translational animal models and systems biology, is a promising approach to enhance the repair of this pathway in PD patients undergoing cell therapy.

Effects of Curcumin in a Mouse Model of Very High Fat Diet-Induced Obesity.

Worldwide rates of Western-diet-induced obesity epidemics are growing dramatically. Being linked with numerous comorbidities and complications, including cardiovascular disease, type 2 diabetes, cancer, chronic inflammation, and osteoarthritis (OA), obesity represents one of the most threatening challenges for modern healthcare. Mouse models are an invaluable tool for investigating the effects of diets and their bioactive components against high fat diet (HFD)-induced obesity and its comorbidities. During recent years, very high fat diets (VHFDs), providing 58-60% kcal fat, have become a popular alternative to more traditional HFDs, providing 40-45% total kcal fat, due to the faster induction of obesity and stronger metabolic responses. This project aims to investigate if the 60% fat VHFD is suitable to evaluate the protective effects of curcumin in diet-induced obesity and osteoarthritis. B6 male mice, prone to diet-induced metabolic dysfunction, were supplemented with VHFD without or with curcumin for 13 weeks. Under these experimental conditions, feeding mice a VHFD for 13 weeks did not result in expected robust manifestations of the targeted pathophysiologic conditions. Supplementing the diet with curcumin, in turn, protected the animals against obesity without significant changes in white adipocyte size, glucose clearance, and knee cartilage integrity. Additional research is needed to optimize diet composition, curcumin dosage, and duration of dietary interventions to establish the VHFD-induced obesity for evaluating the effects of curcumin on metabolic dysfunctions related to obesity and osteoarthritis.

Integrated Transcriptomics, Proteomics, and Glycomics Reveals the Association between Up-regulation of Sialylated N-glycans/Integrin and Breast Cancer Brain Metastasis.

Breast cancer brain metastasis has been recognized as one of the central issues in breast cancer research. The elucidation of the processes and pathways that mediate this step will provide important clues for a better understanding of breast cancer metastasis. Increasing evidence suggests that aberrant glycosylation patterns greatly contribute to cell invasion and cancer metastasis. Herein, we combined next-generation RNA sequencing with liquid chromatography-tandem mass spectrometry-based proteomic and N-glycomic analysis from five breast cancer cell lines and one brain cancer cell line to investigate the possible mechanisms of breast cancer brain metastasis. The genes/proteins associated with cell movement were highlighted in breast cancer brain metastasis. The integrin signaling pathway and the up-regulation of α-integrin (ITGA2, ITGA3) were associated with the brain metastatic process. 12 glycogenes showed unique expression in 231BR, which could result in an increase of sialylation during brain metastasis. In agreement with the changes of glycogenes, 60 out of 63 N-glycans that were identified exhibited differential expression among cell lines. The correlation between glycogenes and glycans revealed the importance of sialylation and sialylated glycans in breast cancer brain metastasis. Highly sialylated N-glycans, which were up-regulated in brain-seeking cell line 231BR, likely play a role in brain metastasis.

Proteome profiling in the aorta and kidney of type 1 diabetic rats.

Diabetes is associated with a number of metabolic and cardiovascular risk factors that contribute to a high rate of microvascular and macrovascular complications. The risk factors and mechanisms that contribute to the development of micro- and macrovascular disease in diabetes are not fully explained. In this study, we employed mass spectrometric analysis using tandem LC-MS/MS to generate a proteomic profile of protein abundance and post-translational modifications (PTM) in the aorta and kidney of diabetic rats. In addition, systems biology analyses were employed to identify key protein markers that can provide insights into molecular pathways and processes that are differentially regulated in the aorta and kidney of type 1 diabetic rats. Our results indicated that 188 (111 downregulated and 77 upregulated) proteins were significantly identified in the aorta of diabetic rats compared to normal controls. A total of 223 (109 downregulated and 114 upregulated) proteins were significantly identified in the kidney of diabetic rats compared to normal controls. When the protein profiles from the kidney and aorta of diabetic and control rats were analyzed by principal component analysis, a distinct separation of the groups was observed. In addition, diabetes resulted in a significant increase in PTM (oxidation, phosphorylation, and acetylation) of proteins in the kidney and aorta and this effect was partially reversed by insulin treatment. Ingenuity pathway analysis performed on the list of differentially expressed proteins depicted mitochondrial dysfunction, oxidative phosphorylation and acute phase response signaling to be among the altered canonical pathways by diabetes in both tissues. The findings of the present study provide a global proteomics view of markers that highlight the mechanisms and putative processes that modulate renal and vascular injury in diabetes.