Conclusion: This paper provides a proof of concept for a novel and specifically developed VTGE process that showed first evidence on the undetectable level of FAAAs in nails of breast cancer patients as metabolite biomarkers. Here, the authors propose the potential use of a VTGE-assisted process to achieve metabolomic discovery in nails of breast cancer and other tumor types.
BACKGROUND: Metabolic reprogramming in breast cancer is depicted as a crucial change in the tumor microenvironment. Besides the molecular understanding of metabolic heterogeneity, appreciable attentions are drawn to characterize metabolite profiles in tumor tissue and derived biological fluids and tissue materials. Several findings reported on the metabolic alterations of free aromatic amino acids (FAAAs) and other metabolites in biological fluids. Furthermore, there is a significant gap in the development of a suitable method for the purification and analysis of metabolite biomarkers in nails of cancer patients. METHODS: To address the metabolite alterations specifically FAAAs level in nails, fingernail clippings of breast cancer patients (N=10), and healthy subjects (N-12) were used for extraction and purification of metabolites. Here, we report a novel and specifically designed vertical tube gel electrophoresis (VTGE) system that helped for the purification of metabolites in the range of (100-1000 Da) from nail materials. The claimed VTGE system uses 15% polyacrylamide under non-denaturing and non-reducing conditions that makes eluted metabolites directly compatible with LC-HRMS and other analytical techniques. Qualitative and quantitative determination of FAAAs in nail lysates were done by positive ESI mode of Agilent LC-HRMS platform. RESULTS: The analysis on collected data of nail metabolites clearly suggests that FAAAs including tryptophan, tyrosine, phenylalanine and histidine are undetectable in nail lysates of breast cancer over healthy subjects. This is a first report that shows the highly reduced levels of FAAAs in nails of breast cancer. Furthermore, present observation is in consonance with previous findings that support that breast cancer patients show cancer cachexia, high amino acid catabolism that drive metabolite driven cancer growth and proliferation. CONCLUSION: This paper provides a proof of concept for a novel and specifically developed VTGE process that assisted to show the first evidence on undetectable level of FAAAs in nails of breast cancer patients as metabolite biomarkers. Here, the authors propose the potential use of VTGE assisted process to achieve metabolomic discovery in nails of breast cancer and other tumor types.
Background:In recent, various human health disorders including cancer, diabetes, neurodegenerative and metabolic diseases are noticed among human populations. Currently, genetic and proteomic approaches are highly reported to detect metabolic disorders that also include inborn error of metabolisms. These existing detection methods are faced with cost issue and time consuming factors. Therefore, metabolites as biomarkers are one of potential avenues to detect metabolic disorders. Further, exploitation of urine as potential source of metabolite biomarkers, there are limitation in this area of research due to abundance of non-metabolite components such as proteins and nucleic acids. Hence, methods and processes are required to precisely fractionate metabolites from urine of inborn error of metabolism patients and then identified by analytical tools such as LC-HRMS and GC-MS.Methods: Sterile filtered urine samples (750 µl) mixed with (250 µl) loading buffer were electrophoresed on VTGE that uses acrylamide gel (acrylamide:bisacrylamide, 30:1) as matrix of 15%. Further, vertical tube gel electrophoresis (VTGE) technique combined with LC-HR-MS to identify metabolites that are known as the biomarkers of metabolic disorders was carried out. Results and Discussion:The authors provide evidence on the use of novel VTGE coupled with LC-HRMS to detect metabolites among metabolic disorders. Data suggest the applicability of VTGE coupled with LC-HRMS technique to detect metabolites such as 2-methyluridine, 2-Methylglutaric acid, 2-Methyl citric acid, 2-Hydroxyglutaric acid in case of metabolic disorders. Conclusion:This preliminary work is suggested to be extended to large clinical samples to validate application of this method to detect metabolic disorders including inborn error of metabolisms.
BACKGROUND: Molecular adaptations in intracellular and extracellular microenvironment of breast cancer cells promote pro-tumor metabolic reprogramming. Hence, metabolic reprogramming is seen as a crucial factor in various tumor hallmarks including drug resistance, invasiveness and metastasis. Among well-known metabolic features of breast carcinoma including Warburg effects, altered amino acid metabolism, lipid remodeling is considered as key factors in achieving pro-tumor microenvironment. Therefore, a better understanding on molecular aspects of lipid remodeling is highly appreciated that may contribute towards future therapeutics and diagnostics purpose including the need of potential biomarkers. The identification and validation of lipid biomarkers are reported in the literature, but evidence on lipid metabolites as biomarkers in nails of breast cancer patients is completely unexplored. METHODS: This study reported a novel and specifically designed vertical tube gel electrophoresis (VTGE) system to assist in the purification of metabolites in the range of (~100-1000 Da) from nail samples. Fingernail clippings of breast cancer patients (N=10), and healthy subjects (N-12) were used for extraction and purification of metabolites. The VTGE system uses 15% polyacrylamide under non-denaturing and non-reducing conditions that makes eluted metabolites directly compatible with LC-HRMS and other analytical techniques. The characterization of lipid metabolites in nail lysates was done by positive ESI mode of Agilent LC-HRMS platform. RESULTS: Data suggest a novel observation that healthy and breast cancer patients show distinct accumulation of lipid metabolites specifically choline-based lipids. This is a first report that suggests that levels of choline, phosphorylcholine and lyso-PC are highly reduced and undetectable in nails of breast cancer patients over healthy subject. Furthermore, the potential use of reduced level of choline, phosphorylcholine and lyso-PC in nails of breast cancer patients is in line with current notion that these lipids are diverted to meet the pro-tumor activities in the tumor microenvironment. CONCLUSION: Data strongly provide a proof of concept for the potential use of lipid metabolites including choline, phosphorylcholine and lyso-PC as a set of biomarkers in nails of breast cancer patients. However, the authors propose that validity of these lipid biomarkers may be extended to large population size of breast cancer patients for future applications in early detection, grading, staging, predicting prognosis and therapeutic targeting of breast carcinoma.
Background The prominent among various existing views on the role of nicotine and nicotine-metabolized products in Oral squamous cell carcinoma (OSSC) is metabolic adaptation that allows the use of methyl-donor S-adenosylmethionine (SAM) for non-epigenetic purpose including the methylation of nicotinamide and nicotine. In fact, channeling of SAM for generation of 1-methylnicotinamide (1-MNA) and methylated nicotine products is seen as a key event in cancer cells that allows favorable epigenetic states by forcing DNA hypomethylation. A better perception of such events can be appreciated by analyzing samples like nail, which represents a perfect biological material for studying long-term metabolic reflections of the body. Methods Potential nicotine-metabolized products and 1-MNA in nails of OSCC patients were analyzed by using a novel approach of Vertical tube gel electrophoresis (VTGE)- assisted purification followed by their identification by LC-HRMS. Further, these identified nicotine metabolized products and 1-MNA were evaluated for their molecular interactions with known methyltransferases including cytosolic nicotinamide methyltransferase (NNMT), DNA methyltransferase (DNMT)1 and histone methyltransferases by molecular docking and molecular dynamics simulation (MDS) analyses. Results Our data suggests the presence of N-methylnicotinium ion and nicotine imine in the nail samples of OSCC patients. Further, 1-MNA is also detected in the nails as a major enzymatic product of a known detoxifying enzyme NNMT. Molecular docking of all nicotine and nicotine metabolized products with DNMT1revealed a specific binding affinity of nicotine imine only with a -6.2 Kcal/Mol docking energy. Importantly, binding of nicotine imine is within the CXCC regulatory domain of DNMT1 and it displays molecular interactions with the key amino acid residues, namely ARG690, PRO574, VAL658, PRO692 and ALA695. Furthermore, MDS data corroborated well with the specific binding affinity of nicotine imine to DNMT1 obtained by docking analysis. Conclusion Identification of N-methylnicotinium ion, nicotine imine and 1-MNA in nail samples indicates their potential as predictive and detectable biomarkers for OSCC. Molecular docking and MDS data lead us to propose a role of nicotine imine in modulating the activity of DNMT1. These data further suggest a novel understanding on the role of nicotine metabolized products in modulating DNA methylation that may contribute to tumorigenicity in oral cancer patients.
Background: Nicotine-metabolized product nicotine imine is suggested to play a role in metabolic changes in oral cancer. There is a significant gap in the detection of oncometabolite nicotine imine in biological fluids and nails of oral cancer patients. Oncometabolites are designated as metabolites those are usually elevated in cancer cells over normal cells. Interestingly, a direct or indirect link is missing that establishes a role of nicotine imine in pro-cancer cellular events including global DNA hypomethylation, a potential metabolic-epigenetic axis in oral cancer. Methods: A novel vertical tube gel electrophoresis (VTGE) system assisted purification and liquid chromatography-high resolution mass spectrometry (LC-HRMS) based identification of nicotine imine in the nails of oral cancer patients. Further, nicotine imine was evaluated for its molecular interactions with various methyltransferases including DNA methyltransferase 1 (DNMT1) by molecular docking and molecular dynamics (MD) simulations. Results: Data suggested the presence of nicotine imine in the nails of oral cancer patients. Molecular docking and MD simulations revealed a specific binding affinity by nicotine imine with DNMT1. Binding by nicotine imine is within the CXCC regulatory domain of DNMT1 including key residues as ARG690, PRO574, VAL658, PRO692 and ALA695. Similar binding residues are displayed by DNMT1 inhibitor 5'-Aza-2'-deoxycytidine. Conclusion : Nicotine imine is suggested as a predictive biomarker for oral cancer patients in nails and this finding is a first report. Molecular docking and dynamics simulation propose the role of nicotine imine as an inhibitor of DNMT1. This work supports the involvement of synergistic pro-tumor metabolic-epigenomic axis by nicotine imine that may contribute towards potential mutagenesis of normal squamous epithelium.
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