ABSTRACT
PURPOSE: Hyperglycaemia-induced oxidative stress and inflammation contribute to vascular cell dysfunction and subsequent cardiovascular events in T2DM. Selective sodium-glucose co-transporter-2 (SGLT-2) inhibitor empagliflozin significantly improves cardiovascular mortality in T2DM patients (EMPA-REG trial). Since SGLT-2 is known to be expressed on cells other than the kidney cells, we investigated the potential ability of empagliflozin to regulate glucose transport and alleviate hyperglycaemia-induced dysfunction of these cells. METHODS: Primary human monocytes were isolated from the peripheral blood of T2DM patients and healthy individuals. Primary human umbilical vein endothelial cells (HUVECs) and primary human coronary artery endothelial cells (HCAECs), and fetoplacental endothelial cells (HPECs) were used as the EC model cells. Cells were exposed to hyperglycaemic conditions in vitro in 40 ng/mL or 100 ng/mL empagliflozin. The expression levels of the relevant molecules were analysed by RT-qPCR and confirmed by FACS. Glucose uptake assays were carried out with a fluorescent derivative of glucose, 2-NBDG. Reactive oxygen species (ROS) accumulation was measured using the H2DFFDA method. Monocyte and endothelial cell chemotaxis were measured using modified Boyden chamber assays. RESULTS: Both primary human monocytes and endothelial cells express SGLT-2. Hyperglycaemic conditions did not significantly alter the SGLT-2 levels in monocytes and ECs in vitro or in T2DM conditions. Glucose uptake assays carried out in the presence of GLUT inhibitors revealed that SGLT-2 inhibition very mildly, but not significantly, suppressed glucose uptake by monocytes and endothelial cells. However, we detected the significant suppression of hyperglycaemia-induced ROS accumulation in monocytes and ECs when empagliflozin was used to inhibit SGLT-2 function. Hyperglycaemic monocytes and endothelial cells readily exhibited impaired chemotaxis behaviour. The co-treatment with empagliflozin reversed the PlGF-1 resistance phenotype of hyperglycaemic monocytes. Similarly, the blunted VEGF-A responses of hyperglycaemic ECs were also restored by empagliflozin, which could be attributed to the restoration of the VEGFR-2 receptor levels on the EC surface. The induction of oxidative stress completely recapitulated most of the aberrant phenotypes exhibited by hyperglycaemic monocytes and endothelial cells, and a general antioxidant N-acetyl-L-cysteine (NAC) was able to mimic the effects of empagliflozin. CONCLUSIONS: This study provides data indicating the beneficial role of empagliflozin in reversing hyperglycaemia-induced vascular cell dysfunction. Even though both monocytes and endothelial cells express functional SGLT-2, SGLT-2 is not the primary glucose transporter in these cells. Therefore, it seems likely that empagliflozin does not directly prevent hyperglycaemia-mediated enhanced glucotoxicity in these cells by inhibiting glucose uptake. We identified the reduction of oxidative stress by empagliflozin as a primary reason for the improved function of monocytes and endothelial cells in hyperglycaemic conditions. In conclusion, empagliflozin reverses vascular cell dysfunction independent of glucose transport but could partially contribute to its beneficial cardiovascular effects.
ABSTRACT
(1) Background: Human milk oligosaccharides (HMOs) are present in maternal serum during pregnancy and their composition is altered in gestational diabetes (GDM). HMOs are also in fetal cord blood and in contact with the feto-placental endothelium, potentially affecting its functions, such as angiogenesis. We hypothesized that cord blood HMOs are changed in GDM and contribute to increased feto-placental angiogenesis, hallmark of GDM. (2) Methods: Using HPLC, we quantified HMOs in cord blood of women with normal glucose tolerance (NGT, n = 25) or GDM (n = 26). We investigated in vitro angiogenesis using primary feto-placental endothelial cells (fpECs) from term placentas after healthy pregnancy (n = 10), in presence or absence of HMOs (100 µg/mL) isolated from human milk, 3'-sialyllactose (3'SL, 30 µg/mL) and lactose (glycan control) and determined network formation (Matrigel assay), proliferation (MTT assays), actin organization (F-actin staining), tube formation (fibrin tube formation assay) and sprouting (spheroid sprouting assay). (3) Results: 3'SL was higher in GDM cord blood. HMOs increased network formation, HMOs and 3'SL increased proliferation and F-actin staining. In fibrin assays, HMOs and 3'SL increased total tube length by 24% and 25% (p < 0.05), in spheroid assays, by 32% (p < 0.05) and 21% (p = 0.056), respectively. Lactose had no effect. (4) Conclusions: Our study suggests a novel role of HMOs in feto-placental angiogenesis and indicates a contribution of HMO composition to altered feto-placental vascularization in GDM.
Subject(s)
Angiogenesis Inducing Agents/blood , Diabetes, Gestational/blood , Fetal Blood/chemistry , Oligosaccharides/blood , Placental Circulation/drug effects , Cell Proliferation/drug effects , Chromatography, High Pressure Liquid , Endothelial Cells/chemistry , Female , Humans , Lactose/blood , Milk, Human/chemistry , Placenta/blood supply , Placenta/cytology , PregnancyABSTRACT
Gestational diabetes (GDM) and preeclampsia (PE) are associated with fetal hyperglycemia, fetal hypoxia, or both. These adverse conditions may compromise fetal and placental endothelial cells. In fact, GDM and PE affect feto-placental endothelial function and also program endothelial function and cardiovascular disease risk of the offspring in the long-term. MicroRNAs are short, non-coding RNAs that regulate protein translation and fine tune biological processes. A group of microRNAs termed angiomiRs is particularly involved in the regulation of endothelial function. We hypothesized that transient hyperglycemia and hypoxia may alter angiomiR expression in feto-placental endothelial cells (fpEC). Thus, we isolated primary fpEC after normal, uncomplicated pregnancy, and induced hyperglycemia (25 mM) and hypoxia (6.5%) for 72 h, followed by reversal to normal conditions for another 72 h. Current vs. transient effects on angiomiR profiles were analyzed by RT-qPCR and subjected to miRNA pathway analyses using DIANA miRPath, MIENTURNET and miRPathDB. Both current and transient hypoxia affected angiomiR profile stronger than current and transient hyperglycemia. Both stimuli altered more angiomiRs transiently, i.e., followed by 72 h culture at control conditions. Pathway analysis revealed that hypoxia significantly altered the pathway 'Proteoglycans in cancer'. Transient hypoxia specifically affected miRNAs related to 'adherens junction'. Our data reveal that hyperglycemia and hypoxia induce memory effects on angiomiR expression in fpEC. Such memory effects may contribute to long-term adaption and maladaption to hyperglycemia and hypoxia.
Subject(s)
MicroRNAs/genetics , Placenta/cytology , Placentation/genetics , Adult , Diabetes, Gestational/metabolism , Endothelial Cells/metabolism , Female , Fetus/metabolism , Gene Expression/genetics , Humans , Hyperglycemia/genetics , Hyperglycemia/metabolism , Hypoxia/genetics , Hypoxia/metabolism , Neovascularization, Physiologic , Placenta/metabolism , Pre-Eclampsia/metabolism , Pregnancy , Primary Cell Culture , Transcriptome/geneticsABSTRACT
Placental hypervascularization has been reported in pregnancy-related pathologies such as gestational diabetes mellitus (GDM). Nevertheless, the underlying causes behind this abnormality are not well understood. In this study, we addressed the expression of SUCNR1 (cognate succinate receptor) in human placental endothelial cells and hypothesized that the succinate-SUCNR1 axis might play a role in the placental hypervascularization reported in GDM. We measured significantly higher succinate levels in placental tissue lysates from women with GDM relative to matched controls. In parallel, SUCNR1 protein expression was upregulated in GDM tissue lysates as well as in isolated diabetic fetoplacental arterial endothelial cells (FpECAds). A positive correlation of SUCNR1 and vascular endothelial growth factor (VEGF) protein levels in tissue lysates indicated a potential link between the succinate-SUCNR1 axis and placental angiogenesis. In our in vitro experiments, succinate prompted hallmarks of angiogenesis in human umbilical vein endothelial cells (HUVECs) such as proliferation, migration and spheroid sprouting. These results were further validated in fetoplacental arterial endothelial cells (FpECAs), where succinate induced endothelial tube formation. VEGF gene expression was increased in response to succinate in both HUVECs and FpECAs. Yet, knockdown of SUCNR1 in HUVECs led to suppression of VEGF gene expression and abrogated the migratory ability and wound healing in response to succinate. In conclusion, our data underline SUCNR1 as a promising metabolic target in human placenta and as a potential driver of enhanced placental angiogenesis in GDM.
Subject(s)
Neovascularization, Physiologic/genetics , Placenta/metabolism , Receptors, G-Protein-Coupled/metabolism , Adult , Case-Control Studies , Cells, Cultured , Diabetes, Gestational/genetics , Diabetes, Gestational/metabolism , Diabetes, Gestational/physiopathology , Endothelium, Vascular/metabolism , Female , Human Umbilical Vein Endothelial Cells , Humans , Neovascularization, Pathologic/genetics , Neovascularization, Pathologic/metabolism , Neovascularization, Pathologic/pathology , Placenta/blood supply , Pregnancy , Receptors, G-Protein-Coupled/genetics , Receptors, G-Protein-Coupled/physiologyABSTRACT
Maternal overweight in pregnancy alters the metabolic environment and generates chronic low-grade inflammation. This affects fetal development and programs the offspring's health for developing cardiovascular and metabolic disease later in life. MME (membrane-metalloendopeptidase, neprilysin) cleaves various peptides regulating vascular tone. Endothelial cells express membrane-bound and soluble MME. In adults, the metabolic environment of overweight and obesity upregulates endothelial and circulating MME. We here hypothesized that maternal overweight increases MME in the feto-placental endothelium. We used primary feto-placental endothelial cells (fpEC) isolated from placentas after normal vs. overweight pregnancies and determined MME mRNA, protein, and release. Additionally, soluble cord blood MME was analyzed. The effect of oxygen and tumor necrosis factor α (TNFα) on MME protein in fpEC was investigated in vitro. Maternal overweight reduced MME mRNA (-39.9%, p < 0.05), protein (-42.5%, p = 0.02), and MME release from fpEC (-64.7%, p = 0.02). Both cellular and released MME protein negatively correlated with maternal pre-pregnancy BMI. Similarly, cord blood MME was negatively associated with pre-pregnancy BMI (r = -0.42, p = 0.02). However, hypoxia and TNFα, potential negative regulators of MME expression, did not affect MME protein. Reduction of MME protein in fpEC and in cord blood may alter the balance of vasoactive peptides. Our study highlights the fetal susceptibility to maternal metabolism and inflammatory state.
Subject(s)
Down-Regulation , Endothelial Cells/enzymology , Fetal Blood/enzymology , Gene Expression Regulation, Developmental , Gene Expression Regulation, Enzymologic , Neprilysin/biosynthesis , Obesity, Maternal/enzymology , Placenta/enzymology , Adult , Cell Line , Endothelial Cells/pathology , Female , Humans , Obesity, Maternal/pathology , Placenta/pathology , PregnancyABSTRACT
BACKGROUND: Diabetes mellitus is an important cardiovascular risk factor characterized by elevated plasma glucose levels. High glucose (HG) negatively influences endothelial cell (EC) function, which is characterized by the inability of ECs to respond to vascular endothelial growth factor (VEGF-A) stimulation. We aimed to identify potential strategies to improve EC function in diabetes. METHODS AND RESULTS: Human umbilical cord endothelial cells (HUVECs) were subjected to hyperglycemic milieu by exposing cells to HG together with glucose metabolite, methylglyoxal (MG) in vitro. Hyperglycemic cells showed reduced chemotactic responses towards VEGF-A as revealed by Boyden chamber migration assays, indicating the development of "VEGF resistance" phenotype. Furthermore, HG/MG-exposed cells were defective in their general migratory and proliferative responses and were in a pro-apoptotic state. Mechanistically, the exposure to HG/MG resulted in reactive oxygen species (ROS) accumulation which is secondary to the impairment of thioredoxin (Trx) activity in these cells. Pharmacological and genetic targeting of Trx recapitulated VEGF resistance. Functional supplementation of Trx using thioredoxin mimetic peptides (TMP) reversed the HG/MG-induced ROS generation, improved the migration, proliferation, survival and restored VEGF-A-induced chemotaxis and sprouting angiogenesis of hyperglycemic ECs. Importantly, TMP treatment reduced ROS accumulation and improved VEGF-A responses of placental arterial endothelial cells isolated from gestational diabetes mellitus patients. CONCLUSIONS: Our findings suggest a putative role for Trx in modulating EC function and its functional impairment in HG conditions contribute to EC dysfunction. Supplementation of TMP could be used as a novel strategy to improve endothelial cell function in diabetes.
Subject(s)
Hyperglycemia , Vascular Endothelial Growth Factor Receptor-2 , Cell Survival , Cells, Cultured , Endothelial Cells , Female , Humans , Hyperglycemia/drug therapy , Pregnancy , Thioredoxins , Vascular Endothelial Growth Factor AABSTRACT
Endothelial function and the risk for endothelial dysfunction differ between males and females. Besides the action of estrogen, sex chromosome gene expression and programming effects also provoke this sexual dimorphism. MicroRNAs (miRNAs) have emerged as regulators of endothelial cell function and dysfunction. We here hypothesized distinct miRNA expression patterns in male versus female human endothelial cells that contribute to the functional differences. We used our well-established model of fetal endothelial cells isolated from placenta (fpEC) and analyzed sexual dimorphic miRNA expression and potentially affected biological functions. Next-generation miRNA sequencing of fpEC isolated after pregnancies with male and female neonates identified sex-dependent miRNA expression patterns. Potential biological pathways regulated by the altered set of miRNAs were determined using mirPath and mirSystem softwares, and suggested differences in barrier function and actin organization. The identified pathways were further investigated by monolayer impedance measurements (ECIS) and analysis of F-actin organization (Phalloidin). Nine miRNAs were differentially expressed in fpEC of male versus female neonates. Functional pathways most significantly regulated by these miRNAs included 'Adherens junction', 'ECM receptor interaction' and 'Focal adhesion'. These pathways control monolayer barrier function and may be paralleled by altered cytoskeletal organization. In fact, monolayer impedance was higher in fpEC of male progeny, and F-actin staining revealed more pronounced peripheral stress fibers in male versus female fpEC. Our data highlight that endothelial cell function differs between males and females already in utero, and that altered miRNAs are associated with sex dependent differences in barrier function and actin organization.
Subject(s)
Actins/metabolism , Endothelial Cells/metabolism , MicroRNAs/genetics , Sex Characteristics , Diabetes, Gestational/genetics , Estrogens/metabolism , Female , Fetus/metabolism , Humans , Male , Placenta/metabolism , PregnancyABSTRACT
Function and dysfunction of endothelial cells are regulated by a multitude of factors. Endothelial cell research often requires in vitro cell culture experiments. Hence, various culture media specifically designed to promote endothelial cell growth are available. These strikingly differ in their composition: complex media contain endothelial cell growth supplement (ECGS), an extract produced of bovine brain with undefined amounts of biologically active compounds, whilst defined media contain selected growth factors in defined concentrations. We here compared the effect of seven purchasable endothelial cell culture media on colony outgrowth, proliferation, viability, in vitro angiogenesis and phenotype of mature primary human endothelial cells using feto-placental endothelial cells isolated from chorionic arteries (fpEC). The effect of media on colony outgrowth was additionally tested on umbilical cord blood-derived endothelial progenitor cells (ECFCs). Outgrowth, purity, proliferation and viability differed between media. Outgrowth of fpEC and ECFCs was best in a defined medium containing EGF, FGF2 and VEGF. By contrast, established fpEC isolations proliferated best in complex media containing ECGS, heparin and ascorbic acid. Also viability of cells was higher in complex media. In vitro angiogenesis was most intense in a defined medium containing the highest number of individual growth factors. FACS analysis of surface markers for endothelial cell subtypes revealed that endothelial phenotype of fpEC was unaffected by media composition. Our data demonstrate the fundamental effect of endothelial cell culture media on primary cell isolation success and behaviour. Whether the composition of supplements is suitable also for individual experiments needs to be tested specifically.
Subject(s)
Culture Media, Conditioned/pharmacology , Endothelial Cells/cytology , Endothelial Cells/drug effects , Neovascularization, Physiologic/drug effects , Animals , Cattle , Cell Proliferation/drug effects , Cell Survival/drug effects , Cells, Cultured , Humans , PhenotypeABSTRACT
MicroRNAs (miRNAs), small non-coding RNAs, have emerged as important, epigenetic regulators of endothelial function. Metabolic disturbances such as diabetes alter miRNA expression. In adults, the miRNA transcriptome as well as endothelial function differ between the sexes. Here, we hypothesized that metabolic disturbances associated with gestational diabetes (GDM) alter miRNA signatures in feto-placental endothelial cells (fpEC), dependent on fetal sex. We isolated human primary fpEC after normal and GDM-complicated pregnancies with male and female neonates and screened for differential miRNA expression using next-generation miRNA sequencing. To test for miRNAs commonly regulated in fpEC of female and male progeny, data were stratified for fetal sex and maternal body mass index (BMI). Analyses were also performed separately for female and male fpEC, again accounting for maternal BMI as covariate. Potential biological pathways regulated by the altered set of miRNAs were determined using mirPath software. Maternal GDM altered 26 miRNA signatures when male and female fpEC were analyzed together. Separate analysis of male versus female fpEC revealed 22 GDM affected miRNAs in the females and only 4 in the males, without overlap. Biological functions potentially modulated by the affected miRNAs related to 'Protein Processing in Endoplasmic Reticulum' and 'Proteoglycans in Cancer'. Maternal GDM alters miRNA signatures in fpEC, and biological functions affected by these miRNAs relate to well-known adverse functional consequences of diabetes on endothelium. GDM effects were highly dependent on fetal sex with miRNA signatures in female fpEC being more susceptible to metabolic derangements of GDM than miRNAs in male fpEC.
Subject(s)
Diabetes, Gestational/genetics , Endothelial Cells/metabolism , MicroRNAs/genetics , Placenta/blood supply , Placental Circulation , Sex Characteristics , Transcriptome , Adult , Case-Control Studies , Cells, Cultured , Diabetes, Gestational/metabolism , Female , Gene Expression Profiling , Gene Expression Regulation, Developmental , Humans , Male , MicroRNAs/metabolism , Pregnancy , Risk Factors , Sex Determination Analysis , Sex Factors , Young AdultABSTRACT
Gestational diabetes mellitus (GDM) is associated with excessive oxidative stress which may affect placental vascular function. Cholesterol homeostasis is crucial for maintaining fetoplacental endothelial function. We aimed to investigate whether and how GDM affects cholesterol metabolism in human fetoplacental endothelial cells (HPEC). HPEC were isolated from fetal term placental arterial vessels of GDM or control subjects. Cellular reactive oxygen species (ROS) were detected by H2DCFDA fluorescent dye. Oxysterols were quantified by gas chromatography-mass spectrometry analysis. Genes and proteins involved in cholesterol homeostasis were detected by real-time PCR and immunoblotting, respectively. Cholesterol efflux was determined from [3H]-cholesterol labeled HPEC and [14C]-acetate was used as cholesterol precursor to measure cholesterol biosynthesis and esterification. We detected enhanced formation of ROS and of specific, ROS-derived oxysterols in HPEC isolated from GDM versus control pregnancies. ROS-generated oxysterols were simultaneously elevated in cord blood of GDM neonates. Liver-X receptor activation in control HPEC by synthetic agonist TO901319, 7-ketocholesterol, or 7ß-hydroxycholesterol upregulated ATP-binding cassette transporters (ABC)A1 and ABCG1 expression, accompanied by increased cellular cholesterol efflux. Upregulation of ABCA1 and ABCG1 and increased cholesterol release to apoA-I and HDL3 (78⯱â¯17%, 40⯱â¯9%, respectively) were also observed in GDM versus control HPEC. The LXR antagonist GGPP reversed ABCA1 and ABCG1 upregulation and reduced the increased cholesterol efflux in GDM HPEC. Similar total cellular cholesterol levels were detected in control and GDM HPEC, while GDM enhanced cholesterol biosynthesis along with upregulated 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) and sterol O-acyltransferase 1 (SOAT1) mRNA and protein levels. Our results suggest that in GDM cellular cholesterol homeostasis in the fetoplacental endothelium is modulated via LXR activation and helps to maintain its proper functionality.
Subject(s)
Cholesterol/metabolism , Diabetes, Gestational/metabolism , Endothelial Cells/metabolism , Endothelium, Vascular/metabolism , Homeostasis/genetics , Liver X Receptors/genetics , ATP Binding Cassette Transporter 1/genetics , ATP Binding Cassette Transporter 1/metabolism , ATP Binding Cassette Transporter, Subfamily G, Member 1/genetics , ATP Binding Cassette Transporter, Subfamily G, Member 1/metabolism , Adult , Case-Control Studies , Cholesterol/pharmacology , Diabetes, Gestational/genetics , Diabetes, Gestational/pathology , Endothelial Cells/drug effects , Endothelial Cells/pathology , Endothelium, Vascular/drug effects , Endothelium, Vascular/pathology , Female , Fetus/blood supply , Fetus/metabolism , Fetus/pathology , Gene Expression Regulation , Humans , Hydroxycholesterols/metabolism , Hydroxycholesterols/pharmacology , Hydroxymethylglutaryl CoA Reductases/genetics , Hydroxymethylglutaryl CoA Reductases/metabolism , Ketocholesterols/metabolism , Ketocholesterols/pharmacology , Lipid Metabolism/drug effects , Liver X Receptors/metabolism , Oxidative Stress , Placenta/blood supply , Placenta/metabolism , Placenta/pathology , Pregnancy , Primary Cell Culture , Sterol O-Acyltransferase/genetics , Sterol O-Acyltransferase/metabolismABSTRACT
PURPOSE: The phenotypical and functional variety of breast cancer cells is well recognized. This variety is evident in primary tumors and in disseminated tumor cells (DTCs) and solid metastases as shown for recognized prognostic factors, such as estrogen receptor, progesterone receptor, or human epidermal growth factor receptor 2/neu and also for cancer stem cell markers such as CD44, CD24, or aldehyde dehydrogenase (ALDH). For the development of new therapeutic strategies, the identification and characterization of disseminated breast cancer cells are needed. This requires the use of multiple antibodies (ie, cytokeratin, Her2/neu, ALDH1, CD44, and CD24) labeled with fluorochromes of different colors and spectral image analysis to separate different color spectra. METHODS: We have focused here on putative breast cancer stem cell markers and evaluated the feasibility of triple and quadruple labeling of breast cancer cells. Using breast cancer cell lines we have developed a method optimized for multimarker analysis by employing novel DyLight Technology. Single marker immunofluorescence was performed in 6 replicates, and reproducible results had to be obtained before proceeding to multimarker immunofluorescence. RESULTS: Three of the markers, CD44, ALDH1, and cytokeratin have been directly conjugated with DyLight dyes. CD24 could not be conjugated directly to the fluorescent dye. A labeled secondary antibody was used for visualization. Single and multimarker immunofluorescence gave consistent results throughout the replicates. CONCLUSIONS: This novel protocol will facilitate detection and phenotypical characterization of disseminated tumor cells. In addition, by adding additional markers, distinct subpopulations could be evaluated for the expression of particular therapeutic targets.
Subject(s)
Antibodies, Neoplasm/chemistry , Biomarkers, Tumor/metabolism , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Fluorescent Antibody Technique, Indirect/methods , Neoplastic Stem Cells/metabolism , Antibodies, Neoplasm/immunology , Biomarkers, Tumor/immunology , Female , Fluorescent Antibody Technique, Indirect/standards , Hep G2 Cells , Humans , Neoplastic Stem Cells/immunologyABSTRACT
OBJECTIVES: The aim of the present study was to evaluate the precision and reproducibility of the LINE-1 high-resolution melting (HRM) assay to detect LINE-1 hypomethylation. DESIGN AND METHODS: We first evaluated a methylated DNA dilution matrix and a panel of human cancer cell lines. We then applied this LINE-1 HRM assay to a set of 37 archival prostate cancer tissue samples. RESULTS: Our LINE-1 HRM assay revealed small and reproducible run-to-run and bisulfite-to-bisulfite variations. As expected, we found a large variation in methylation levels between different cancer cell lines. All results were confirmed with MethyLight and pyrosequencing as indicated by the high correlation coefficient. Finally, we successfully applied the LINE-1 HRM assay to archival prostate cancer tissues. CONCLUSIONS: The present LINE-1 HRM assay represents a novel, accurate, and cost-effective method to measure global hypomethylation, which makes it suitable for high- and low-throughput laboratories.
Subject(s)
Biological Assay/methods , DNA Methylation , DNA, Neoplasm/analysis , Long Interspersed Nucleotide Elements , Nucleic Acid Denaturation , Cell Line, Tumor , Humans , Neoplasms/genetics , Neoplasms/metabolismABSTRACT
High-resolution melting (HRM) analysis is a novel tool for analysis of promoter methylation. The aim of the present study was to establish and validate HRM analysis for detection of promoter methylation on archival formalin-fixed paraffin-embedded tissues from colorectal cancer patients. We first evaluated HRM assays for O(6)-methylguanine-DNA methyltransferase (MGMT) and adenomatous polyposis coli (APC) promoter methylation on a methylated DNA dilution matrix and DNA extracted from eight fresh or formalin-fixed paraffin-embedded human cancer cell lines. Then we used these assays for the analysis of MGMT and APC promoter methylation in a subset of archival formalin-fixed paraffin-embedded colorectal tumor specimens. All samples with promoter methylation of MGMT or APC and randomly selected samples without promoter methylation were analyzed twice. All results generated by HRM were validated with MGMT and APC MethyLight assays. APC and MGMT promoter methylation data were consistent and reproducible throughout the dilutions and all three replicates in the methylated DNA dilution matrix and between two experiments in clinical samples. There was high concordance between HRM and MethyLight results. HRM for APC promoter methylation revealed consistent results between fresh and formalin-fixed paraffin-embedded human cancer cell line DNA. The methylation status in archival tumor specimens from patients with colorectal cancer can therefore be determined with high quality by HRM. The ability to analyze archival tissues greatly facilitates further research and its clinical implementation.