Exploring the feasibility of using long-term stored newborn dried blood spots to identify metabolic features for congenital heart disease screening.

Congenital heart disease (CHD) represents a significant contributor to both morbidity and mortality in neonates and children. There's currently no analogous dried blood spot (DBS) screening for CHD immediately after birth. This study was set to assess the feasibility of using DBS to identify reliable metabolite biomarkers with clinical relevance, with the aim to screen and classify CHD utilizing the DBS. We assembled a cohort of DBS datasets from the California Department of Public Health (CDPH) Biobank, encompassing both normal controls and three pre-defined CHD categories. A DBS-based quantitative metabolomics method was developed using liquid chromatography with tandem mass spectrometry (LC-MS/MS). We conducted a correlation analysis comparing the absolute quantitated metabolite concentration in DBS against the CDPH NBS records to verify the reliability of metabolic profiling. For hydrophilic and hydrophobic metabolites, we executed significant pathway and metabolite analyses respectively. Logistic and LightGBM models were established to aid in CHD discrimination and classification. Consistent and reliable quantification of metabolites were demonstrated in DBS samples stored for up to 15 years. We discerned dysregulated metabolic pathways in CHD patients, including deviations in lipid and energy metabolism, as well as oxidative stress pathways. Furthermore, we identified three metabolites and twelve metabolites as potential biomarkers for CHD assessment and subtypes classifying. This study is the first to confirm the feasibility of validating metabolite profiling results using long-term stored DBS samples. Our findings highlight the potential clinical applications of our DBS-based methods for CHD screening and subtype classification.

Serological Phenotyping Analysis Uncovers a Unique Metabolomic Pattern Associated With Early Onset of Type 2 Diabetes Mellitus.

Background: Type 2 diabetes mellitus (T2DM) is a multifaceted disorder affecting epidemic proportion at global scope. Defective insulin secretion by pancreatic ß-cells and the inability of insulin-sensitive tissues to respond effectively to insulin are the underlying biology of T2DM. However, circulating biomarkers indicative of early diabetic onset at the asymptomatic stage have not been well described. We hypothesized that global and targeted mass spectrometry (MS) based metabolomic discovery can identify novel serological metabolic biomarkers specifically associated with T2DM. We further hypothesized that these markers can have a unique pattern associated with latent or early asymptomatic stage, promising an effective liquid biopsy approach for population T2DM risk stratification and screening. Methods: Four independent cohorts were assembled for the study. The T2DM cohort included sera from 25 patients with T2DM and 25 healthy individuals for the biomarker discovery and sera from 15 patients with T2DM and 15 healthy controls for the testing. The Pre-T2DM cohort included sera from 76 with prediabetes and 62 healthy controls for the model training and sera from 35 patients with prediabetes and 27 healthy controls for the model testing. Both global and targeted (amino acid, acylcarnitine, and fatty acid) approaches were used to deep phenotype the serological metabolome by high performance liquid chromatography-high resolution mass spectrometry. Different machine learning approaches (Random Forest, XGBoost, and ElasticNet) were applied to model the unique T2DM/Pre-T2DM metabolic patterns and contrasted with their effectiness to differentiate T2DM/Pre-T2DM from controls. Results: The univariate analysis identified unique panel of metabolites (n = 22) significantly associated with T2DM. Global metabolomics and subsequent structure determination led to the identification of 8 T2DM biomarkers while targeted LCMS profiling discovered 14 T2DM biomarkers. Our panel can effectively differentiate T2DM (ROC AUC = 1.00) or Pre-T2DM (ROC AUC = 0.84) from the controls in the respective testing cohort. Conclusion: Our serological metabolite panel can be utilized to identifiy asymptomatic population at risk of T2DM, which may provide utility in identifying population at risk at an early stage of diabetic development to allow for clinical intervention. This early detection would guide ehanced levels of care and accelerate development of clinical strategies to prevent T2DM.

Multi-Omics Signatures Link to Ticagrelor Effects on Vascular Function in Patients With Acute Coronary Syndrome.

BACKGROUND: Long-term antiplatelet agents including the potent P2Y12 antagonist ticagrelor are indicated in patients with a previous history of acute coronary syndrome. We sought to compare the effect of ticagrelor with that of aspirin monotherapy on vascular endothelial function in patients with prior acute coronary syndrome. METHODS: This was a prospective, single center, parallel group, investigator-blinded randomized controlled trial. We randomized 200 patients on long-term aspirin monotherapy with prior acute coronary syndrome in a 1:1 fashion to receive ticagrelor 60 mg BD (n=100) or aspirin 100 mg OD (n=100). The primary end point was change from baseline in brachial artery flow-mediated dilation at 12 weeks. Secondary end points were changes to platelet activation marker (CD41_62p) and endothelial progenitor cell (CD34/133) count measured by flow cytometry, plasma level of adenosine, IL-6 (interleukin-6) and EGF (epidermal growth factor), and multi-omics profiling at 12 weeks. RESULTS: After 12 weeks, brachial flow-mediated dilation was significantly increased in the ticagrelor group compared with the aspirin group (ticagrelor: 3.48±3.48% versus aspirin: -1.26±2.85%, treatment effect 4.73 [95% CI, 3.85-5.62], P<0.001). Nevertheless ticagrelor treatment for 12 weeks had no significant effect on platelet activation markers, circulating endothelial progenitor cell count or plasma level of adenosine, IL-6, and EGF (all P>0.05). Multi-omics pathway assessment revealed that changes in the metabolism and biosynthesis of amino acids (cysteine and methionine metabolism; phenylalanine, tyrosine, and tryptophan biosynthesis) and phospholipids (glycerophosphoethanolamines and glycerophosphoserines) were associated with improved brachial artery flow-mediated dilation in the ticagrelor group. CONCLUSIONS: In patients with prior acute coronary syndrome, ticagrelor 60 mg BD monotherapy significantly improved brachial flow-mediated dilation compared with aspirin monotherapy and was associated with significant changes in metabolomic and lipidomic signatures. REGISTRATION: URL: https://www. CLINICALTRIALS: gov; Unique identifier: NCT03881943.

Downregulation of SHIP2 by Hepatitis B Virus X Promotes the Metastasis and Chemoresistance of Hepatocellular Carcinoma through SKP2.

Hepatitis B virus (HBV)-encoded X protein (HBx) plays an important role in the development of hepatocellular carcinoma (HCC). The protein SH2 domain containing inositol 5-phosphatase 2 (SHIP2) belongs to the family of enzymes that dephosphorylate the 5 position of PI(3,4,5)P3 to produce PI(3,4)P2. Expression of SHIP2 has been associated with several cancers including HCC. However, its role in the development of HBV-related HCC remains elusive. In this study, we performed tissue microarray analysis using 49 cases of HCC to explore SHIP2 expression changes and found that SHIP2 was downregulated in HBV-positive HCC. In addition, S-phase kinase-associated protein 2 (SKP2), a component of the E3 ubiquitin-ligase complex, was increased in HCC cell lines that overexpressed HBx, which also showed a notable accumulation of polyubiquitinated SHIP2. Moreover, HCC cells with silenced SHIP2 had increased expression of mesenchymal markers, which promotes cell migration, enhances glucose uptake, and leads to resistance to the chemotherapy drug (5-Fluorouracil, 5-FU). Taken together, our results demonstrate that HBx downregulates SHIP2 through SKP2 and suggest a potential role for SHIP2 in HBx-mediated HCC migration.

Combinations of FUT2 gene polymorphisms and environmental factors are associated with oral cancer risk.

In humans, fucosyltransferase-2 (FUT2) plays an important role in α1,2- linkage of fucose and participates in complex cellular processes such as fertilization, embryogenesis, and immune responses. However, little information is available concerning the FUT2 expression in tumorigenesis. The aim of this work was to investigate the combined effect of FUT2 gene polymorphisms and exposure to environmental carcinogens on the susceptibility and clinic pathological characteristics of oral cancer. Four SNPs of the FUT2 gene (rs281377, rs1047781, rs601338, and rs602662) from 1200 non-cancer controls and 700 oral squamous cell carcinoma (OSCC) patients were analyzed by real-time polymerase chain reaction (PCR). The samples were further analyzed to clarify the associations between these gene polymorphisms and the risk of OSCC, and the impact of these SNPs on the susceptibility and clinic pathological characteristics of OSCC. After adjusting for other covariant, we observed that betel quid chewing among 1255 smokers who carrying at least one C genotype (TC and CC) at rs281377 and least one T genotype (TA and TT) at rs1047781 were exhibited synergistic effects of environmental factors (betel quid and cigarette use) on the susceptibility of oral cancer. Taken together, our results support gene-environment interactions of FUT2 polymorphisms with smoking and betel quid chewing habits possibly altering oral cancer susceptibility. Furthermore, to our knowledge, this is the first study of association between FUT2 gene variants and OSCC risk.

Effects of EZH2 promoter polymorphisms and methylation status on oral squamous cell carcinoma susceptibility and pathology.

Oral squamous cell carcinoma (OSCC), which is malignant tumors in oral cavity, is the fourth most common male cancer in Taiwan. EZH2 plays a key role in transcriptional repression through chromatin remodeling and in cancer development. Although the EZH2 expression in OSCC is highly correlated with tumorigenesis, it has not been determined if specific EZH2 genetic variants are associated with OSCC risk. The aim of this study was to investigate the relationship between genetic polymorphisms of EZH2 and susceptibility to OSCC in Taiwan. Here, four SNPs of EZH2 (rs6950683, rs2302427, rs3757441, and rs41277434) were analyzed by a real-time PCR genotyping in 576 patients with oral cancer and 552 cancer-free controls. After adjusting for other co-variants, we found that carrying CC genotype at EZH2 rs6950683 and rs3757441 had a lower risk of developing OSCC than did wild-type carriers. The CCCA or CCTA haplotype among the four EZH2 sites was also associated with a reduced risk of OSCC. Furthermore, OSCC patients who carried CC genotype at EZH2 rs6950683 had a higher methylation than TC genotype. Our results suggest that the two SNPs of EZH2 (rs6950683 and rs3757441) might contribute to the prediction of OSCC susceptibility. Moreover, rs6950683 CC genotype exhibits hypermethylation in EZH2 promoter. This is the first study to provide insight into risk factors associated with EZH2 variants and epigenetic changes in carcinogenesis of OSCC in Taiwan.

Impact of EZH2 polymorphisms on urothelial cell carcinoma susceptibility and clinicopathologic features.

BACKGROUND: The gene EZH2, the polycomb group protein enhancer of zeste 2, encodes a transcriptional repressor that also serves as a histone methyltransferase that is associated with progression to more advanced disease in a variety of malignancies. EZH2 expression level in urothelial cell carcinoma (UCC) is highly correlated with tumor aggressiveness, but it has not been determined if specific EZH2 genetic variants are associated with UCC risk. This study investigated the potential associations of EZH2 single-nucleotide polymorphisms with UCC susceptibility and its clinicopathologic characteristics. METHODOLOGY/PRINCIPAL FINDINGS: A total of 233 UCC patients and 552 cancer-free controls, all of whom were from Taiwan, were analyzed for four EZH2 single-nucleotide polymorphisms (rs6950683, rs2302427, rs3757441, and rs41277434) using real-time PCR genotyping. After adjusting for other co-variants, we found that individuals carrying at least one C allele at EZH2 rs6950683 had a lower risk of developing UCC than did major allele carriers. The CCCA or TGTA haplotype among the four EZH2 sites was also associated with a reduced risk of UCC. Furthermore, UCC patients who carried at least one G allele at rs2302427 had a lower invasive tumor stage than did patients carrying the major allele. CONCLUSIONS: The rs6950683 SNPs of EZH2 might contribute to the prediction of UCC susceptibility. This is the first study to provide insight into risk factors associated with EZH2 variants in carcinogenesis of UCC in Taiwan.

Effects of EZH2 polymorphisms on susceptibility to and pathological development of hepatocellular carcinoma.

BACKGROUND: The enhancer of zeste 2 (EZH2) gene encodes the histone methyltransferase that is the catalytic component of the polycomb repressive complex-2, which initiates epigenetic silencing of genes. The expression level of EZH2 in hepatocellular carcinoma (HCC) is highly correlated with tumor progression; however, it has not been determined if specific EZH2 genetic variants are associated with the risk of HCC. This study investigated the potential associations of EZH2 single-nucleotide polymorphisms with HCC susceptibility and its clinicopathologic characteristics. METHODOLOGY/PRINCIPAL FINDINGS: A total of 220 HCC patients and 552 cancer-free controls were analyzed for four EZH2 single-nucleotide polymorphisms (rs6950683, rs2302427, rs3757441, and rs41277434) using real-time PCR genotyping. After adjusting for other co-variants, the individuals carrying at least one C allele at EZH2 rs6950683 and rs3757441 had a 0.611-fold and a 0.660-fold lower risk of developing HCC than did wild-type (TT) carriers, respectively. The CCCA or CCTA haplotype among the four EZH2 sites (rs6950683, rs2302427, rs3757441, and rs41277434), respectively, was also associated with a reduced risk of HCC. Furthermore, HCC patients who carried at least one C allele at rs6950683 or rs3757441 had a higher lymph-node-metastasis risk but a lower liver-cirrhosis risk than did patients carrying the wild-type allele. CONCLUSIONS: The rs6950683 and rs3757441 polymorphic genotypes of EZH2 might contribute to the prediction of susceptibility to and pathological development of HCC. This is the first study to provide insight into risk factors associated with EZH2 variants in carcinogenesis of HCC in Taiwan.

Targeting the EGFR/PCNA signaling suppresses tumor growth of triple-negative breast cancer cells with cell-penetrating PCNA peptides.

Tyrosine 211 (Y211) phosphorylation of proliferation cell nuclear antigen (PCNA) coincides with pronounced cancer cell proliferation and correlates with poor survival of breast cancer patients. In epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI)-resistant cells, both nuclear EGFR (nEGFR) expression and PCNA Y211 phosphorylation are increased. Moreover, the resistance to EGFR TKI is a major clinical problem in treating EGFR-overexpressing triple-negative breast cancer (TNBC). Thus, effective treatment to combat resistance is urgently needed. Here, we show that treatment of cell-penetrating PCNA peptide (CPPP) inhibits growth and induces apoptosis of human TNBC cells. The Y211F CPPP specifically targets EGFR and competes directly for PCNA tyrosine Y211 phosphorylation and prevents nEGFR from binding PCNA in vivo; it also suppresses tumor growth by sensitizing EGFR TKI resistant cells, which have enhanced nEGFR function and abrogated classical EGFR membrane signaling. Furthermore, we identify an active motif of CPPP, RFLNFF (RF6 CPPP), which is necessary and sufficient to inhibit TKI-resistant TNBC cell growth of orthotopic implanted tumor in mice. Finally, the activity of its synthetic retro-inverted derivative, D-RF6 CPPP, on an equimolar basis, is more potent than RF6 CPPP. Our study reveals a drug candidate with translational potential for the future development of safe and effective therapeutic for EGFR TKI resistance in TNBC.

Rutin, a Flavonoid That Is a Main Component of Saussurea involucrata, Attenuates the Senescence Effect in D-Galactose Aging Mouse Model.

Saussurea involucrata (Kar. et Kir.), known as the snow lotus, grows in the Tian Shan and A'er Tai areas of China. It has recently been reported that the ethyl acetate extract of S. involucrata (SI-2) can inhibit proliferation and induce apoptosis in PC-3 human prostate cancer cells. This study investigated the protective effect of ethyl acetate extract of S. involucrata (SI-2) or rutin, a flavonoid extracted from ethyl acetate extract of S. involucrata (SI-2), on D-galactose- (D-gal-) induced brain injury in mice. Administering SI-2 or rutin (30 mg/kg/d and 30 mg/kg/d) for 6 weeks, concomitant with D-gal injection, significantly increased superoxide dismutase and glutathione peroxidase activities and decreased the MDA level in plasma. Furthermore, the result showed that the percentages of cleaved caspase-3 and PARP in the D-gal-treated mice were much higher than those in the control. Pretreatment using SI-2 or rutin decreased the expression of cyclooxygenase-2 via downregulation of NF-kappaB, resulting in a decrease in lipid peroxidation. Furthermore, our results also showed that oral administration of rutin to these mice significantly improved behavioral performance in a step-through passive avoidance task and these results suggest that SI-2 or rutin exerts potent antiaging effects on D-gal in mice via antioxidative mechanisms.

The cytotoxic protein can induce autophagocytosis in addition to apoptosis in MCF-7 human breast cancer cells.

Phagocytic clearance of dying cells is found in many phagocytes. It has been shown that dying cells can be phagocytosed by other phagocytic cells through autophagic or apoptotic cellular death. To date, whether cancer cells have such phagocytic activity has not been studied. In this study, our data shows that RC-RNase can trigger cell death in human breast cancer MCF-7 cells through the apoptotic pathway. Interestingly, when treated with cytotoxic protein, the remaining MCF-7 cells can phagocytose the dying MCF-7 cells via autophagocytic activity, demonstrated directly by real-time image observation and electron microscopy analysis. To sum up, this study demonstrates for the first time that RC-RNase can trigger apoptosis and autophagocytosis in MCF-7 cancer cells.

Synergistic anti-tumor activity of isochaihulactone and paclitaxel on human lung cancer cells.

Drug resistance frequently develops in tumors during chemotherapy. Therefore, to improve the clinical outcome, more effective and tolerable combination treatment strategies are needed. Here, we show that isochaihulactone (K8) enhanced paclitaxel-induced apoptotic death in human lung cancer cells, and the enhancing effect was related to increased NSAID-activated gene-1 (NAG-1) expression. CalcuSyn software was used to evaluate the synergistic interaction of K8 and paclitaxel on human lung cancer cells; the synergistic effect of K8 in combination with paclitaxel was increased more than either of these drugs alone. Furthermore, the activity of ERK1/2 was enhanced by the combination of K8 and paclitaxel, and an ERK1/2 inhibitor dramatically inhibited NAG-1 expression in human lung cancer cells. Therefore, this synergistic apoptotic effect in human lung cancer cells may be directly associated with K8-induced NAG-1 expression through ERK1/2 activation. Moreover, over-expression of NAG-1 enhanced K8/paclitaxel-induced apoptosis in human lung cancer cells. In addition, treatment of nude mice with K8 combined with paclitaxel induced phospho-ERK1/2 and NAG-1 expression in vivo. Targeting of NAG-1 signaling could enhance therapeutic efficacy in lung cancer. Our results reveal that activation of NAG-1 by K8 enhanced the therapeutic efficacy of paclitaxel in human lung cancer cells via the ERK1/2 signaling pathway.

Isochaihulactone protects PC12 cell against H(2)O(2) induced oxidative stress and exerts the potent anti-aging effects in D-galactose aging mouse model.

AIM: to investigate the effect of isochaihulactone (also known as K8), a lignan compound of Bupleurum scorzonerifolium, on H(2)O(2)-induced cytotoxicity in neuronally differentiated PC12 cells (nPC12). METHODS: viability of neuronal PC12 cells was measured using MTT assay. Protein expression was determined by Western blot. Apoptotic cells was determined using TUNEL assay. D-galactose aging mice were used as a model system to study the anti-oxidant effects of isochaihulactone in vivo. RESULTS: pretreatment with isochaihulactone (5-10 micromol/L) increased cell viability and decreased membrane damage, generation of reactive oxygen species and degradation of poly (ADP-ribose) polymerase in H(2)O(2)-treated nPC12 cells and also decreased the expression of cyclooxygenase-2, via downregulation of NF-kappaB, resulting in a decrease in lipid peroxidation. The results suggest that isochaihulactone is a potential antioxidant agent. In a murine aging model, in which chronic systemic exposure to D-galactose (D-gal) causes the acceleration of senescence, administration of isochaihulactone (10 mgxkg(-1)xd(-1), sc) for 7 weeks concomitant with D-gal injection significantly increased superoxide dismutase and glutathione peroxidase activities and decreased the MDA level in plasma. Furthermore, H&E staining to quantify cell death within hippocampus showed that percentage of pyknotic nuclei in the D-gal-treated mice were much higher than in control. CONCLUSION: the results suggest that isochaihulactone exerts potent anti-aging effects against D-gal in mice possibly via antioxidative mechanisms.

Extended O6-methylguanine methyltransferase promoter hypermethylation following n-butylidenephthalide combined with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) on inhibition of human hepatocellular carcinoma cell growth.

Epigenetic alteration of DNA methylation plays an important role in the regulation of gene expression associated with chemosensitivity of human hepatocellular (HCC) carcinoma cells. With the aim of improving the chemotherapeutic efficacy for HCC, the effect of the naturally occurring compound n-butylidenephthalide (BP), which is isolated from a chloroform extract of Angelica sinensis, was investigated. In both HepG2 and J5 HCC cell lines, a synergistic antiproliferative effect was observed when a low dosage of BP was combined with the chemotherapeutic drug 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). BCNU is an alkylating agent, and it prompts us to examine one of DNA repair genes, O(6)-methylguanine methyltransferase (MGMT). It was evident from methylation-specific polymerase chain reaction (PCR) analysis that BP/BCNU combined treatment caused a time- and concentration-dependent enhancement of MGMT promoter methylation. Overexpression of MGMT could abolish BP-induced growth inhibition in the J5 tumor cell line as measured by colony formation assay. When BP was combined with BCNU and administered, it showed significant antitumor effects in both HepG2 and J5 xenograft tumors as compared with the use of only one of these drugs. The BCNU-induced apoptosis and inhibited MGMT protein expression in HCC cells, both in vitro and in vivo, resulting from the combination treatment of BP and BCNU suggest a potential clinical use of this compound for improving the prognosis for HCCs.