Transcriptomic Analysis of Alzheimer's Disease Pathways in a Pakistani Population.

Background: Alzheimer's disease (AD) is a multifactorial neurodegenerative disorder that is most prevalent in elderly individuals, especially in developed countries, and its prevalence is now increasing in developing countries like Pakistan. Objective: Our goal was to characterize key genes and their levels of expression and related molecular transcriptome networks associated with AD pathogenesis in a pilot case-control study in a Pakistani population. Methods: To obtain the spectrum of molecular networks associated with pathogenesis in AD patients in Pakistan (comparing cases and controls), we used high-throughput qRT-PCR (TaqMan Low-Density Array; n = 33 subjects) coupled with Affymetrix Arrays (n = 8) and Ingenuity Pathway Analysis (IPA) to identify signature genes associated with Amyloid processing and disease pathways. Results: We confirmed 16 differentially expressed AD-related genes, including maximum fold changes observed in CAPNS2 and CAPN1. The global gene expression study observed that 61% and 39% of genes were significantly (p-value 0.05) up- and downregulated, respectively, in AD patients compared to healthy controls. The key pathways include, e.g., Amyloid Processing, Neuroinflammation Signaling, and ErbB4 Signaling. The top-scoring networks in Diseases and Disorders Development were Neurological Disease, Organismal Injury and Abnormalities, and Psychological Disorders. Conclusions: Our pilot study offers a non-invasive and efficient way of investigating gene expression patterns by combining TLDA and global gene expression method in AD patients by utilizing whole blood. This provides valuable insights into the expression status of genes related to Amyloid Processing, which could play potential role in future studies to identify sensitive, early biomarkers of AD in general.

Transcriptomics of MASLD Pathobiology in African American Patients in the Washington DC Area †.

Metabolic-dysfunction-associated steatotic liver disease (MASLD) is becoming the most common chronic liver disease worldwide and is of concern among African Americans (AA) in the United States. This pilot study evaluated the differential gene expressions and identified the signature genes in the disease pathways of AA individuals with MASLD. Blood samples were obtained from MASLD patients (n = 23) and non-MASLD controls (n = 24) along with their sociodemographic and medical details. Whole-blood transcriptomic analysis was carried out using Affymetrix Clarion-S Assay. A validation study was performed utilizing TaqMan Arrays coupled with Ingenuity Pathway Analysis (IPA) to identify the major disease pathways. Out of 21,448 genes in total, 535 genes (2.5%) were significantly (p < 0.05) and differentially expressed when we compared the cases and controls. A significant overlap in the predominant differentially expressed genes and pathways identified in previous studies using hepatic tissue was observed. Of note, TGFB1 and E2F1 genes were upregulated, and HMBS was downregulated significantly. Hepatic fibrosis signaling is the top canonical pathway, and its corresponding biofunction contributes to the development of hepatocellular carcinoma. The findings address the knowledge gaps regarding how signature genes and functional pathways can be detected in blood samples ('liquid biopsy') in AA MASLD patients, demonstrating the potential of the blood samples as an alternative non-invasive source of material for future studies.

Comparative and integrative analysis of transcriptomic and epigenomic-wide DNA methylation changes in African American prostate cancer.

African American (AA) men have the highest incidence and mortality rate from Prostate cancer (PCa) than any other racial/ethnic group. To date, PCa genomic studies have largely under-represented tumour samples from AA men. We measured genome-wide DNA methylation in benign and tumor prostate tissues from AA men using the Illumina Infunium 850 K EPIC array. mRNA expression database from a subset of the AA biospecimen were used to assess correlation of transcriptome and methylation datasets. Genome-wide methylation analysis identified 11,460 probes that were significant (p < 0.01) and differentially methylated in AA PCa compared to normal prostate tissues and showed significant (p < 0.01) inverse-correlation with mRNA expression. Ingenuity pathway analysis and Gene Ontology analysis in our AA dataset compared with TCGA dataset showed similarities in methylation patterns: top candidate genes with significant hypermethylation and corresponding down-regulated gene expression were associated with biological pathways in hemidesmosome assembly, mammary gland development, epidermis development, hormone biosynthesis, and cell communication. In addition, top candidate genes with significant hypomethylation and corresponding up-regulated gene expression were associated with biological pathways in macrophage differentiation, cAMP-dependent protein kinase activity, protein destabilization, transcription co-repression, and fatty acid biosynthesis. In contrast, differences in genome-wide methylation in our AA dataset compared with TCGA dataset were enriched for genes in steroid signalling, immune signalling, chromatin structure remodelling and RNA processing. Overall, differential methylation of AMIGO3, IER3, UPB1, GRM7, TFAP2C, TOX2, PLSCR2, ZNF292, ESR2, MIXL1, BOLL, and FGF6 were significant and uniquely associated with PCa progression in our AA cohort.

Mechanism of Antitumor Effects of Saffron in Human Prostate Cancer Cells.

Prostate cancer is the most common cancer and the second leading cause of cancer deaths among men in the USA. Several studies have demonstrated the antitumor properties of saffron in different types of cancers, including prostate cancer. The oral administration of saffron extract has been reported to have antitumor effects on aggressive prostate-cancer-cell-line-derived xenografts in nude male mice. The objective of this study was to carry out in vitro studies of saffron-treated prostate cancer cells to ascertain the effects of saffron on key intermediates in prostate carcinogenesis. Our studies demonstrated the significant inhibition of cell proliferation for androgen-sensitive prostate cancer cell lines via apoptotic pathways. We also demonstrate the statistically significant down-regulation of DNA methyltransferases (COMT, MGMT, EHMT2, and SIRT1 deacetylase) in saffron-treated prostate cancer cells. In addition, saffron-treated prostate cancer cells displayed a statistically significant dysregulation of DNA repair intermediates (WRN, p53, RECQ5, MST1R, and WDR70) in a time-dependent manner. Furthermore, Western blot analysis demonstrated that saffron treatment induced changes in the expression of other key genes (DNMT1, DNMT3b, MBD2, CD44, HDAC3, c-Myc, NF-kB, TNFα, AR, N-RAS, and PTEN) in prostate cancer cells. Collectively, our findings demonstrate the important mechanisms by which saffron mediates anti-tumor properties in prostate cancer. These findings suggest that the use of saffron supplements alongside standard treatment protocols may yield beneficial effects for individuals with prostate cancer.

Sex differences in saliva-based DNA methylation changes and environmental stressor in young African American adults.

BACKGROUND: Low socioeconomic status neighborhood exposure to stress and violence may be sources of negative stimuli that poses significant health risks for children, adolescents and throughout the life course of an individual. The study aims to investigate if aberrant epigenetic DNA methylation changes may be a potential mechanism for regulating neighborhood exposures and health outcomes. METHODS: Exposure to environmental stressors identified in 98 young African American (AA) adults aged 18-25 years old from the Washington D.C., area were used in the study. We correlated the association between stress markers; cortisol, CRP, IgG, IGA, IgM, and self-reported exposure to violence and stress, with quantitative DNA methylation changes in a panel of gene-specific loci using saliva DNA. RESULTS: In all participants studied, the exposure to violence was significant and negatively correlated with DNA methylation of MST1R loci (p = 0.032; r = -0.971) and nominally significant with NR3C1 loci (p = 0.053; r = -0.948). In addition, we observed significant and negative correlation of DNA methylation changes of LINE1 (p = 0.044; r = -0.248); NR3C1 (p = 0.017; r = -0.186); MSTR1 (p = 0.022; r = -0.192); and DRD2 (p = 0.056; r = -0.184; albeit nominal significant correlation) with IgA expression. On the other hand, we observed a significant and position correlation of DNA methylation changes in DRD2 (p = 0.037; r = 0.184) with IgG expression. When participants were stratified by sex, we observed in AA young male adults, significant DNA methylation changes of MST1R (p< 0.05) and association with exposure to violence and IgG level. We also observed significant DNA methylation levels of DRD2 (p< 0.05) and association with IgA, IgG, and cortisol level. Furthermore, we observed significant DNA methylation changes of NR3C1 (p< 0.05) with stress, IgA, and IgG in the male participants only. On the other hand, we only observed significant and a positive association of IgG with DNA methylation levels of ESR1 (p = 0.041) in the young AA female participants. CONCLUSION: Our preliminary observation of significant DNA methylation changes in neuronal and immune genes in saliva samples supports our recently published genome-wide DNA methylations changes in blood samples from young AA male adults indicating that saliva offers a non-invasive means for DNA methylation prediction of exposure to environmental stressors in a gender-specific manner.

Specific Combinations of Inflammatory, Angiogenesis and Vascular Integrity Biomarkers Are Associated with Clinical Severity, Coma and Mortality in Beninese Children with Plasmodium Falciparum Malaria.

Malaria-related deaths could be prevented if powerful diagnostic and reliable prognostic biomarkers were available to allow rapid prediction of the clinical severity allowing adequate treatment. Using quantitative ELISA, we assessed the plasma concentrations of Procalcitonin, Pentraxine-3, Ang-2, sTie-2, suPAR, sEPCR and sICAM-1 in a cohort of Beninese children with malaria to investigate their potential association with clinical manifestations of malaria. We found that all molecules showed higher levels in children with severe or cerebral malaria compared to those with uncomplicated malaria (p-value < 0.005). Plasma concentrations of Pentraxine-3, Procalcitonin, Ang-2 and the soluble receptors were significantly higher in children with coma as defined by a Blantyre Coma Score < 3 (p < 0.001 for Pentraxine-3, suPAR, and sTie-2, p = 0.004 for PCT, p = 0.005 for sICAM-1, p = 0.04 for Ang-2). Moreover, except for the PCT level, the concentrations of Pentraxine-3, suPAR, sEPCR, sICAM-1, sTie-2 and Ang-2 were higher among children who died from severe malaria compared to those who survived (p = 0.037, p = 0.035, p < 0.0001, p= 0.0008, p = 0.01 and p = 0.02, respectively). These findings indicate the ability of these molecules to accurately discriminate among clinical manifestations of malaria, thus, they might be potentially useful for the early prognostic of severe and fatal malaria, and to improve management of severe cases.

Interactions Between Obesity and One-Carbon Metabolism Genes in Predicting Prostate Cancer Outcomes Among White and Black Patients.

BACKGROUND: One-carbon metabolism genes are linked to several cancers, but the association with prostate cancer (PCa) is less clear. Studies examining the relationship have not accounted for obesity, a risk factor for advanced PCa and altered methylation patterns. We hypothesized that obesity could moderate the association between one-carbon metabolism genes and PCa outcomes. METHODS: We conducted secondary data analyses of the Study of Clinical Outcomes, Risk and Ethnicity. Obesity was included as a primary exposure and modifier (interacting with genetic polymorphisms) in the analytic models. We used logistic regression to determine associations of common one-carbon metabolism genotypes with odds of high stage (T3/T4) and high grade (Gleason score ≥ 7). We used Cox regression to examine associations of genotypes with biochemical recurrence. RESULTS: There were 808 patients (632 White and 176 Black.) Among White men, we observed associations of TCN2_R259P with increased odds of high stage (OR = 0.64, 95% CI = 0.41-1.00), but no significant interactions with obesity. Among Black men, the SCL19A1_61bpdel and CBS_68bpINS variants were associated with high grade (OR = 2.61, 95% CI = 1.39-4.89 and OR = 0.29, 95% CI = 0.09-0.91, respectively.) Both the CBS_68bpINS and MTHFR_E429A variants interacted with obesity in Black men, where the highest risk for biochemical failure and odds of high grade, respectively, occurred among obese patients with variants. CONCLUSIONS: We observed associations of one-carbon metabolism genes with different associations by race. We also observed interactions with obesity related to PCa outcomes in Black men only. Therefore, the involvement of one-carbon metabolism on PCa was dependent upon obesity status for Black men. These novel results could help identify patients that might benefit from effective weight management targeting one-carbon metabolism effects.

RASAL2 suppresses the proliferative and invasive ability of PC3 prostate cancer cells.

The RAS protein activator like 2 (RASAL2) negatively regulates RAS proto-oncogene which is activated by high mutation rate in cancer. Thus, RASAL2 expression could potentially limit the function of RAS in prostate cancer (PCa). Genome-wide DNA methylation analysis demonstrated that RASAL2 is differentially hypermethylated in PCa tissues compared to benign prostate tissues. The PCR analysis of RASAL2 mRNA transcript showed differential expression in a panel of prostate cell lines with most PCa showing lower RASAL2 expression compared to benign prostatic epithelial cells. In PCa PC3 cells, the ectopic expression of RASAL2 significantly inhibited cell proliferation and invasion and induced an S phase plus G2/M phase cell cycle arrest. Ingenuity Pathway Analysis (IPA) demonstrated a cross talk between RASAL2 and TNFα, a key cytokine in immune signaling pathway that is relevant in PCa. Over-expression of RASAL2 downregulated TNFα expression whereas the knockdown of RASAL2 caused increased expression of TNFα. Taken together, our data demonstrates tumor suppressor role for RASAL2 in human PCa cells, despite increased RAS oncogenic activity. Our observation provides a new mechanistic insight of RASAL2 expression in aberrant Ras expression and immune signaling in PCa cells suggesting a potential novel therapeutic target for PCa.

DNA methylation and exposure to violence among African American young adult males.

Exposure to violence (ETV) has been linked to epigenomics mechanisms such as DNA methylation (DNAm). We used epigenetic profiling of blood collected from 32 African American young adult males who lived in Washington DC to determine if changes in DNAm at CpG sites affiliated with nervous and immune system were associated with exposure to violence. Pathway analysis of differentially methylated regions comparing high and low ETV groups revealed an enrichment of gene sets annotated to nervous system and immune ontologies. Many of these genes are known to interact with each other which suggests DNAm alters gene function in the nervous and immune system in response to ETV. Using data from a unique age group, young African American adult males, we provide evidence that lifetime ETV could impact DNA methylation in genes impacted at Central Nervous System and Immune Function sites. METHOD: Methylation analysis was performed on DNA collected from the blood of participants classified with either high or low lifetime ETV. Illumina®MethylationEPIC Beadchips (~850k CpG sites) were processed on the iScan System to examine whole-genome methylation differences. Differentially methylated CpG-sites between high (n â€‹= â€‹19) and low (n â€‹= â€‹13) groups were identified using linear regression with violence and substance abuse as model covariates. Gene ontology analysis was used to identify enrichment categories from probes annotated to the nearest gene. RESULTS: A total of 595 probes (279 hypermethylated; 316 hypomethylated) annotated to 383 genes were considered differentially methylated in association with ETV. Males with high ETV showed elevated methylation in several signaling pathways but were most impacted at Central Nervous System and Immune Function affiliated sites. Eight candidate genes were identified that play important biological roles in stress response to violence with HDAC4 (10%), NR4A3 (11%), NR4A2 (12%), DSCAML1(12%), and ELAVL3 (13%) exhibiting higher levels in the low ETV group and DLGAP1 (10%), SHANK2 (10%), and NRG1(11%) having increased methylation in the high ETV group. These findings suggest that individuals subjected to high ETV may be at risk for poor health outcomes that have not been reported previously.

Aerobic Exercise Training-Induced Changes on DNA Methylation in Mild Cognitively Impaired Elderly African Americans: Gene, Exercise, and Memory Study - GEMS-I.

BACKGROUND: DNA methylation at CpG sites is a vital epigenetic modification of the human genome affecting gene expression, and potentially, health outcomes. However, evidence is just budding on the effects of aerobic exercise-induced adaptation on DNA methylation in older mild cognitively impaired (MCI) elderly African American (AAs). Therefore, we examined the effects of a 6-month aerobic exercise-intervention on genome-wide DNA methylation in elderly AA MCI volunteers. DESIGN: Elderly AA volunteers confirmed MCI assigned into a 6-month program of aerobic exercise (eleven participants) underwent a 40-min supervised-training 3-times/week and controls (eight participants) performed stretch training. Participants had maximal oxygen consumption (VO2max) test and Genome-wide methylation levels at CpG sites using the Infinium HumanMethylation450 BeadChip assay at baseline and after a 6-month exercise program. We computed false discovery rates (FDR) using Sidak to account for multiplicity of tests and performed quantitative real-time polymerase chain-reaction (qRT-PCR) to confirm the effects of DNA methylations on expression levels of the top 5 genes among the aerobic participants. CpG sites identified from aerobic-exercise participants were similarly analyzed by the stretch group to quantify the effects of exercise-induced methylation changes among the group of stretch participants. RESULTS: Eleven MCI participants (aerobic: 73% females; mean age 72.3 ± 6.6 years) and eight MCI participants (stretch: 75% female; mean age 70.6 ± 6.7 years) completed the training. Aerobic exercise-training was associated with increases in VO2max and with global hypo- and hypermethylation changes. The most notable finding was CpG hypomethylation within the body of the VPS52 gene (P = 5.4 × 10-26), a Golgi-associated protein, involved in intracellular protein trafficking including amyloid precursor protein. qRT-PCR confirmed a nearly twofold increased expression of VPS52. Other top findings with FDR q-value < 10-5, include hypomethylations of SCARB1 (8.8 × 10-25), ARTN (6.1 × 10-25), NR1H2 (2.1 × 10-18) and PPP2R5D (9.8 × 10-18). CONCLUSION: We conclude that genome-wide DNA methylation patterns is associated with exercise training-induced methylation changes. Identification of methylation changes around genes previously shown to interact with amyloid biology, intracellular protein trafficking, and lipoprotein regulations provide further support to the likely protective effect of exercise in MCI. Future studies in larger samples are needed to confirm our findings.

Androgen-induced Epigenetic Profiles of Polycomb and Trithorax Genes in Prostate Cancer Cells.

BACKGROUND/AIM: Androgens are essential for the growth of most prostate cancers (PCa). As a result, androgen ablation is the mainstay of the treatment of PCa. Proteins of the polycomb and trithorax family are master epigenetic regulators of cell type specific gene expression including androgen receptor. MATERIALS AND METHODS: We interrogated epigenetic changes of a 24-gene panel corresponding to polycomb and trithorax genes by PCR array and differential gene expression by quantitative real time-PCR on prostate cancer cell line (LNCaP) treated with the synthetic ligand R1881. RESULTS: We observed the highest methylation for CBX2, PCGF6, PHC2, EZH2 and TRIM27 genes and the lowest methylation for CBX8 and PCGF2 (p<0.05), and a modest decrease in the expression of EZH2. CONCLUSION: Differential methylation profiles of polycomb and trithorax genes may contribute to the dynamics of prostate carcinogenesis.

Association of Genetic Ancestry With DNA Methylation Changes in Prostate Cancer Disparity.

BACKGROUND: We hypothesized that ancestry-mediated methylated DNA changes may drive racial and ethnic disparity in prostate cancer (PCa). To test this hypothesis, we analyzed genetic ancestry and association with DNA methylation changes in PCa disparity. MATERIALS AND METHODS: Pyrosequencing and ancestry informative markers were used for DNA methylation and genetic ancestry testing, respectively. RESULTS: Using Spearman rho rank correlation test, the data demonstrated significant (p<0.05) and variable association between African-American ancestry and DNA methylation for all genes investigated in prostate tissues. CONCLUSION: Genetic ancestry influences DNA methylation and this modifying factor must be considered in epigenetic association studies in populations of admixed patients.

Correlating blood-based DNA methylation markers and prostate cancer risk in African-American men.

The objective of this work was to investigate the clinical significance of promoter gene DNA methylation changes in whole blood from African-American (AA) men with prostate cancer (PCa). We used high throughput pyrosequencing analysis to quantify percentage DNA methylation levels in a panel of 8 genes (RARß2, TIMP3, SPARC, CDH13, HIN1, LINE1, CYB5R2 and DRD2) in blood DNA obtained from PCa and non-cancerous controls cases. Correlations of methylation status and various clinicopathological features were evaluated. Six genes tested achieved significant difference in DNA methylation levels between the PCa compared to control cases (P < 0.05). The TIMP3 loci demonstrated significant correlation of DNA methylation with age for all cases analyzed (p < 0.05). We observed an inverse correlation between CDH13 methylation (p = 0.045; r = -0.21) and serum vitamin D level whereas TIMP3 methylation (p = 0.021; r = -0.24) and DRD2 methylation (p = 0.056; r = -0.201) showed inverse correlation with supplementary vitamin D in the cancer cases. We also observed a direct correlation between methylation of RARß2 (p = 0.0036; r = 0.293) and SPARC (p = 0.0134; r = 0.20) loci with PSA level in the controls but not the cancer cases. In addition, alcohol cases significantly correlated with higher RARß2 methylation (p = 0.0314) in comparison with non-alcohol cases. Furthermore, we observed an inverse correlation of DRD2 methylation (p = 0.0349; r = -0.343) and Gleason score. Our data suggests that promoter methylation occurred more frequently in the blood of AA PCa and is associated with various clinicopathological features in AA men with PCa.

Sex steroid-induced DNA methylation changes and inflammation response in prostate cancer.

BACKGROUND: Sex steroid hormones have been reported to induce inflammation causing dysregulation of cytokines in prostate cancer cells. However, the underlying epigenetic mechanism has not well been studied. The objective of this study was to evaluate the effect of sex steroid hormones on epigenetic DNA methylation changes in prostate cancer cells using a signature PCR methylation array panel that correspond to 96 genes with biological function in the human inflammatory and autoimmune signals in prostate cancer. Of the 96-gene panel, 32 genes showed at least 10% differentially methylation level in response to hormonal treatment when compared to untreated cells. Genes that were hypomethylated included CXCL12, CXCL5, CCL25, IL1F8, IL13RAI, STAT5A, CXCR4 and TLR5; and genes that were hypermethylated included ELA2, TOLLIP, LAG3, CD276 and MALT1. Quantitative RT-PCR analysis of select genes represented in a cytokine expression array panel showed inverse association between DNA methylation and gene expression for TOLLIP, CXCL5, CCL18 and IL5 genes and treatment of prostate cancer cells with 5'-aza-2'-deoxycytidine with or without trichostatin A induced up-regulation of TOLLIP expression. Further analysis of relative gene expression of matched prostate cancer tissues when compared to benign tissues from individual patients with prostate cancer showed increased and significant expression for CCL18 (2.6-fold; p<0.001), a modest yet significant increase in IL5 expression (1.17-fold; p=0.015), and a modest increase in CXCL5 expression (1.4-fold; p=0.25). In conclusion, our studies demonstrate that sex steroid hormones can induce aberrant gene expression via differential methylation changes in prostate carcinogenesis.

Design, synthesis and cytotoxicity studies of dithiocarbamate ester derivatives of emetine in prostate cancer cell lines.

A small library of emetine dithiocarbamate ester derivatives were synthesized in 25-86% yield via derivatization of the N2'- position of emetine. Anticancer evaluation of these compounds in androgen receptor positive LNCaP and androgen receptor negative PC3 and DU145 prostate cancer cell lines revealed time dependent and dose-dependent cytotoxicity. With the exception of compound 4c, all the dithiocarbamate ester analogs in this study showed appreciable potency in all the prostate cancer cell lines (regardless of whether it is androgen receptor positive or negative) with a cytotoxicity IC50 value ranging from 1.312 ± 0.032 µM to 5.201 ± 0.125 µM by day 7 of treatment. Compared to the sodium dithiocarbamate salt 1, all the dithiocarbamate ester analogs (2 and 4a-4 g) displayed lower cytotoxicity than compound 1 (PC3, IC50 = 0.087 ± 0.005 µM; DU145, IC50 = 0.079 ± 0.003 µM and LNCaP, IC50 = 0.079 ± 0.003 µM) on day 7 of treatment. Consequently, it appears that S-alkylation of compound 1 leads to a more stable dithiocarbamate ester derivative that resulted in lower anticancer activity in the prostate cancer cell lines.

DNA methylation profile associated with rapid decline in kidney function: findings from the CRIC study.

BACKGROUND: Epigenetic mechanisms may be important in the progression of chronic kidney disease (CKD). METHODS: We studied the genome-wide DNA methylation pattern associated with rapid loss of kidney function using the Infinium HumanMethylation 450 K BeadChip in 40 Chronic Renal Insufficiency (CRIC) study participants (n = 3939) with the highest and lowest rates of decline in estimated glomerular filtration rate. RESULTS: The mean eGFR slope was 2.2 (1.4) and -5.1 (1.2) mL/min/1.73 m(2) in the stable kidney function group and the rapid progression group, respectively. CpG islands in NPHP4, IQSEC1 and TCF3 were hypermethylated to a larger extent in subjects with stable kidney function (P-values of 7.8E-05 to 9.5E-05). These genes are involved in pathways known to promote the epithelial to mesenchymal transition and renal fibrosis. Other CKD-related genes that were differentially methylated are NOS3, NFKBIL2, CLU, NFKBIB, TGFB3 and TGFBI, which are involved in oxidative stress and inflammatory pathways (P-values of 4.5E-03 to 0.046). Pathway analysis using Ingenuity Pathway Analysis showed that gene networks related to cell signaling, carbohydrate metabolism and human behavior are epigenetically regulated in CKD. CONCLUSIONS: Epigenetic modifications may be important in determining the rate of loss of kidney function in patients with established CKD.

Design, synthesis, and evaluation of pH-dependent hydrolyzable emetine analogues as treatment for prostate cancer.

The N-2' position of the natural product emetine has been derivatized to thiourea, urea, sulfonamide, dithiocarbamate, carbamate, and pH responsive hydrolyzable amide analogues. In vitro studies of these analogues in PC3 and LNCaP prostate cancer cell lines showed that the analogues are generally less cytotoxic (average IC(50) ranging from 0.079 to 10 µM) than emetine (IC(50) ranging from 0.0237 to 0.0329 µM). The pH sensitive sodium dithiocarbamate salt 13 and the amide analogues 21, 22, 26 (obtained from maleic and citraconic anhydrides) showed the most promise as acid-activatable prodrugs under mildly acidic conditions found in the cancer microenvironment. These prodrugs released 12-83% of emetine at pH 6.5 and 41-95% emetine at pH 5.5. Compounds 13 and 26 were further shown to exhibit increased cytotoxicity in PC3 cell culture medium that was already below pH 7.0 at the time of treatment.

Relationship between tumor DNA methylation status and patient characteristics in African-American and European-American women with breast cancer.

Aberrant DNA methylation is critical for development and progression of breast cancer. We investigated the association of CpG island methylation in candidate genes and clinicopathological features in 65 African-American (AA) and European-American (EA) breast cancer patients. Quantitative methylation analysis was carried out on bisulfite modified genomic DNA and sequencing (pyrosequencing) for promoter CpG islands of p16, ESR1, RASSF1A, RARß2, CDH13, HIN1, SFRP1 genes and the LINE1 repetitive element using matched paired non-cancerous and breast tumor specimen (32 AA and 33 EA women). Five of the genes, all known tumor suppressor genes (RASSF1A, RARß2, CDH13, HIN1 and SFRP1), were found to be frequently hypermethylated in breast tumor tissues but not in the adjacent non-cancerous tissues. Significant differences in the CDH13 methylation status were observed by comparing DNA methylation between AA and EA patients, with more obvious CDH13 methylation differences between the two patient groups in the ER- disease and among young patients (age<50). In addition, we observed associations between CDH13, SFRP1, and RASSF1A methylation and breast cancer subtypes and between SFRP1 methylation and patient's age. Furthermore, tumors that received neoadjuvant therapy tended to have reduced RASSF1A methylation when compared with chemotherapy naïve tumors. Finally, Kaplan Meier survival analysis showed a significant association between methylation at 3 loci (RASSF1A, RARß2 and CDH13) and reduced overall disease survival. In conclusion, the DNA methylation status of breast tumors was found to be significantly associated with clinicopathological features and race/ethnicity of the patients.

Humanizing π-class glutathione S-transferase regulation in a mouse model alters liver toxicity in response to acetaminophen overdose.

BACKGROUND: Glutathione S-transferases (GSTs) metabolize drugs and xenobiotics. Yet despite high protein sequence homology, expression of π-class GSTs, the most abundant of the enzymes, varies significantly between species. In mouse liver, hepatocytes exhibit high mGstp expression, while in human liver, hepatocytes contain little or no hGSTP1 mRNA or hGSTP1 protein. π-class GSTs are known to be critical determinants of liver responses to drugs and toxins: when treated with high doses of acetaminophen, mGstp1/2+/+ mice suffer marked liver damage, while mGstp1/2-/- mice escape liver injury. METHODOLOGY/PRINCIPAL FINDINGS: To more faithfully model the contribution of π-class GSTs to human liver toxicology, we introduced hGSTP1, with its exons, introns, and flanking sequences, into the germline of mice carrying disrupted mGstp genes. In the resultant hGSTP1+mGstp1/2-/- strain, π-class GSTs were regulated differently than in wild-type mice. In the liver, enzyme expression was restricted to bile duct cells, Kupffer cells, macrophages, and endothelial cells, reminiscent of human liver, while in the prostate, enzyme production was limited to basal epithelial cells, reminiscent of human prostate. The human patterns of hGSTP1 transgene regulation were accompanied by human patterns of DNA methylation, with bisulfite genomic sequencing revealing establishment of an unmethylated CpG island sequence encompassing the gene promoter. Unlike wild-type or mGstp1/2-/- mice, when hGSTP1+mGstp1/2-/- mice were overdosed with acetaminophen, liver tissues showed limited centrilobular necrosis, suggesting that π-class GSTs may be critical determinants of toxin-induced hepatocyte injury even when not expressed by hepatocytes. CONCLUSIONS: By recapitulating human π-class GST expression, hGSTP1+mGstp1/2-/- mice may better model human drug and xenobiotic toxicology.

Identification of differentially methylated genes in normal prostate tissues from African American and Caucasian men.

PURPOSE: Aberrant DNA methylation changes are common somatic alterations in prostate carcinogenesis. We examined the methylation status of six genes in prostate tissue specimens from African American (AA) and Caucasian (Cau) males. EXPERIMENTAL DESIGN: We used pyrosequencing to quantitatively measure the methylation status of GSTP1, AR, RARbeta2, SPARC, TIMP3, and NKX2-5. Real-time PCR was used to determine gene expression, and gene reactivation was analyzed by 5-aza-2'-deoxycytidine and/or trichostatin A treatment. RESULTS: Statistical analysis showed significantly higher methylation in the prostate cancer tissue samples in comparison with matched normal samples for GSTP1 (P = 0.0001 for AA; P = 0.0008 for Cau), RARbeta2 (P < 0.001 for AA and Cau), SPARC (P < 0.0001 for AA and Cau), TIMP3 (P < 0.0001 for AA and Cau), and NKX2-5 (P < 0.0001 for AA; P = 0.003 for Cau). Overall, we observed significant differences (P < 0.05) in the methylation level for all genes, except GSTP1, in the AA samples in comparison with the Cau samples. Furthermore, regression analysis revealed significantly higher methylation for NKX2-5 (P = 0.008) and TIMP3 (P = 0.039) in normal prostate tissue samples from AA in comparison with Cau, and a statistically significant association of methylation with age for NKX2-5 (P = 0.03) after adjusting for race. CONCLUSION: Our findings show higher methylation of several genes in prostate tissue samples from AA in comparison with Cau and may potentially contribute to the racial differences that are observed in prostate cancer pathogenesis.