catechol-O-methyltransferase Expression Research Articles

Targeting Catechol-O-Methyltransferase Induces Mitochondrial Dysfunction and Enhances the Efficacy of Radiotherapy in Glioma.

Radiotherapy (RT) is commonly used to try to eliminate any remaining tumor cells following surgical resection of glioma. However, tumor recurrence is prevalent, highlighting the unmet medical need to develop therapeutic strategies to enhance the efficacy of RT in glioma. Focusing on the radiosensitizing potential of the currently approved drugs known to cross the blood-brain barrier can facilitate rapid clinical translation. Here, we assessed the role of catechol-O-methyltransferase (COMT), a key enzyme to degrade catecholamines and a drug target for Parkinson's disease, in glioma treatment. Analysis of The Cancer Genome Atlas data showed significantly higher COMT expression levels in both low-grade glioma and glioblastoma compared to normal brain tissues. Inhibition of COMT by genetic knockout or FDA-approved COMT inhibitors significantly sensitized glioma cells to RT in vitro and in vivo. Mechanistically, COMT inhibition in glioma cells led to mitochondria dysfunction and increased mitochondrial RNA release into the cytoplasm, activating the cellular antiviral double-stranded RNA sensing pathway and type I interferon (IFN) response. Elevated type I IFNs stimulated the phagocytic capacity of microglial cells, enhancing RT efficacy. Given the long-established safety record of the COMT inhibitors, these findings provide a solid rationale to evaluate them in combination with RT in patients with glioma. Significance: Inhibition of catechol-O-methyltransferase, a well-established drug target in Parkinson's disease, interferes with mitochondrial electron transport and induces mitochondrial double-stranded RNA leakage, activating type I interferon signaling and sensitizing glioma to radiotherapy.

Sex-Specific Regulation of Catecholamine Signaling in Rats Exposed to Dexamethasone In Utero and Angiotensin in Adulthood

Pregnant women who are at risk of pre-term delivery are given the synthetic glucocorticoid dexamethasone (DEX) to stimulate fetal lung development as it is not degraded by 11ß-hydroxysteroid dehydrogenase, enabling DEX to freely cross through the placenta and act on the developing fetus. Our lab has previously found that in-utero DEX exposure increases stress-induced blood pressure and heart rate responses in female, as well as autonomic dysregulation in the adult offspring. Angiotensin II (Ang II) induces hypertension and cardiac remodeling in part through activation of the sympathetic nervous system. Given these prior findings, this project explores whether prenatal DEX exposure alters the response of Ang II on cardiac sympathetic nervous system regulation. Pregnant rats were administered either vehicle (20% w/v 2-hydroxypropyl ß-cyclodextran) or the glucocorticoid DEX (0.4 mg/kg body weight, i.p) on gestation days 18-21. When offspring reached ~15 weeks of age, groups were administered either subcutaneous AngII (200 ng/kg per min) as a cardiovascular challenge or saline as experimental control for four weeks. To measure local sympathetic signaling, tyrosine hydroxylase (TH) was analyzed as it serves as the rate limiting step of catecholamine synthesis while catechol-O-methyltransferase (COMT) was selected as it catalyzes catecholamine breakdown. Gene expression of adrenoreceptor ß1 ( Adrb1) was measured to assess receptor availability for sympathetic activity. Data were analyzed by 3-way ANOVA with sex, prenatal treatment (i.e., DEX vs. Vehicle), and postnatal treatment (i.e., saline vs. AngII) as the variables. There was an overall main effect of prenatal DEX treatment to reduce TH expression in the LV (p=0.018), an effect that was more pronounced in males. COMT expression was found to be significantly impacted by sex, prenatal treatment, and post-natal treatment (3-way interaction, p=0.016). Thus, the degree to which DEX and Ang II impacted COMT expression differed by sex. There was a sex x postnatal treatment interaction (p=0.013) on ADRB1 expression whereby expression was increased by Ang II only in female hearts. Although prior studies have shown that Ang II can increase local sympathetic activity in the heart, the present data suggest that prenatal treatments disrupt this response in a sex-specific manner. Moreover, sex and prenatal treatment were greater drivers of local sympathetic regulation than Ang II. NIMH/ORWH 1U54MH118919. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Sex dimorphism controls dysbindin-related cognitive dysfunctions in mice and humans with the contribution of COMT.

Cognitive dysfunctions are core-enduring symptoms of schizophrenia, with important sex-related differences. Genetic variants of the DTBPN1 gene associated with reduced dysbindin-1 protein (Dys) expression negatively impact cognitive functions in schizophrenia through a functional epistatic interaction with Catechol-O-methyltransferase (COMT). Dys is involved in the trafficking of dopaminergic receptors, crucial for prefrontal cortex (PFC) signaling regulation. Moreover, dopamine signaling is modulated by estrogens via inhibition of COMT expression. We hypothesized a sex dimorphism in Dys-related cognitive functions dependent on COMT and estrogen levels. Our multidisciplinary approach combined behavioral-molecular findings on genetically modified mice, human postmortem Dys expression data, and in vivo fMRI during a working memory task performance. We found cognitive impairments in male mice related to genetic variants characterized by reduced Dys protein expression (pBonferroni = 0.0001), as well as in male humans through a COMT/Dys functional epistatic interaction involving PFC brain activity during working memory (t(23) = -3.21; pFDR = 0.004). Dorsolateral PFC activity was associated with lower working memory performance in males only (p = 0.04). Also, male humans showed decreased Dys expression in dorsolateral PFC during adulthood (pFDR = 0.05). Female Dys mice showed preserved cognitive performances with deficits only with a lack of estrogen tested in an ovariectomy model (pBonferroni = 0.0001), suggesting that genetic variants reducing Dys protein expression could probably become functional in females when the protective effect of estrogens is attenuated, i.e., during menopause. Overall, our results show the differential impact of functional variants of the DTBPN1 gene interacting with COMT on cognitive functions across sexes in mice and humans, underlying the importance of considering sex as a target for patient stratification and precision medicine in schizophrenia.

Abstract 7629: Association between COMT expression and therapeutic efficacy of androgen receptor-axis targeted in prostate cancer

Abstract Background: The enzyme of catechol-O-methyltransferase (COMT) inhibits the proliferation of prostate cancer (PCa). There has been no obvious biomarker that contributes to the selection of a therapeutic agent among new-generation androgen receptor-axis targeted (ARAT) therapy, such as abiraterone (ABI) or enzalutamide (ENZ). We aimed to assess the possibility of using COMT expression as a biomarker for PCa progression and the criteria for selecting ABI or ENZ. Methods: We retrospectively evaluated 60 patients with PCa treated with ABI or ENZ via needle biopsy or radical prostatectomy. Immunostaining of COMT was performed using formalin-fixed, paraffin-embedded blocks obtained from the initial prostate biopsy for diagnosis or prostatectomy. Immunostaining evaluations were scored by immunostaining intensity (0-3) and staining area (0-3), and the total score ≥4 was defined as "high expression" and the total score <4 was defined as "low expression.” The associations between COMT expression levels and clinical outcomes, including the duration of ENZ or ABI response, were also assessed. Results: Of the 60 cases, nine (15%) showed low expression of COMT. Overall survival was significantly shorter in patients with lower COMT expression (hazard ratio [HR] = 0.24, 95% confidence interval [CI] 0.07-0.83, P= 0.024). When ENZ was administered as the first ARAT, progression-free survival (PFS) was significantly different between patients with COMT expression scores <4 and ≥4 (median 5.9 and 23.3 months, respectively). However, when ABI was administered as the first ARAT, there was no significant difference in the PFS of ARAT between COMT expression scores <4 and ≥4. Conclusion: Patients with lower COMT expression have a significantly poorer prognosis for PCa. In addition, PFS after ENZ administration, as the first ARAT, correlated with COMT expression levels. To our knowledge, this is the first report of an association between COMT expression and the duration of ARAT. Citation Format: Shigekatsu Maekawa, Ryo Takata, Ei Shiomi, Daiki Ikarashi, Tomohiko Matsuura, Renpei Kato, Mitsugu Kanehira, Jun Sugimura, Wataru Obara. Association between COMT expression and therapeutic efficacy of androgen receptor-axis targeted in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7629.

Integrated multiplexed assays of variant effect reveal determinants of catechol-O-methyltransferase gene expression

Multiplexed assays of variant effect are powerful methods to profile the consequences of rare variants on gene expression and organismal fitness. Yet, few studies have integrated several multiplexed assays to map variant effects on gene expression in coding sequences. Here, we pioneered a multiplexed assay based on polysome profiling to measure variant effects on translation at scale, uncovering single-nucleotide variants that increase or decrease ribosome load. By combining high-throughput ribosome load data with multiplexed mRNA and protein abundance readouts, we mapped the cis-regulatory landscape of thousands of catechol-O-methyltransferase (COMT) variants from RNA to protein and found numerous coding variants that alter COMT expression. Finally, we trained machine learning models to map signatures of variant effects on COMT gene expression and uncovered both directional and divergent impacts across expression layers. Our analyses reveal expression phenotypes for thousands of variants in COMT and highlight variant effects on both single and multiple layers of expression. Our findings prompt future studies that integrate several multiplexed assays for the readout of gene expression.

Dopamine receptors gene overexpression in the microenvironment of invasive gastric cancer and its potential implications.

Gastric cancer (GC) is the fifth most common cancer worldwide and the most commonly diagnosed cancer in Iran. The nervous system provides proximity to tumor cells by releasing neurotransmitters such as dopamine and presenting them to the corresponding receptor-bearing tumors. While nerve fibers infiltrate the tumor microenvironment, little is known about the expression levels of dopamine (DA), dopamine receptors (DRs), and catechol-O-methyltransferase (COMT) in GC patients. DRs and COMT expression were analyzed in 45 peripheral blood mononuclear cells (PBMCs) and 20 paired tumor and adjacent tissue of GC patients by quantitative polymerase chain reaction. DA was measured in plasma specimens using enzyme-linked immunosorbent assay. Protein-protein interaction analysis was carried out to identify GC-related hub genes. Increased expression of DRD1-DRD3 was found in tumor specimens compared with adjacent non-cancerous specimens (P < 0.05). A positive correlation was found between DRD1 and DRD3 expression (P = 0.009); DRD2 and DRD3 expression (P = 0.04). Plasma levels of dopamine were significantly lower in patients (1298 pg/ml) than in controls (4651 pg/ml). DRD1-DRD4 and COMT were up-regulated in PBMCs of patients compared with controls (P < 0.0001). Bioinformatic analyses showed 30 hub genes associated with Protein kinase A and extracellular signal-regulated kinase signaling pathways. The findings indicated dysregulation of DRs and COMT mRNA expression in GC and suggest that the brain- gastrointestinal axis may mediate gastric cancer development. Network analysis revealed that combination treatments could be considered for optimizing and improving the precision treatment of GC.

Sex-specific responses to juvenile stress on the dopaminergic system in an animal model of attention-deficit hyperactivity disorder

The etiology of attention-deficit hyperactivity disorder (ADHD) strongly suggests a genetic component as the main cause; however, environmental factors such as early adverse experiences in childhood may play an interactive role with the genetic susceptibility. Spontaneously hypertensive rats (SHRs), a genetic ADHD model, and control Wistar Kyoto rats (WKYs) were subjected to chronic unpredictable mild stress during the juvenile period. The behavioral characteristics were monitored, and dopamine-related factors in the core regions of dopaminergic pathways were measured. Higher ADHD symptom-related behaviors were observed in response to juvenile stress in male SHRs than control WKYs. For the SHRs subjected to juvenile stress, hyperactivity in males, recognition in females, and depressant potential in both sexes were markedly observed. In the expression of 17 dopamine-related genes and proteins, greater changes were detected in male SHRs subjected to juvenile stress, especially in dopamine metabolic factors. Dopamine clearance factors involved in dopamine degradation and transport, especially catechol-O-methyltransferase (COMT) and dopamine transporter (DAT), showed sex-specific differences induced by juvenile stress in dopamine metabolite assays. Moreover, stressed male SHRs treated with methylphenidate showed better improvement in behavior than the females, resulting in different levels of COMT and DAT amelioration. These results suggest that juvenile stress potentially increased the incidence of ADHD in a genetic rat model, which showed sex-specific differences based on the expression of COMT and DAT. Therefore, our results could help develop gender-specific diagnostics and healthcare options for juvenile stress in patients with ADHD.

Catechol-O-Methyltransferase Loss Drives Cell-Specific Nociceptive Signaling via the Enteric Catechol-O-Methyltransferase/microRNA-155/Tumor Necrosis Factor α Axis

The etiology of abdominal pain in postinfectious, diarrhea-predominant irritable bowel syndrome (PI-IBS-D) is unknown, and few treatment options exist. Catechol-O-methyltransferase (COMT), an enzyme that inactivates and degrades biologically active catecholamines, plays an important role in numerous physiologic processes, including modulation of pain perception. Our objective was to determine the mechanism(s) of how decreased colonic COMT in PI-IBS-D patients contributes to the chronic abdominal pain phenotype after enteric infections. Colon neurons, epithelial cells, and macrophages were procured with laser capture microdissection from PI-IBS-D patients to evaluate cell-specific colonic COMT, microRNA-155 (miR-155), and tumor necrosis factor (TNF) α expression levels compared to recovered patients (infection cleared: did not develop PI-IBS-D) and control individuals. COMT-/-, colon-specific COMT-/-, and miR-155-/- mice and human colonoids were used to model phenotypic expression of COMT in PI-IBS-D patients and to investigate signaling pathways linking abdominal pain. Citrobacter rodentium and trinitrobenzene sulfonic acid animal models were used to model postinflammatory changes seen in PI-IBS-D patients. Colonic COMT levels were significantly decreased and correlated with increased visual analog scale abdominal pain ratings in PI-IBS-D patients compared to recovered patients and control individuals. Colonic miR-155 and TNF-α were increased in PI-IBS-D patients with diminished colonic COMT. COMT-/- mice had significantly increased expression of miR-155 and TNF-α in both colon tissues and dorsal root ganglia. Introduction of cV1q antibody (anti-TNF-α) into mice reversed visceral hypersensitivity after C rodentium and trinitrobenzene sulfonic acid. Decreased colonic COMT in PI-IBS-D patients drives abdominal pain phenotypes via the COMT/miR-155/TNF-α axis. These important findings will allow new treatment paradigms and more targeted and personalized medicine approaches for gastrointestinal disorders after enteric infections.

An integrated targeted metabolomics and network pharmacology approach to exploring the mechanism of ellagic acid against sleep deprivation-induced memory impairment and anxiety.

As a neuroprotective agent, ellagic acid (EA) is extremely beneficial. Our previous study found that EA can alleviate sleep deprivation (SD)-induced abnormal behaviors, although the mechanisms underlying this protective effect have not yet been fully elucidated. An integrated network pharmacology and targeted metabolomics approach was utilized in this study to investigate the mechanism of EA against SD-induced memory impairment and anxiety. Behavioral tests were conducted on mice after 72 h of SD. Hematoxylin and eosin staining and nissl staining were then carried out. Integration of network pharmacology and targeted metabolomics was performed. Eventually, the putative targets were further verified using molecular docking analyses and immunoblotting assays. The present study findings confirmed that EA ameliorated the behavioral abnormalities induced by SD and prevented histopathological and morphological damage to hippocampal neurons. Through multivariate analysis, clear clustering was obtained among different groups, and potential biomarkers were identified. Four key targets, catechol-O-methyltransferase (COMT), cytochrome P450 1B1 (CYP1B1), glutathione S-transferase A2 (GSTA2), and glutathione S-transferase P1 (GSTP1), as well as the related potential metabolites and metabolic pathways, were determined by further integrated analysis. Meanwhile, in-silico studies revealed that EA is well located inside the binding site of CYP1B1 and COMT. The experimental results further demonstrated that EA significantly reduced the increased expression of CYP1B1 and COMT caused by SD. The findings of this study extended our understanding of the underlying mechanisms by which EA treats SD-induced memory impairment and anxiety, and suggested a novel approach to address the increased health risks associated with sleep loss.

The potential protective effects of estradiol and 2-methoxyestradiol in ischemia reperfusion-induced kidney injury in ovariectomized female rats

AimsInvestigating the impact of 17β estradiol (E2) and its endogenous non-hormonal metabolite 2-methoxyestradiol (2ME) on renal ischemia-reperfusion (RIR) induced kidney injury in ovariectomized (OVX) rats and the role of catechol-O-methyltransferase (COMT) in their effects. Main methodsEighty female rats were allocated into eight groups. Control group, Sham group, OVX group, OVX and RIR group, OVX + RIR + E2 group, OVX + RIR + 2ME group, OVX + RIR + E2 + Entacapone group and OVX + RIR + 2ME + Entacapone group, respectively.Twenty-four hours post RIR, creatinine (Cr) and blood urea nitrogen (BUN) were determined in serum, while malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT), Glutathione (GSH), myeloperoxidase (MPO), as well as the expressions of COMT, hypoxia inducible factor-1α (HIF-1α) and tyrosine hydroxylase (TH) were assessed in the kidney tissues. Key findingsSerum Cr, BUN, MPO, as well as HIF-1α and TH expressions were significantly higher with concomitant decrease in COMT expression, SOD and CAT activities and GSH content observed in OVX and RIR group compared to sham group. E2 and 2ME treatment significantly ameliorated all parameters measured in OVX and RIR rats. On the other hand, Entacapone significantly decreased the effect of E2, with no effect on 2ME treatment. SignificanceE2 ameliorates RIR-induced kidney injury and this effect is mediated, at least in part, via its COMT-mediated conversion to 2ME. Thus, 2ME by the virtue of its pleiotropic pharmacological effects can be used as a safe and effective treatment of RIR injury.

Suppressor effect of catechol-O-methyltransferase gene in prostate cancer.

Catechol-estrogens can cause genetic mutations and to counteract their oncogenicity, the catechol-O-methyltransferase (COMT) gene is capable of neutralizing these reactive compounds. In this study, we determined the functional effects and regulation of COMT in prostate cancer. Both the Cancer Genome Atlas (TCGA) and immunohistochemical analysis of clinical specimens demonstrated a reduction of COMT expression in prostate cancer. Also, western analyses of prostate cancer cell lines show COMT levels to be minimal in DuPro and DU145 and thus, these cells were used for further analyses. Re-expression of COMT led to suppressed migration ability (wound healing assay) and enhanced apoptosis (flow cytometric analyses), and when challenged with 4-hydroxyestradiol, a marked reduction of cell proliferation (MTT assay) was observed. Xenograft growth in athymic mice also resulted in inhibition due to COMT. As a mechanism, western analyses show cleaved CASP3 and BID were increased whereas XIAP and cIAP2 were reduced due to COMT. As COMT expression is low in prostate cancer, its regulation was determined. Databases identified several miRNAs capable of binding COMT and of these, miR-195 was observed to be increased in prostate cancer according to TCGA. Real-time PCR validated upregulation of miR-195 in clinical prostate cancer specimens as well as DuPro and DU145 and interestingly, luciferase reporter showed miR-195 capable of binding COMT and overexpressing miR-195 could reduce COMT in cells. These results demonstrate COMT to play a protective role by activating the apoptosis pathway and for miR-195 to regulate its expression. COMT may thus be a potential biomarker and gene of interest for therapeutic development for prostate cancer.

Genetic and Proteinic Linkage of MAO and COMT with Oral Potentially Malignant Disorders and Cancers of the Oral Cavity and Pharynx

Simple SummaryThe prevention and treatment of cancers of the oral cavity and pharynx are currently important issues for national health. Currently, the incidence of oral cavity and pharynx cancers is globally the highest in Taiwanese men. Regarding the occurrence of oral cavity and pharynx cancers and oral potentially malignant disorders (OPMD), no report has ascertained how betel quid (BQ) can induce the expression of monoamine oxidase (MAO) and catechol-O-methyltransferase (COMT). We aimed to explore the role and clinical significance of specific markers of BQ exposure and human susceptibility to MAO and COMT. Our findings highlight the association of MAO and COMT biomarkers to risks of oral and pharyngeal cancers and OPMD. These novel findings will provide important strategies for disease prevention, early clinical diagnosis, and treatment effectiveness, and will offer a strong foundation to reduce BQ-related cancers of the oral cavity and pharynx and OPMD.Betel quid (BQ), a group I human carcinogen, strongly contributes to an increased risk of oral potentially malignant disorders (OPMD) and cancers of the oral cavity and pharynx. This study was conducted to discover whether monoamine oxidase (MAO) and catechol-O-methyltransferase (COMT) variants play a potential role in the risk assessment of oral cavity and pharynx cancers and OPMD, particularly among BQ users. We applied a case–control study to confirm the polymorphism of MAO and COMT using single-nucleotide polymorphisms. We used qRT-PCR, Western blotting, and immunohistochemistry (IHC) to determine MAO and COMT expression. Carriers of the MAOA rs6323 G-allele, MAOB rs6324 G-allele, and COMT rs4633 C/C-genotype had a prominently increased risk of oral cavity and pharynx cancers (AOR = 56.99; p < 0.001). Compared to adjacent noncancerous tissues, a significant downregulation of MAO and COMT expression was exhibited in cancerous tissues (p < 0.01). Furthermore, in different cell models, MAO and COMT expression was significantly downregulated with an increased dose of arecoline (p < 0.01). In personalized preventive medicine for oral and pharyngeal cancers, our findings are the first to demonstrate the potential role of lower MAO and COMT expression levels, with the risk polymorphisms utilized as clinical biomarkers.

Association Analysis Between Catechol-O-Methyltransferase Expression and Cognitive Function in Patients with Schizophrenia, Bipolar Disorder, or Major Depression.

IntroductionSchizophrenia, bipolar disorder (BD), and major depressive disorder are three common mental disorders. Although their diagnosis and treatment differ, they partially overlap.MethodsTo explore the similarities and characteristics of these three psychiatric diseases, an intelligence quotient (IQ) assessment was performed to evaluate cognitive deficits. Relative catechol-O-methyltransferase (COMT) expression in peripheral blood mononuclear cells was examined in all three groups compared with healthy controls (HCs).ResultsThe results indicated that patients with any of the three psychiatric diseases presented IQ deficits, and that the first-episode schizophrenia (FES) group had even lower cognitive function than the other two groups. The relative COMT expression decreased in the FES group and increased in the BD group compared with the HC group. The correlation analysis of COMT expression level and IQ scores showed a positive correlation between relative COMT expression and full-scale IQ in the HC group. However, this correlation disappeared in all three psychiatric diseases studied.ConclusionIn conclusion, this cross-disease strategy provided important clues to explain lower IQ scores and dysregulated COMT expression among three common mental illnesses.

Accurate and sensitive detection of Catechol-O-methyltransferase activity by liquid chromatography with fluorescence detection

Catechol-O-methyltransferase (COMT) is a major drug metabolizing enzyme in humans. COMT expression is directedly associated with various mental diseases and cancers due to its essential role in catalyzing metabolic inactivation of endogenous catecholamines and catechol estrogens. However, a practical method to precisely measure COMT activities in biological samples is lacking. In the current study, we established a liquid chromatography-fluorescence detection (LC-FD) method based on fluorometric detection of the methylated product of 3-BTD, a fluorescent probe for COMT, to sensitively quantify COMT activities in human erythrocytes and cell homogenates. Assay validation of the established LC-FD based method was conducted for selectivity and sensitivity, range of linearity, precision and accuracy, recovery, biological matrices effect and stability. The limit of quantification for 3-BTMD (the methylated product of 3-BTD by COMT) of this method was 0.0083 nM, which is nearly 10-fold lower than that for previously published methods. The method was precise with intra- and inter-day relative standard deviation (RSD) lower than 5%. In addition, this method showed an excellent anti-interference ability with no effects of the endogenous substances on the fluorometric detection of 3-BTMD. The practical use of this method was established by its successful application for the measurement of COMT activities in individual human erythrocytes (n = 13), and in cell homogenates generated from four different human cell lines. Our results suggest that this method will be of great value in accurately determining the native activity of COMT in biological samples, which is beneficial for a complete understand of the role of COMT both in physiological and pathological conditions.

Pioglitazone ameliorates high fat diet-induced hypertension and induces catechol o-methyl transferase expression in rats

Our study aimed to investigate the effect of pioglitazone (PIO) on the obesity-associated metabolic effects and whether this effect is associated with modulation of catechol O-methyl transferase (COMT) expression in the high fat diet (HFD) induced obese rats. Male Wistar rats fed HFD were used to evaluate the effect of PIO on obesity-associated hypertension and the expression of COMT. The HFD-induced obesity was confirmed by the change in body weights, the fasting serum insulin (FSI) which assessed by ELISA, homeostasis model assessment - insulin resistance (HOMA-IR), fasting blood glucose (FBG), oral glucose tolerance test (OGTT) and lipid profile which were determined by colorimetric methods. Plasma epinephrine (EP) and norepinephrine (NE) were determined by ELISA and the systolic blood pressure (SBP) was recorded using the tail-cuff method. COMT expression was assessed by quantitative real time-polymerase chain reaction (qRT-PCR), and western blotting. The HFD-induced obesity was associated with glucose intolerance, derangement of the lipid profile, increased SBP, reduced COMT expression with a concomitant increase in plasma catecholamines. Most importantly, treatment with PIO ameliorated the HFD-induced metabolic changes, improved the lipid profile, reduced SBP, increased COMT expression, and reduced plasma catecholamines. Treatment with PIO reversed HFD-induced glucose intolerance and the associated metabolic derangement. In addition, these effects of PIO were associated with up-regulating COMT expression with a subsequent reduction in plasma catecholamines levels.

Schizophrenia‑associated microRNA‑148b‑3p regulates COMT and PRSS16 expression by targeting the ZNF804A gene in human neuroblastoma cells.

Zinc finger protein 804A (ZNF804A) has been identified by genome-wide association studies as a robust risk gene in schizophrenia, but how ZNF804A contributes to schizophrenia and its upstream regulation remains unknown. Previous studies have indicated that microRNAs (miRs) are key factors that regulate the expression levels of their target genes. The present study revealed significantly increased expression of miR-148b-3p in the peripheral blood of patients with first-onset schizophrenia compared with healthy controls, and bioinformatics analysis predicted that the ZNF804A gene is a target of miR-148b-3p. Therefore, the present study investigated the possible upstream regulation of ZNF804A by miR-148b-3p in the human neuroblastoma SH-SY5Y cell line, and assessed the implications for schizophrenia. The results revealed significantly reversed expression levels of miR-148b-3p (P=0.0051) and ZNF804A (P=0.0218) in the peripheral blood of patients with first-onset schizophrenia compared with healthy individuals. Furthermore, it was demonstrated that miR-148b-3p directly targeted ZNF804A via binding to conserved target sites in the 3′-untranslated region of ZNF804A mRNA, where it inhibited the endogenous expression of ZNF804A at both the mRNA (P=0.048) and protein levels (P=0.013) in SH-SY5Y cells. Furthermore, miR-148b-3p was revealed to regulate the expression levels of catechol-O-methyltransferase (COMT) and serine protease 16 (PRSS16) by targeting ZNF804A in SH-SY5Y cells. Collectively, the present results indicated that there was a direct upstream regulation of the schizophrenia risk gene ZNF804A by miR-148b-3p, which contributed to the regulation of the downstream genes COMT and PRSS16. Thus, the miR-148b-3p/ZNF804A/COMT/PRSS16 pathway may play an important role in the pathophysiology of schizophrenia, and may serve as a potential target in drug discovery and gene therapy for this disorder.

2-Methoxyestradiol ameliorates metabolic syndrome-induced hypertension and catechol-O-methyltransferase inhibited expression and activity in rats

Metabolic syndrome (MetS) is a common disorder that is associated with hypertension and poses a significant cardiovascular risk. Deactivation of extra-neuronal norepinephrine is mediated by catechol-O-methyltransferase (COMT). Endogenous 2-methoxyestradiol (2ME) is a product of COMT activity. The current study investigated the impact of 2ME on MetS-induced hypertension and the possible alterations in COMT expression and activity in rats. Animals were randomly divided into 4 groups. Group 1 received drinking water and standard food pellets. Groups 2, 3 and 4 were subjected to experimental induction of MetS. Animals in groups 3 and 4 were given daily IP injection of 2ME (25 and 50 mg/kg, respectively). MetS animals showed significant increases in body weight gain and visceral fat, fasting blood glucose and serum insulin and insulin resistance. Meanwhile, MetS was associated with a significant hypertriglyceridemia. Further, MetS significantly increased systolic, diastolic and mean arterial blood pressure. These effects were associated with significant reduction in COMT expression in the liver, kidneys and aorta as well as reduced hepatic activity. 2ME inhibited the alterations in body weight gain, visceral fat accumulation, fasting blood glucose and serum insulin, insulin resistance and serum triglycerides. Elevations in blood pressure were significantly inhibited by 2ME. Also, it attenuated the decrease in liver, kidney and aorta COMT expression and hepatic COMT activity. MetS was associated with elevated epinephrine and norepinephrine levels. Only the higher dose of 2ME significantly mitigated the rise in epinephrine level. In conclusion, 2ME protects against MetS-induced hypertension and averts COMT inhibited expression and activity.

Chronic persistent surgical pain is strongly associated with COMT alleles in patients undergoing cardiac surgery with median sternotomy.

Catechol-O-methyl transferase (COMT), a catechol-dependent enzyme, plays pivotal role in the development of pain. In different ethnic populations, it is associated with chronic persistent surgical pain (CPSP). In this context, the present study is aimed to assess involvement of COMT allele (Val158Met) in the development of CPSP. The patients (n = 216) underwent cardiac surgery with median sternotomy were selected to assess the magnitude of the CPSP evaluated with pain questionnaires' after 3months from surgery. The exon 4 of COMT gene was PCR amplified and sequenced. The quantitative gene expression of COMT using RT-PCR corroborated the COMT enzyme activity. Among 216 patients who underwent sternotomy procedure, 54 patients showed CPSP even after 3months from surgery. The sequence analysis revealed that, in 25% (54/216) patients having following one or more alleles: c.472G>A (Val158Met) (reported), and novel c.382C>G;c.383G>C (Arg128Ala), c.373C>G (Arg125Gly), c.370G>A (Val124Met), c.359G>C (Gly120Ala), c.349G>A, c.350G>A(Ala117Ser), c.349G>C, c.351C>A (Ala117Pro), c.349G>A (Ala117Thr), c.350G>C (Ala117Gly), and c.405G>C (Ala135Ser) were observed for the first time in Indian population. Distinct CPSP (≥ 4 NRS pain score) was observed in these patients correlating with COMT enzyme activity (7.80 ± 0.92 units/mg) which is 14 times lowered when compared with non-CPSP patient's (n = 162) 110.15 ± 6.41units/mg. The findings of COMT gene expression using quantitative RT-PCR corroborated the COMT enzyme activity. The dominant effect of mutant COMT alleles connecting with low enzyme activity resulted in CPSP, warrants COMT genetic analysis prior to surgery was useful to predict the occurrence of CPSP.

Prenatal zinc supplementation attenuates lipopolysaccharide-induced behavioral impairments in maternal immune activation model

Maternal infection during pregnancy is considered a key risk factor for developing schizophrenia in offspring. There is evidence that maternal exposure to infectious agents is associated with fetal zinc deficiency. Due to the essential role of zinc in brain function and development, in the present study, we activated maternal immune system using lipopolysaccharide (LPS) as a model of schizophrenia to examine whether zinc supplementation throughout pregnancy can reverse LPS-induced deleterious effects.To test the hypothesis, pregnant rats were treated with intraperitoneal injection of either saline or LPS (0.5 mg/kg) at gestational day 15 and 16, and zinc supplementation (30 mg/kg) was administered throughout pregnancy by gavage. At postnatal day 60, Y-maze was used to evaluate working memory of offspring. Moreover, the expression levels of catechol O-methyltransferase (COMT) and glutamate decarboxylase 67 (GAD67) were measured in the frontal cortex of the brain samples.Only male offspring prenatally exposed to LPS showed a significant impairment in working memory. In addition, prenatal LPS exposure causes a moderate decrease in GAD67 expression level in the male pups, while COMT expression was found unchanged. Interestingly, zinc supplementation restored the alterations in working memory as well as GAD67 mRNA level in the male rats. No alteration was detected for neither working memory nor COMT/GAD67 genes expression in female offspring.This study demonstrates that zinc supplementation during pregnancy can attenuate LPS-induced impairments in male pups. These results support the idea to consume zinc supplementation during pregnancy to limit neurodevelopmental deficits induced by infections in offspring.

COMT Effects on Vitamin E and Colorectal Cancer, in-vitro and in Two Randomized Trials (P15-005-19)

ObjectivesDespite promising observational data and compelling mechanisms of action, vitamin E has failed to demonstrate evidence of benefit in randomized clinical trials (RCTs). In two large long-term placebo-controlled RCTs, we reported that vitamin E effects on total cancer were modified by genetic variation in catechol-O-methyltransferase (COMT), an enzyme that metabolizes catecholamines. Here we investigate COMT effects on colorectal cancer (CRC) in the two RCTs and a CRC cell line. MethodsWe analyzed COMT rs4680 association with rates of CRC in the Women’s Health Study (WHS), N = 23,294 and a case/control (N = 2396/2235) subset of the Alpha-Tocopherol Beta-Carotene Cancer Prevention Study (ATBC). Cell survival and apoptosis were examined in-vitro in HCT116 cells treated with increasing doses of vitamin E when COMT gene expression was inhibited by silencing RNA (siRNA). ResultsRates of CRC were higher with randomized vitamin E compared to placebo among COMT high-activity val/val homozygotes in ATBC (HR, [CI] = 3.00, [1.48–6.09]), but not WHS (HR, [CI] = 0.99, [0.63–1.57]). Among low-activity met/met homozygotes randomized to vitamin E compared to placebo, rates of CRC were borderline lower in WHS (HR, [CI] = 0.66, [0.44–1.01]), but not in ATBC (HR, [CI] = 0.93, [0.63–1.62]).In cell culture, vitamin E at 3 μg/ml and 10 μg/mL had no effect on cell viability or apoptosis. However, silencing COMT resulted in a modest apoptotic effect that vitamin E enhanced in a dose-dependent manner. Human apoptosis arrays indicated that in the absence of COMT expression, vitamin E induced protein expression related to the intrinsic apoptotic pathway through p53 activation, dysregulation of Bcl-2 family protein expression and down-regulation of IAP family protein expression. ConclusionsDifferential COMT effects on vitamin E and CRC were similar to those previously reported for all invasive cancers, but were only significant for val/val homozygotes. Further, inhibiting COMT in the presence of vitamin E in a CRC in-vitro model, recapitulated the RCT observation that among individuals homozygous for the low-activity allele (met/met) vitamin E tended to reduce invasive cancer and here CRC. Funding SourcesNational Institutes of Health: NCI and NHLBI.