Nicotinamide N-methyltransferase mRNA Research Articles

The dual GCGR/GLP-1R agonist survodutide: Biomarkers and pharmacological profiling for clinical candidate selection.

To describe the biomarker strategy that was applied to select survodutide (BI 456906), BI 456908 and BI 456897 from 19 dual glucagon receptor (GCGR)/ glucagon-like peptide-1 receptor (GLP-1R) agonists for in-depth pharmacological profiling, which led to the qualification of survodutide as the clinical development candidate. Potencies to increase cyclic adenosine monophosphate (cAMP) were determined in Chinese hamster ovary (CHO)-K1 cells stably expressing human GCGR and GLP-1R. Agonism for endogenously expressed receptors was investigated in insulinoma cells (MIN6) for mouse GLP-1R, and in rat primary hepatocytes for the GCGR. In vivo potencies to engage the GLP-1R or GCGR were determined, measuring improvement in oral glucose tolerance (30 nmol/kg) and increase in plasma fibroblast growth factor-21 (FGF21) and liver nicotinamide N-methyltransferase (NNMT) mRNA expression (100 nmol/kg), respectively. Body weight- and glucose-lowering efficacies were investigated in diet-induced obese (DIO) mice and diabetic db/db mice, respectively. Upon acute dosing in lean mice, target engagement biomarkers for the GCGR and GLP-1R demonstrated a significant correlation (Spearman correlation coefficient with p < 0.05) to the in vitro GCGR and GLP-1R potencies for the 19 dual agonists investigated. Survodutide, BI 456908 and BI 456897 were selected for in-depth pharmacological profiling based on the significant improvement in acute oral glucose tolerance achieved (area under the curve [AUC] of 54%, 57% and 60% vs. vehicle) that was comparable to semaglutide (AUC of 45% vs. vehicle), while showing different degrees of in vivo GCGR engagement, as determined by hepatic NNMT mRNA expression (increased by 15- to 17-fold vs. vehicle) and plasma FGF21 concentrations (increased by up to sevenfold vs. vehicle). In DIO mice, survodutide (30 nmol/kg/once daily), BI 456908 (30 nmol/kg/once daily) and BI 456897 (10 nmol/kg/once daily) achieved a body weight-lowering efficacy from baseline of 25%, 27% and 26%, respectively. In db/db mice, survodutide and BI 456908 (10 and 20 nmol/kg/once daily) significantly lowered glycated haemoglobin (0.4%-0.6%); no significant effect was observed for BI 456897 (3 and 7 nmol/kg/once daily). Survodutide was selected as the clinical candidate based on its balanced dual GCGR/GLP-1R pharmacology, engaging the GCGR for robust body weight-lowering efficacy exceeding that of selective GLP-1R agonists, while achieving antidiabetic efficacy that was comparable to selective GLP-1R agonism. Survodutide is currently being investigated in Phase 3 clinical trials in people living with obesity.

Knockdown of nicotinamide N-methyltransferase suppresses proliferation, migration, and chemoresistance of Merkel cell carcinoma cells in vitro.

Merkel cell carcinoma (MCC) is an aggressive skin cancer, with a propensity for early metastasis. Therefore, early diagnosis and the identification of novel targets become fundamental. The enzyme nicotinamide N-methyltransferase (NNMT) catalyzes the reaction of N-methylation of nicotinamide and other analogous compounds. Although NNMT overexpression was reported in many malignancies, the significance of its dysregulation in cancer cell phenotype was partly clarified. Several works demonstrated that NNMT promotes cancer cell proliferation, migration, and chemoresistance. In this study, we investigated the possible involvement of this enzyme in MCC. Preliminary immunohistochemical analyses were performed to evaluate NNMT expression in MCC tissue specimens. To explore the enzyme function in tumor cell metabolism, MCC cell lines have been transfected with plasmids encoding for short hairpin RNAs (shRNAs) targeting NNMT mRNA. Preliminary immunohistochemical analyses showed elevated NNMT expression in MCC tissue specimens. The effect of enzyme downregulation on cell proliferation, migration, and chemosensitivity was then evaluated through MTT, trypan blue, and wound healing assays. Data obtained clearly demonstrated that NNMT knockdown is associated with a decrease of cell proliferation, viability, and migration, as well as with enhanced sensitivity to treatment with chemotherapeutic drugs. Taken together, these results suggest that NNMT could represent an interesting MCC biomarker and a promising target for targeted anti-cancer therapy.

Targeting nicotinamide N-methyltransferase decreased aggressiveness of osteosarcoma cells.

Osteosarcoma (OS) is a primary bone malignancy that mostly affects young people, characterized by high metastatic potential, and a marked chemoresistance that is responsible for disease relapse in most patients. Therefore, it is necessary to identify novel molecules to setup targeted strategies to improve the clinical outcome. The enzyme nicotinamide N-methyltransferase (NNMT) catalyses the N-methylation of nicotinamide and other analogs, playing a crucial role in the biotransformation of drugs and xenobiotics. NNMT overexpression was reported in a wide variety of cancers, and several studies demonstrated that is able to promote cell proliferation, migration and resistance to chemotherapy. The aim of this study was to explore the potential involvement of NNMT in OS. Immunohistochemical analyses have been performed to evaluate NNMT expression in selected OS and healthy bone tissue samples. Subsequently, OS cell lines have been transfected with vectors targeting NNMT mRNA (shRNAs) and the impact of this downregulation on migration, cell proliferation, and response to chemotherapeutic treatment was also analysed by wound healing, MTT, SRB and Trypan blue assays, respectively. Results showed that OS samples display a significantly higher NNMT expression compared with healthy tissue. Preliminary results suggest that NNMT silencing in OS cell lines is associated to a decrease of cell proliferation and migration, as well as to enhanced sensitivity to chemotherapy. Data obtained showed that NNMT may represent an interesting marker for OS detection and a promising target for effective anti-cancer therapy.

Correlation of NNMT and DKK1 Protein Expression With Clinicopathological Characteristics and Prognosis of Breast Cancer.

Nicotinamide N-methyltransferase (NNMT) and Dickkopf-1 (DKK1) play an important role in the development of breast cancer, and the purpose of this study was designed to examine the clinical and prognostic significance of NNMT and DKK1 in breast cancer. The GEPIA2 database was used to evaluate the expression and survival of NNMT mRNA and DKK1 mRNA of breast cancer. Then an immunohistochemical study was carried out on 374 cases of breast tissue to identify the protein expression and significance of NNMT and DKK1. Next, the prognostic significance of DKK1 in breast cancer was explored by COX and Kaplan-Meier models. Protein NNMT expression was correlated with lymph node metastasis and histological grade (P < .05) while protein DKK1 expression was related to tumor size, pT stage, histological grade, and Ki-67 (P < .05). Protein DKK1 was related to disease-specific survival (DSS), and low DKK1 expression indicated a poor prognosis of breast cancer patients (P < .05). Combined expression of protein NNMT and protein DKK1 predicted different prognosis of DSS (P < .05). Nicotinamide N-methyltransferase and DKK1 were linked to breast cancer malignancy and invasion. Breast cancer patients with low DKK1 expression had a worse prognosis. Oncotypes of NNMT and DKK1 expression predicted patient outcomes.

Tumor stromal nicotinamide N-methyltransferase overexpression as a prognostic biomarker for poor clinical outcome in early-stage colorectal cancer

In a quest for prognostic biomarkers in early-stage colorectal cancer, we investigated NNMT (nicotinamide N-methyltransferase) in large cohorts of patients. Immunohistochemical examination of 679 patients illustrates that NNMT protein is predominantly expressed in the cancer stroma at varying levels, and about 20% of cancer tissues overexpress NNMT when compared to levels observed in normal colorectal mucosa. Clinical correlation analyses of 572 patients with early-stage cancers reveal that NNMT protein overexpression is significantly associated with shorter overall and disease-free survival, but no such correlation is found in late-stage colorectal cancer. Analyses of TCGA and CPTAC colorectal cancer cohorts show that NNMT mRNA expression is positively correlated with protein levels, is significantly higher in CIMP-high or MSI subtypes than in CIMP-low or MSS subtypes, and is positively correlated with its paralog INMT but not with its interaction partners such as PNMT, ADK, APP, ATF6, BMF, BRD4, CDC37, or CRYZ. In early-stage cancers, NNMT expression is higher in BRAF-mutated than in BRAF wild type tumors but is not affected by KRAS or PIK3CA mutation status. As a cancer stromal protein with important roles in metabolism and cancer epigenetics, NNMT is emerging as a promising biomarker for risk stratification of early-stage cancers.

Nicotinamide N-Methyltransferase Suppression Participates in Nickel-Induced Histone H3 Lysine9 Dimethylation in BEAS-2B Cells

Background: Nickel compounds are well-established human carcinogens with weak mutagenic activity. Histone methylation has been proposed to play an important role in nickel-induced carcinogenesis. Nicotinamide N-methyltransferase (NNMT) decreases histone methylation in several cancer cells by altering the cellular ratio of S-adenosylmethionine (SAM) to S-adenosylhomocysteine (SAH). However, the role of NNMT in nickel-induced histone methylation remains unclear. Methods: BEAS-2B cells were exposed to different concentrations of nickel chloride (NiCl₂) for 72 h or 200 μM NiCl₂ for different time periods. Histone H3 on lysine 9 (H3K9) mono-, di-, and trimethylation and NNMT protein levels were measured by western blot analysis. Expressions of NNMT mRNA and the H3k9me2-associated genes, mitogen-activated protein kinase 3 (MAP2K3) and dickkopf1 (DKK1), were determined by qPCR analysis. The cellular ratio of nicotinamide adenine dinucleotide (NAD⁺) to reduced NAD (NADH) and SAM/SAH ratio were determined. Results: Exposure of BEAS-2B cells to nickel increased H3K9 dimethylation (H3K9me2), suppressed the expressions of H3K9me2-associated genes (MAP2K3 and DKK1), and induced NNMT repression at both the protein and mRNA levels. Furthermore, over-expression of NNMT inhibited nickel-induced H3K9me2 and altered the cellular SAM/SAH ratio. Additionally, the NADH oxidant phenazine methosulfate (PMS) not only reversed the nickel-induced reduction in NAD⁺/NADH but also inhibited the increase in H3K9me2. Conclusions: These findings indicate that the repression of NNMT may underlie nickel-induced H3K9 dimethylation by altering the cellular SAM/SAH ratio.

Abstract 1693: Nicotinamide N-methyltransferase in clear cell renal cell carcinoma primary tumors and metastases

Abstract Background/Purpose: Nicotinamide N-methyltransferase (NNMT) is an enzyme involved in the biotransformation of many drugs and xenobiotic compounds. It is overexpressed in different cancers and has been shown to enhance the migratory and invasive activity of cancer cells. Additionally, NNMT is an important regulator of the methylation potential in cells by consumption of S-adenosyl methionine (SAM), an important substrate for methyltransferases. By this mechanism NNMT reduces methylation of proteins such as certain histones leading to epigenetic remodeling and induced expression of tumor-promoting genes (Ulanovskaya et al, Nat. Chem. Biol. 2013). In clear cell renal cell carcinoma (ccRCC), the dominant histological subtype of renal cell cancer, NNMT is expressed at high levels compared to normal kidney tissue and could represent a novel therapeutic target, especially for the treatment of metastatic disease which is marked by a five-year survival rate of below 10% with currently available therapies. Methods: We analyzed NNMT in a ccRCC patient cohort from The Cancer Genome Atlas (TCGA) concerning mRNA expression (n = 463), somatic variations (n = 410) and association with clinical parameters and patient survival. In an independent collection of ccRCC tissues (n = 64), corresponding normal kidney tissues and metastatic tissues from different organs (n = 129), NNMT mRNA and protein expression were quantified via Real-Time PCR and immunohistochemical staining of tissue microarrays, respectively. Results: In both cohorts, NNMT mRNA expression was significantly upregulated in tumor compared to non-tumor tissue. Somatic mutations and copy number alterations in the NNMT gene locus were observed in only a small number of patients. NNMT mRNA expression correlated weakly with tumor stage and grading in both cohorts, but not with the presence of lymph node or distant metastases. In Kaplan-Meier analyses high NNMT expression was associated with worse overall and cancer-specific survival. In ccRCC metastases from different organ sites, NNMT mRNA and protein expression were also induced compared to normal kidney tissue and high expression was again associated with worse patient survival in Kaplan-Meier analysis (HR = 2.15, Plog-rank = 0.05). Conclusion: NNMT mRNA and protein are highly expressed not only in primary tissue of ccRCC, but also in metastases derived from different organs, offering it as a potential drug-target for metastatic ccRCC. Although NNMT expression is a good prognostic indicator for patient survival, its molecular function in ccRCC tumorigenesis and metastasis formation has to be further evaluated. Supported by the Robert Bosch Foundation, Stuttgart, Germany and the ICEPHA Grant Tuebingen-Stuttgart, Germany Citation Format: Anna Reustle, Steffen Rausch, Stefan Winter, Florian Büttner, Stephan Kruck, Joerg Hennenlotter, Marcus Scharpf, Falko Fend, Jens Bedke, Arnulf Stenzl, Matthias Schwab, Elke Schaeffeler. Nicotinamide N-methyltransferase in clear cell renal cell carcinoma primary tumors and metastases. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1693.

Potential involvement of nicotinamide N-methyltransferase in the pathogenesis of metabolic syndrome.

Metabolic syndrome is a complex disorder characterized by the presence of insulin resistance (IR), type 2 diabetes mellitus (T2DM), impaired glucose tolerance (IGT), or impaired fasting glucose (IFG), plus at least two of the following conditions--hypertension, hyperlipidemia, obesity, and microalbuminuria. Metabolic syndrome exposes patients to a greater risk of developing cardiovascular disease (CVD) and is often associated with elevated levels of homocysteine (Hcy). In the current work, we analyzed the expression of nicotinamide N-methyltransferase (NNMT). Because NNMT is involved in Hcy metabolism and participates in the regulation of the cellular and plasma levels of this compound, we explored the role played by the enzyme in metabolic syndrome. Real-time PCR, immunohistochemistry, western blot analysis, and catalytic activity assay were performed to evaluate NNMT expression levels in adipose tissue from 10 Wistar Ottawa Karlsburg W (WOKW) rats, which are an animal model for metabolic syndrome, and from 10 Dark Agouti (DA) rats as the disease-resistant control strain. NNMT mRNA, protein, and activity levels were significantly higher in adipose tissue obtained from WOKW rats compared with those observed in adipose tissue of DA rats. Data reported in this study represent the first evidence supporting the hypothesis that NNMT could play an important role in the pathogenesis of metabolic syndrome and could have a potential for the development of a targeted therapy.

N-methylnicotinamide and nicotinamide N-methyltransferase are associated with microRNA-1291-altered pancreatic carcinoma cell metabolome and suppressed tumorigenesis.

The cell metabolome comprises abundant information that may be predictive of cell functions in response to epigenetic or genetic changes at different stages of cell proliferation and metastasis. An unbiased ultra-performance liquid chromatography-mass spectrometry-based metabolomics study revealed a significantly altered metabolome for human pancreatic carcinoma PANC-1 cells with gain-of-function non-coding microRNA-1291 (miR-1291), which led to a lower migration and invasion capacity as well as suppressed tumorigenesis in a xenograft tumor mouse model. A number of metabolites, including N-methylnicotinamide, involved in nicotinamide metabolism, and l-carnitine, isobutyryl-carnitine and isovaleryl-carnitine, involved in fatty acid metabolism, were elevated in miR-1291-expressing PANC-1. Notably, N-methylnicotinamide was elevated to the greatest extent, and this was associated with a sharp increase in nicotinamide N-methyltransferase (NNMT) mRNA level in miR-1291-expressing PANC-1 cells. In addition, expression of NNMT mRNA was inversely correlated with pancreatic tumor size in the xenograft mouse model. These results indicate that miR-1291-altered PANC-1 cell function is associated with the increase in N-methylnicotinamide level and NNMT expression, and in turn NNMT may be indicative of the extent of pancreatic carcinogenesis.

Metabolomics reveals 1‐methylnicotinamide and nicotinamide N‐methyltransferase as crucial biomarkers in microRNA‐1291‐altered pancreatic carcinoma cell metabolism (1147.1)

Our recent studies have demonstrated that microRNA miR‐1291 may play an important role in the regulation of tumorigenesis of pancreatic cancer cells. To understand the underlying mechanisms, we employed a LC/MS‐based metabolomics approach to determine the impact of miR‐1291 on pancreatic carcinoma cell metabolism. Our data showed that the miR‐1291 stably transfected and control PANC‐1 cells are readily segregated based upon their metabolomic profiles. A number of significantly altered cellular metabolites were identified, including 1‐methylnicotinamide (MNA), taurine, L‐carnitine, isobutyryl‐carnitine, isovaleryl‐carnitine and hydroxybutyryl‐carnitine. Among them, MNA was altered to the greatest extent, and the increase in cellular MNA was associated with a sharply elevated expression of nicotinamide N‐methyltransferase (NNMT). In addition, NNMT mRNA expression was inversely related to the size of PANC‐1 derived xenograft tumors. Our results suggest that MNA and NNMT are possible biomarkers in miR‐1291‐altered pancreatic carcinoma cell metabolism. The findings may help identify key metabolic pathways in PANC‐1 cells perturbed by miR‐1291 and understand their involvement in pancreatic tumorigenesis.

Upregulation of Tissue and Urinary Nicotinamide N-Methyltransferase in Bladder Cancer: Potential for the Development of a Urine-Based Diagnostic Test

Carcinoma of the bladder is one of the most common urologic malignancies occurring worldwide. Diagnosis and monitoring of bladder urothelial carcinoma (UC) are based on cystoscopy and urinary cytology. However, these diagnostic methods still have some limitations, mainly related to invasive nature and lack of sensitivity. New reliable and non-invasive biomarkers for bladder cancer detection are therefore required. To explore the involvement of enzymes of drug metabolism in bladder cancer, in the present study, we analyzed the gene expression profiles of tumor and normal looking tissues obtained from the same patient by cDNA macroarray. The enzyme nicotinamide N-methyltransferase (NNMT) was identified as a highly expressed gene in bladder cancer. RT-PCR, Real-Time PCR, Western blot analysis, and catalytic activity assay, performed on a large cohort of patients with bladder UC, confirmed NNMT upregulation. NNMT mRNA and protein levels were also determined in urine specimens obtained from patients with bladder UC and healthy subjects. We found that NNMT expression levels were significantly higher in patients with bladder tumor compared to controls that showed very low or undetectable amounts of NNMT transcript and protein. Our results indicate that a marked NNMT increase is a peculiar feature of bladder UC and suggest the potential suitability of urine NNMT expression levels determination for early and non-invasive diagnosis of bladder cancer.

Inhibiting Proliferation in KB Cancer Cells by RNA Interference-Mediated Knockdown of Nicotinamide N-Methyltransferase Expression

The enzyme Nicotinamide N-methyltransferase (NNMT) catalyzes the methylation of nicotinamide and other pyridines, playing a pivotal role in the biotransformation and detoxification of many drugs and xenobiotic compounds. Several tumours have been associated with abnormal NNMT expression, however its role in tumour development remains largely unknown. In this study we investigated expression levels of Nicotinamide N-methyltransferase in a cancer cell line and we evaluated the effect of shRNA-mediated silencing of NNMT on cell proliferation. Cancer cells were examined for NNMT expression by semiquantitative RT-PCR and Western blot analysis. A HPLC-based catalytic assay was performed to assess enzyme activity. Cells were transfected with four shRNA plasmids against NNMT and control cells were treated with transfection reagent only (mock). The efficiency of gene silencing was detected by Real-Time PCR and Western blot analysis. MTT cell proliferation assay and the soft agar colony formation assay were then applied to investigate the functional changes in cancerous cell. NNMT mRNA was detected in cancer cells, showing a very high expression level. In keeping with the results of RT-PCR analysis, the protein level and NNMT enzyme activity were particularly high in KB cells. ShRNA vectors targeted against NNMT efficiently suppressed gene expression, showing inhibition observed at both the mRNA and protein levels. Down-regulation of NNMT significantly inhibited cell proliferation and decreased colony formation ability on soft agar. The present data support the hypothesis that the enzyme plays a role in tumour expansion and its inhibition could represent a possible molecular approach to the treatment of cancer.

Hyperhomocysteinemia does not affect global DNA methylation and nicotinamide N-methyltransferase expression in mice

DNA methylation is coupled with one-carbon metabolism involving homocysteine/methionine interconversion. Correlation between plasma homocysteine levels and leukocyte global DNA methylation was reported but not always replicated. Nicotinamide N-methyltransferase (NNMT) is a determinant of plasma homocysteine levels. Findings suggest alteration of one-carbon metabolism in schizophrenia etiology; hyperhomocysteinemia was observed in schizophrenia. A recent study carried out by the authors of this paper found an association between NNMT and schizophrenia and decreased post-mortem brain NNMT mRNA levels. The present study assessed the interrelationship between brain and leukocytes global DNA methylation and plasma homocysteine levels, and between hyperhomocysteinemia and brain NNMT expression. Mice were administered homocysteine in drinking water. Percentage global genome DNA methylation was measured using the cytosine-extension method, and NNMT expression was measured using real-time quantitative reverse transcriptase PCR (qRT-PCR). Homocysteine administration resulted in a 10-fold increase in plasma homocysteine. However, there was no change in global DNA methylation in lymphocytes or in the frontal cortex. No significant intra-individual correlation was found between global DNA methylation in leukocytes and frontal cortex, suggesting that leukocyte global DNA methylation may not serve as a marker for brain global DNA methylation. No difference was found in NNMT expression in homocysteine-treated mice compared with control mice. In conclusion, relatively short-term hyperhomocysteinemia in mice does not reproduce or lead to alterations reported in one-carbon metabolism in disorders associated with lifelong elevated plasma homocysteine.

Expression of nicotinamide N-methyltransferase in hepatocellular carcinoma is associated with poor prognosis

BackgroundHepatocellular carcinoma (HCC) is the most common tumor in the adult liver, with high relapse and mortality rates despite diverse treatment modalities. In this study, nicotinamide N-methyltransferase (NNMT), a key enzyme in drug metabolism, was investigated as a potential prognostic factor.MethodsFrozen tumors and non-cancerous surrounding tissues from 120 patients with primary HCC were studied. Expressions of NNMT and internal control genes were measured by real-time reverse-transcription PCR (RT-PCR). The relationship of NNMT mRNA level with clinicopathologic parameters and clinical outcome was evaluated.ResultsNNMT mRNA level is markedly reduced in HCCs compared to non-cancerous surrounding tissues (P < 0.0001), and NNMT expression in tumors was significantly correlated with tumor stage (P = 0.010). Moreover, stratification of patients based on tumor NNMT mRNA levels revealed that the patients who expressed higher NNMT mRNA levels tended to have a shorter overall survival (OS) time (P = 0.053) and a significantly shorter disease-free survival (DFS) time (P = 0.016). Both NNMT expression (P = 0.0096) and tumor stage (P = 0.0017) were found to be significant prognostic factors for DFS in a multivariate analysis.ConclusionThe results of this study indicated that NNMT gene expression is associated with tumor stage and DFS time in HCC cases. Because of the broad substrate specificity of NNMT, which could alter the efficacy and adverse effects of chemotherapy, NNMT merits further investigation regarding its role as a prognostic factor with a larger cohort of HCC patients.

Adipose tissue as a source of nicotinamide N-methyltransferase and homocysteine

Nicotinamide N-methyltransferase (NNMT) catalyses the conversion of nicotinamide to 1-methylnicotinamide and plays an important role in hepatic detoxification reactions. Here we show that, in addition to the liver, 3T3-L1 adipocytes as well as human and murine adipose tissue explants express high amounts of enzymatically active NNMT. NNMT mRNA levels and enzyme activity increased in 3T3-L1 cells in a differentiation-dependent manner. Homocysteine, the atherogenic product of the NNMT-catalyzed reaction, was secreted from 3T3-L1 cells or adipose tissue cultures. Homocysteine release increased during 3T3-L1 differentiation and was reduced when adipose tissue was treated with the NNMT inhibitor 1-methylnicotinamide. Nicotinic acid (NA), a widely used drug to lower elevated plasma lipid levels, induced NNMT enzyme activity in white adipose tissue of mice. In tissue culture nicotinamide treatment led to an increase in adipose tissue homocysteine secretion. These data support the concept that adipose tissue NNMT contributes to the increased plasma homocysteine levels in patients treated with NA.

Profiling of mRNA Expression in Quadriceps of Patients with COPD and Muscle Wasting

Peripheral muscle wasting is a feature of chronic obstructive pulmonary disease (COPD). Potent therapeutic strategies are needed to improve peripheral muscle mass in these patients. We hypothesized that the evaluation of the mRNA expression profile of quadriceps muscle could be useful in identifying key biochemical pathways involved in the wasting process. We monitored mRNA expression profile of quadriceps muscle in four patients with COPD with muscle atrophy (age: 71.3 ± 2.1 years, mean SD; FEV1 28.3 ± 10.8 % predicted) and four control subjects (age: 66.5 ± 1.3 years) using HuU95v2 gene chips. Fifty-seven mRNAs transcripts (0.5%) were found to be differentially expressed in muscles of COPD patients (i.e., p < 0.01). Among them, forkhead box O -1 and -3 and insulin-like growth factor-1 expressions being significantly elevated in COPD subjects. Concomitantly, a significant reduction in mRNA expression of two myofilament proteins was observed. Energy production appears to be impaired as indicated by the significant rise in nicotinamide N-methyltransferase mRNA expression. This study provides for the first time evidence that genes are selectively expressed in limb muscles of COPD patients and further research need to focus on their functional roles in the pathogenesis of muscle dysfunction.

Histone Deacetylase Inhibitor Depsipeptide Represses Nicotinamide N-Methyltransferase and Hepatocyte Nuclear Factor-1βGene Expression in Human Papillary Thyroid Cancer Cells

Nicotinamide N-methyltransferase (NNMT) catalyzes N-methylation of nicotinamide and other structural analogues. NNMT gene expression is enhanced in many papillary thyroid cancer cells and activated by hepatocyte nuclear factor (HNF)-1beta. In this work, we studied the effects of depsipeptide, a histone deacetylase inhibitor, on NNMT gene expression in BHP 18-21 papillary thyroid cancer cells. Depsipeptide reduced NNMT mRNA level in a dose-dependent and time-dependent manner as determined by semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR). In contrast, expression of the sodium iodide symporter (NIS), a gene with differentiated function, waas enhanced in the treated cells. NNMT protein level determined by Western blot analysis and NNMT catalytic activity was also reduced significantly in the depsipeptide-treated cells. To study the mechanism of NNMT gene repression by depsipeptide, effects of depsipeptide on NNMT promoter activity were determined by luciferase reporter gene assay. NNMT promoter activity was significantly reduced in the HNF-1beta-positive BHP 18-21 cells but not in the HNF-1beta-negative BHP 14-9 papillary cancer cells. A mutant reporter construct with mutations in a HNF-1 site in the NNMT basal promoter region did not respond to depsipeptide in both HNF-1beta protein levels, and abolished activity of DNA binding to the HNF-1 site in the NNMT promoter region. Protein synthesis inhibitor cycloheximide and proteasome inhibitor MG-132 enhanced HNF-1beta stability in the depsipeptide-treated cells. In summary, depsipeptide represses NNMT and HNF-1beta gene expression in some papillary thyroid cancer cells. the repression of NNMT by depsipeptide is at the transcription level through downregulation of transcription activator HNF-1beta.

Gene expression analysis of renal carcinoma: adipose differentiation‐related protein as a potential diagnostic and prognostic biomarker for clear‐cell renal carcinoma

The gene expression profiles of 33 renal cell carcinomas (RCCs) and nine normal kidney samples were examined using high-density oligonucleotide microarrays in an attempt to identify biomolecular markers for the diagnosis of tumour subtypes and also for prediction of prognosis. Hierarchical clustering demonstrated that clear-cell RCC, chromophobe RCC, and normal kidney tissue showed distinctive gene expression profiles. The mean expression levels of 149 of 12 500 genes were more than three times higher in clear-cell RCC than in chromophobe RCC and normal kidney tissue. Among the genes whose expression was upregulated in clear-cell RCC, adipose differentiation-related protein (ADFP) and nicotinamide N-methyltransferase (NNMT) were selected for further analysis. Consistent with the results of the microarray, increased levels of ADFP and NNMT mRNA were found more frequently in clear-cell RCCs than in other non-clear-cell tumour subtypes using real-time quantitative PCR. Immunohistochemistry for ADFP showed strong and unique tumour cell staining patterns in the majority of clear-cell RCCs. More importantly, patients bearing tumours with higher AFDP mRNA levels showed significantly better survival in both univariate and multivariate analyses. ADFP is a lipid storage droplet-associated protein and its transcription is considered to be regulated by the von Hippel-Lindau/hypoxia-inducible factor pathway. It is known that clear-cell RCC contains abundant lipids and cholesterols. Thus it is likely that sustained upregulation of ADFP following VHL inactivation is involved in the morphological appearance of clear-cell RCC. Moreover ADFP expression status may provide useful prognostic information as a biomolecular marker in patients with clear-cell RCC.