Inhibiting proliferation in KB cancer cell by RNA interference-mediated knockdown of nicotinamide N-Methyltransferase expression

Department of Biochemistry, Biology and Genetics
International journal of immunopathology and pharmacology (Impact Factor: 1.62). 01/2011; 24(1):69-77.
Source: PubMed


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.

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    • "NNMT encodes nicotinamide N-methyltransferase , an enzyme that catabolizes nicotinamide and other pyridine compounds in a reaction that uses the methyl group generated during the conversion of SAM to S-adenosylhomocysteine , involved in the biotransformation of many drugs and xenobiotic compounds (Aksoy et al. 1994). A recent study found that downregulation of NNMT inhibited proliferation in KB cancer cells, suggesting NNMT might be a target for therapeutics and could alter the efficacy of standard chemotherapeutic drugs (Pozzi et al. 2011). Furthermore, in MDA-MB-435 cells, CDC42EP5 was hypomethylated and upregulated in GGH overexpression, while it was hypermethylated and downregulated in GGH inhibition. "
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    ABSTRACT: γ-Glutamyl hydrolase (GGH) plays an important role in folate homeostasis by catalyzing hydrolysis of polyglutamylated folate into monoglutamates. Polyglutamylated folates are better substrates for several enzymes involved in the generation of S-adenosylmethionine, the primary methyl group donor, and hence, GGH modulation may affect DNA methylation. DNA methylation is an important epigenetic determinant in gene expression, in the maintenance of DNA integrity and stability, and in chromatin modifications, and aberrant or dysregulation of DNA methylation has been mechanistically linked to the development of human diseases including cancer. Using a recently developed in vitro model of GGH modulation in HCT116 colon and MDA-MB-435 breast cancer cells, we investigated whether GGH modulation would affect global and gene-specific DNA methylation and whether these alterations were associated with significant gene expression changes. In both cell lines, GGH overexpression decreased global DNA methylation and DNA methyltransferase (DNMT) activity, while GGH inhibition increased global DNA methylation and DNMT activity. Epigenomic and gene expression analyses revealed that GGH modulation influenced CpG promoter DNA methylation and gene expression involved in important biological pathways including cell cycle, cellular development, and cellular growth and proliferation. Some of the observed altered gene expression appeared to be regulated by changes in CpG promoter DNA methylation. Our data suggest that the GGH modulation-induced changes in total intracellular folate concentrations and content of long-chain folylpolyglutamates are associated with functionally significant DNA methylation alterations in several important biological pathways.
    Genes & Nutrition 12/2014; 10(1). DOI:10.1007/s12263-014-0444-0 · 2.79 Impact Factor
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    • "In addition, we reported an overexpression of salivary and urinary NNMT in OSCC and BUC, respectively, indicating that the enzyme could represent a potential biomarker for early and noninvasive diagnosis of these malignancies ( Sartini et al., 2012 , 2013a). Moreover, NNMT knockdown led to reduced tumorigenicity of KB and PE/CA-PJ15 cancer cells, suggesting the possibility of the enzyme as a molecular target for anti-cancer therapy ( Pozzi et al., 2011 , 2013). "
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    ABSTRACT: Abstract Lung cancer is the second most commonly diagnosed neoplasm, and represents the leading cause of tumor death worldwide. Since patients are often diagnosed at a late stage, current therapeutic strategies have limited effectiveness and the prognosis remains poor. Successful treatment depends on early diagnosis and knowledge concerning molecular mechanisms underlying lung carcinogenesis. In the present study, we focused on nicotinamide N-methyltransferase (NNMT), which is overexpressed in several malignancies. First, we analysed NNMT expression in a cohort of 36 patients with non-small cell lung cancer (NSCLC) by immunohistochemistry. Subsequently, we examined NNMT expression levels in the human lung cancer cell line A549 by Real-Time PCR, Western blot and catalytic activity assay, and evaluated the effect of NNMT knockdown on cell proliferation and anchorage independent cell growth by MTT and soft-agar colony formation assays, respectively. NSCLC displayed higher NNMT expression levels compared to both tumor-adjacent and surrounding tissue. Moreover, shRNA-mediated gene silencing of NNMT led to a significant inhibition of cell proliferation and colony formation ability on soft agar. Our results show that the downregulation of NNMT significantly reduced in vitro tumorigenicity of A549 cells and suggest that NNMT could represent an interesting molecular target for lung cancer therapy.
    Biological Chemistry 09/2014; 396(3). DOI:10.1515/hsz-2014-0231 · 3.27 Impact Factor
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    • "Our data support the hypothesis that the enzyme plays a role in tumor expansion and may have a potential as prognostic marker for OSCC [6], [7]. Moreover, downregulation of NNMT led to decreased KB cancer cell growth, suggesting the possibility of NNMT as a therapeutic target for the treatment of cancer [11]. In a recent study, a marked increase in enzyme activity in oral cancer and an up-regulation of salivary NNMT have been shown. "
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    ABSTRACT: Oral squamous cell carcinoma (OSCC) is the most common type of oral cancer. Despite progress in the treatment of OSCC, overall survival has not improved substantially in the last three decades. Therefore, identification of reliable biomarkers becomes essential to develop effective anti-cancer therapy. In this study, we focused on the enzyme Nicotinamide N-methyltransferase (NNMT), which plays a fundamental role in the biotransformation of many xenobiotics. Although several tumors have been associated with abnormal NNMT expression, its role in cancer cell metabolism remains largely unknown. In this report, 7 human oral cancer cell lines were examined for NNMT expression by Real-Time PCR, Western blot and HPLC-based catalytic assay. Subsequently, we evaluated the in vitro effect of shRNA-mediated silencing of NNMT on cell proliferation. In vivo tumorigenicity of oral cancer cells with stable knockdown of NNMT was assayed by using xenograft models. High expression levels of NNMT were found in PE/CA PJ-15 cells, in keeping with the results of Western blot and catalytic activity assay. PE/CA PJ-15 cell line was stably transfected with shRNA plasmids against NNMT and analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and soft agar Assays. Transfected and control cells were injected into athymic mice in order to evaluate the effect of NNMT silencing on tumor growth. NNMT downregulation resulted in decreased cell proliferation and colony formation ability on soft agar. In athymic mice, NNMT silencing induced a marked reduction in tumour volume. Our results show that the downregulation of NNMT expression in human oral carcinoma cells significantly inhibits cell growth in vitro and tumorigenicity in vivo. All these experimental data seem to suggest that NNMT plays a critical role in the proliferation and tumorigenic capacity of oral cancer cells, and its inhibition could represent a potential molecular approach to the treatment of oral carcinoma.
    PLoS ONE 08/2013; 8(8):e71272. DOI:10.1371/journal.pone.0071272 · 3.23 Impact Factor
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