title: Inhibiting proliferation in KB cancer cells by RNA interference-mediated knockdown of nicotinamide N-methyltransferase expression.
abstract: 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.
authors.affiliation: Department of Biochemistry, Biology and Genetics; Polytechnic University of Marche, Ancona, Italy.
authors.forename: V
authors.initials: V
authors.lastname: Pozzi
authors.forename: M
authors.initials: M
authors.lastname: Mazzotta
authors.forename: L
authors.initials: L
authors.lastname: Lo Muzio
authors.forename: D
authors.initials: D
authors.lastname: Sartini
authors.forename: A
authors.initials: A
authors.lastname: Santarelli
authors.forename: E
authors.initials: E
authors.lastname: Renzi
authors.forename: R
authors.initials: R
authors.lastname: Rocchetti
authors.forename: M
authors.initials: M
authors.lastname: Tomasetti
authors.forename: D
authors.initials: D
authors.lastname: Ciavarella
authors.forename: M
authors.initials: M
authors.lastname: Emanuelli
mesh_terms: D015153:Blotting, Western; D049109:Cell Proliferation; D006801:Humans; D007624:KB Cells; D050903:Nicotinamide N-Methyltransferase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000502:physiology*; D034622:RNA Interference; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D050903:Nicotinamide N-Methyltransferase