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.