RNA-Mediated Gene Silencing of Nicotinamide N-Methyltransferase Is Associated with Decreased Tumorigenicity in Human Oral Carcinoma Cells

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.

Identification and Characterization of Cancer Stem Cells from Head and Neck Squamous Cell Carcinoma Cell Lines

“…Although several cancers have been associated with abnormal NNMT expression, its role in cancer cell metabolism remains largely unknown. We previously demonstrated that downregulation of NNMT expression in human oral and lung cancer cell lines significantly inhibited cell growth and tumorigenicity, suggesting that NNMT could play a critical role in the proliferation and tumorigenic capacity of cancer cells, and the possibility of NNMT as a therapeutic target for the treatment of tumor [62][63][64]. In this work, since CSCs appear to be responsible for tumor initiation and progression (for tumor onset, self-renewal/ maintenance, and metastasis), we decided to analyze the mRNA expression and catalytic activity of NNMT in CSCs enriched populations.…”

Nicotinamide N-methyltransferase enhances the capacity of tumorigenesis associated with the promotion of cell cycle progression in human colorectal cancer cells

“…The HPLC-UV method used for the determination of NNMT activity was based on the method developed by Valentina Pozzi et al [13]. The cell pellet (5 Â 10 6 cells) samples were prepared by homogenization in 200 ll of cold lysis buffer as previously described followed by centrifugation at 16,000g for 10 min at 4°C.…”

“…One hundred microliters of the neutralized supernatant was injected into a high-performance liquid chromatography system with a Hewlett-Packard 1100 photodiode array detector (Waldbronn, Germany) using a 250 Â 4.6-mm-inner-diameter Agilent TC-C18 5-lm reversed-phase column. The chromatographic analysis of the extracted supernatant was performed as previously described [13]. The peak areas corresponding to 1-MNA were integrated, and the enzyme activities were tested by measuring the amount of 1-methylnicotinamide (1-MNA) produced.…”

Bioinformatics Analysis Reveals Significant Genes and Pathways to Targetfor Oral Squamous Cell Carcinoma

“…It has been reported that the NNMT expression is related with differentiation of oral cancer cells and may be a biomarker for OSCC treatment (Emanuelli et al, 2010). A recent study revealed that the abnormal expression of NNMT was closely associated with the metabolism of oral squamous carcinoma cells (Pozzi et al, 2013). The decreased expression of NNMT was able to suppress cancer cell proliferation and tumor development.…”

“…The decreased expression of NNMT was able to suppress cancer cell proliferation and tumor development. The inhibition of NNMT showed potential capacity for oral cancer treatment by molecular approach (Pozzi et al, 2013). Besides, MMP9 (Matrix Metalloproteinases-9) is reported to be significantly activated in malignant tissues of patients with oral cancer (Patel et al, 2007).…”