Background/Aims: Head and neck squamous cell carcinoma (HNSCC) ranks sixth worldwide for tumor-related mortality. A subpopulation of tumor cells, termed cancer stem cells (CSCs), has the ability to support cancer growth. Therefore, profiling CSC-enriched populations could be a reliable tool to study cancer biology. Methods: We performed phenotypic characterization of 7 HNSCC cell lines and evaluated the presence of CSCs. CSCs from Hep-2 cell line and HNSCC primary cultures were enriched through sphere formation and sphere-forming cells have been characterized both in vitro and in vivo. In addition, we investigated the expression levels of Nicotinamide N-methyltransferase (NNMT), an enzyme overexpressed in several malignancies. Results: CSC markers were markedly expressed in Hep-2 cell line, which was found to be highly tumorigenic. CSC-enriched populations displayed increased expression of CSC markers and a strong capability to form tumors in vivo. We also found an overexpression of CSC markers in tumor formed by CSC-enriched populations. Interestingly, NNMT levels were significantly higher in CSC-enriched populations compared with parental cells. Conclusion: Our study provides an useful procedure for CSC identification and enrichment in HNSCC. Moreover, results obtained seem to suggest that CSCs may represent a promising target for an anticancer therapy.
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.
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.
Metallic indium and zinc readily add to 3-bromopropenyl acetate (5) and benzoate (6) either in THF or in water, affording the corresponding 3-acyloxyallyl organometallic compounds. Nucleophilic addition to aldehydes opens a route to alk-1-ene-3,4-diols 2 in good to excellent yields. Two synthetic protocols were developed, the former involving indium in THF under Grignard conditions and the latter involving zinc in aqueous ammonium chloride under Barbier conditions. The diastereoselectivity, under all the conditions examined, mainly depends on the nature of the carbonyl compound; conjugated aldehydes afford syn adducts 2, while unconjugated aldehydes display the opposite anti stereopreference.
Lung cancer, predominantly non-small cell lung cancer (NSCLC), is currently the most common cause of malignancy-related death in the world. Despite advances in both detection and treatment, its incidence rate is still increasing. Therefore, effective strategies for early detection as well as molecular therapeutic targets are urgently needed. We focused on the enzyme nicotinamide N-methyltransferase (NNMT). NNMT expression levels were investigated in tumor, tumor-adjacent, and surrounding tissue samples of 25 patients with NSCLC by Real-Time PCR, Western blot analysis, and catalytic activity assay. NNMT enzyme activity in NSCLC was then correlated with clinicopathological characteristics. Results obtained showed NNMT upregulation (mRNA and protein) in tumor compared with both tumor-adjacent and surrounding tissue. Moreover, NSCLC displayed significantly higher activity levels than those determined in both tumor-adjacent and surrounding tissue. Interestingly, both tumor-adjacent and surrounding tissue samples of unfavorable cases (N+) seem to display higher activity levels than those of favorable NSCLCs (N0). The present work shows a marked increase of NNMT enzyme activity in NSCLC and suggests that normal-looking tissue of unfavorable cases seems to change toward cancer. Further studies may establish whether NNMT could represent a target for an effective anti-cancer therapy.
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