Role Played by Paraoxonase-2 Enzyme in Cell Viability, Proliferation and Sensitivity to Chemotherapy of Oral Squamous Cell Carcinoma Cell Lines.

Oral squamous cell carcinoma represents the most aggressive and frequent form of head and neck cancer. Due to drug resistance, the 5-year survival rate of patients with advanced disease is less than 50%. In order to identify molecular targets for effective oral cancer treatment, we focused on paraoxonase-2 enzyme. Indeed, based on data previously obtained from preliminary immunohistochemistry and Western blot analyses performed on tissue specimens, the enzyme was found to be upregulated in tumor compared with normal oral mucosa. Therefore, paraoxonase-2 gene silencing was achieved in HSC-3 and HOC621 oral cancer cell lines, and the effect on cell proliferation, viability, apoptosis induction and sensitivity to cisplatin and 5-fluorouracil treatment was evaluated. Fourier Transform InfraRed Microspectroscopy analyzed alterations of cellular macromolecules upon treatment. Enzyme level and cell proliferation were also determined in cisplatin-resistant clones obtained from HOC621 cell line, as well as in parental cells. Reported data showed that paraoxonase-2 knockdown led to a reduction of cell proliferation and viability, as well as to an enhancement of sensitivity to cisplatin, together with the activation of apoptosis pathway. Spectroscopical data demonstrated that, under treatment with cisplatin, oxidative damage exerted on lipids and proteins was markedly more evident in cells down-regulating paraoxonase-2 compared to controls. Interestingly, enzyme expression, as well as cell proliferation were significantly higher in cisplatin-resistant compared with control HOC621 cells. Taken together these results seem to candidate the enzyme as a promising target for molecular treatment of this neoplasm.

Effects of Fermented Wheat Germ Extract on Oral Cancer Cells: An In Vitro Study.

Oral carcinoma is one of the most aggressive cancers, and despite the advances in the therapy, its mortality is still high. An attention in cancer treatment has focused on natural compounds due to their potential beneficial effects on human health. In this study, the effects of dietary supplement Fermented Wheat Germ Extract (FWGE) on oral tongue squamous cell carcinoma (OTSCC) cells were investigated In Vitro using three cell lines (HSC-3, SAS, SCC-25) with variable aggressiveness. The cell viability was significantly decreased by the treatment with high concentration of FWGE in every cell line. Regarding migration and invasion, HSC-3 and SCC-25 cells were most sensitive to FWGE since their movement was significantly reduced with 5 and 10 mg/ml FWGE, while SAS was inhibited only with 10 mg/ml FWGE. Chemotherapeutic compounds (cisplatin and 5-fluorouracil) significantly reduced all OTSCC cells viability. Importantly, combination of these drugs with 10 mg/ml FWGE significantly decreased the cell viability compared to the treatment with the chemotherapeutics or FWGE alone. Based on these In Vitro experiments, the use of FWGE seems to improve the anticancer effects on OTSCC cells. Further In Vivo and clinical studies should be conducted to verify the positive effects of FWGE for OTSCC patients.

Nicotinamide N-methyltransferase gene silencing enhances chemosensitivity of melanoma cell lines.

Melanoma accounts for less than 5% of all cutaneous neoplasms but is responsible for the greater part of skin cancer-related deaths. Therefore, the identification of molecules that could serve as the therapeutic target is urgent. This study focused on the enzyme nicotinamide N-methyltransferase (NNMT). The effect of NNMT knockdown on cell proliferation and migration of A375 melanoma cells was evaluated by MTT and wound healing assays, respectively. Viability of A375 cells downregulating NNMT was also explored under treatment with dacarbazine, a chemotherapeutic drug approved for advanced melanoma treatment. The impact of enzyme knockdown on cell proliferation and chemosensitivity was also investigated in WM-115 melanoma cells. Results obtained demonstrated that NNMT silencing led to a significant reduction of cell proliferation and migration of A375 cells. Moreover, enzyme downregulation was associated with an increase of melanoma cells sensitivity to treatment with dacarbazine. Analogous effects induced by enzyme knockdown on cell proliferation and chemosensitivity were also found in the WM-115 cell line. Our data seem to demonstrate that NNMT could represent a promising molecular target for the effective treatment of this form of skin cancer.

Cancer stem cell enrichment is associated with enhancement of nicotinamide N-methyltransferase expression.

The cancer stem cell theory states that a subset of tumor cells, termed cancer stem cells (CSCs), has the ability to self-renew and differentiate within the tumors. According to this theory, CSCs would be mainly responsible for tumor initiation, progression, resistance to therapy, recurrence, and metastasis. In this study, a culture system was setup to enrich CSCs from bladder cancer (T24), lung cancer (A549), colorectal cancer (CaCo-2), and osteosarcoma (MG63) cell lines, through sphere formation. Magnetic-activated cell sorting was also used to further increase CSC enrichment. Subsequently, molecular characterization of CSC-enriched cell populations and parental cells was carried out, by exploring the expression levels of stem markers and the enzyme nicotinamide N-methyltransferase (NNMT). Results obtained showed a significant upregulation of stem cell markers in CSC-enriched populations, obtained upon sphere formation, compared with parental counterparts. Moreover, NNMT expression levels were markedly increased in samples enriched with CSCs with respect to control cells. Considering the fundamental role played by CSCs in carcinogenesis, reported data strengthen the hypothesis that sustains a pivotal role of NNMT in cancer growth and metastasis. In addition, these findings could represent an important achievement for the development of new and effective anticancer therapies, based on CSC-associated targets.

Bisubstrate Inhibitors of Nicotinamide N-Methyltransferase (NNMT) with Enhanced Activity.

Nicotinamide N-methyltransferase (NNMT) catalyzes the methylation of nicotinamide to form N-methylnicotinamide. Overexpression of NNMT is associated with a variety of diseases, including a number of cancers and metabolic disorders, suggesting a role for NNMT as a potential therapeutic target. By structural modification of a lead NNMT inhibitor previously developed in our group, we prepared a diverse library of inhibitors to probe the different regions of the enzyme's active site. This investigation revealed that incorporation of a naphthalene moiety, intended to bind the hydrophobic nicotinamide binding pocket via π-π stacking interactions, significantly increases the activity of bisubstrate-like NNMT inhibitors (half-maximal inhibitory concentration 1.41 µM). These findings are further supported by isothermal titration calorimetry binding assays as well as modeling studies. The most active NNMT inhibitor identified in the present study demonstrated a dose-dependent inhibitory effect on the cell proliferation of the HSC-2 human oral cancer cell line.