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Nicotinamide N-methyltransferase (NNMT) catalyzes the N-methylation reaction of nicotinamide, using S-adenosyl-L-methionine as the methyl donor. Enzyme overexpression has been described in many non-neoplastic diseases, as well as in a wide range of solid malignancies. This review aims to report and discuss evidence available in scientific literature, dealing with NNMT expression and the potential involvement in main urologic neoplasms, namely, renal, bladder and prostate cancers. Data illustrated in the cited studies clearly demonstrated NNMT upregulation (pathological vs. normal tissue) in association with these aforementioned tumors. In addition to this, enzyme levels were also found to correlate with key prognostic parameters and patient survival. Interestingly, NNMT overexpression also emerged in peripheral body fluids, such as blood and urine, thus leading to candidate the enzyme as promising biomarker for the early and non-invasive detection of these cancers. Examined results undoubtedly showed NNMT as having the capacity to promote cell proliferation, migration and invasiveness, as well as its potential participation in fundamental events highlighting cancer progression, metastasis and resistance to chemo- and radiotherapy. In the light of this evidence, it is reasonable to attribute to NNMT a promising role as a potential biomarker for the diagnosis and prognosis of urologic neoplasms, as well as a molecular target for effective anti-cancer treatment. [ABSTRACT FROM AUTHOR]

Saliva is easy to access, non-invasive and a useful source of information useful for the diagnosis of serval inflammatory and immune-mediated diseases. Following the advent of genomic technologies and -omic research, studies based on saliva testing have rapidly increased and human salivary proteome has been partially characterized. As a proteomic protocol to analyze the whole saliva proteome is not currently available, the most common aim of the proteomic analysis is to discriminate between physiological and pathological conditions. The salivary proteome has been initially investigated in several diseases: oral squamous cell carcinoma and oral leukoplakia, chronic graft-versus-host disease, and Sjögren's syndrome. Otherwise, salivary proteomics studies in the dermatological field are still in the initial phase, thus the aim of this review is to collect the best research evidence on the role of saliva proteomics analysis in immune-mediated skin diseases to understand the direction of research in this field. The results of PRISMA analysis reported herein suggest that human saliva analysis could provide significant data for the diagnosis and prognosis of several immune-mediated and inflammatory skin diseases in the next future. [ABSTRACT FROM AUTHOR]

(1) Background: Hyperglycemia leads to several biochemical and physiological consequences, such as the generation of advanced glycation end products (AGEs) and reactive oxygen species (ROS), which are involved in the development of several human diseases. Intestinal cells are continuously exposed to pro-oxidants and lipid peroxidation products from ingested foods, and also to glyco-oxidative damage. It has been reported that free radical generation may be linked to the development of inflammation-related gastrointestinal diseases. (2) Methods: The effects of high glucose (HG) treatment (50 mM) were assessed in terms of free radical production, lipid peroxidation, and AGEs formation. Furthermore, the expression and the antiapoptotic and antioxidant activity of the paraoxonase-2 (PON2) enzyme in intestinal cells has been investigated. (3) Results: Caco-2 cells treated with media supplied with high glucose (HG) (50 mM) showed, with respect to physiological glucose concentration (25 mM), an increase in ROS production, lipid peroxidation, and AGEs formation. Moreover, a lower PON2 expression and activity in HG-treated cells was related to activation of the apoptotic pathways. (4) Conclusions: Our results demonstrated that high glucose concentrations triggered glyco-oxidative stress in intestinal cells; the downregulation of PON2 could result in a higher oxidative stress and might contribute to intestinal dysfunction. [ABSTRACT FROM AUTHOR]

NAD + -dependent deacetylase sirtuin-1 (Sirt1) belongs to the sirtuins family, known to be longevity regulators, and exerts a key role in the prevention of vascular aging. By aging, the expression levels of Sirt1 decline with a severe impact on vascular function, such as the rise of endothelial dysfunction, which in turn promotes the development of cardiovascular diseases. In this context, the impact of Sirt1 activity in preventing endothelial senescence is particularly important. Given the key role of Sirt1 in counteracting endothelial senescence, great efforts have been made to deepen the knowledge about the intricate cross-talks and interactions of Sirt1 with other molecules, in order to set up possible strategies to boost Sirt1 activity to prevent or treat vascular aging. The aim of this review is to provide a proper background on the regulation and function of Sirt1 in the vascular endothelium and to discuss the recent advances regarding the therapeutic strategies of targeting Sirt1 to counteract vascular aging.

Clear cell renal cell carcinoma (ccRCC) represents the most common subtype of renal tumor. Despite recent advances in identifying novel target molecules, the prognosis of patients with ccRCC continues to be poor, mainly due to the lack of sensitivity to chemo- and radiotherapy and because of one-third of renal cell carcinoma patients displays metastatic disease at diagnosis. Thus, identifying new molecules for early detection and for developing effective targeted therapies is mandatory. In this work, we focused on paraoxonase-2 (PON2), an intracellular membrane-bound enzyme ubiquitously expressed in human tissues, whose upregulation has been reported in a variety of malignancies, thus suggesting its possible role in cancer cell survival and proliferation. To investigate PON2 involvement in tumor cell metabolism, human ccRCC cell lines were transfected with plasmid vectors coding short harpin RNAs targeting PON2 transcript and the impact of PON2 silencing on cell viability, migration, and response to chemotherapeutic treatment was then explored. Our results showed that PON2 downregulation was able to trigger a decrease in proliferation and migration of ccRCC cells, as well as an enhancement of cell sensitivity to chemotherapy. Thus, taken together, data reported in this study suggest that the enzyme may represent an interesting therapeutic target for ccRCC., (© 2024 Wiley Periodicals LLC.)

Oral Squamous Cell Carcinoma (OSCC) is the most common cancer arising from squamous epithelium in the oral cavity and is characterized by high aggressiveness and metastatic potential, which together with a late diagnosis results in a 5-year survival rate of only 50% of patients. The therapeutic options for OSCC management are limited and largely influenced by the cancer stage. While radical surgery can be curative in early stage of disease, most cases require adjuvant therapies, including chemotherapy and radiotherapy which, however, often achieve poor curative rates and are associated with important negative effects. Therefore, there is an urgent need to discover new alternative treatment strategies to improve patients' outcomes. Several medicinal herbs are being studied for their preventive or therapeutic effect in several diseases, including cancer. In particular, the Indian spice curcumin, largely used in oriental countries, has been studied as a chemopreventive or adjuvant agent for different malignancies. Indeed, curcumin is characterized by important biological properties, including antioxidant, anti-inflammatory, and anticancer effects, which could also be exploited in OSCC. However, due to its limited bioavailability and poor aqueous solubility, this review is focused on studies designing new synthetic analogues and developing novel types of curcumin delivery systems to improve its pharmacokinetic and biological properties. Thus, this review analyses the potential therapeutic role of curcumin in OSCC by providing an overview of current in vitro and in vivo studies demonstrating the beneficial effects of curcumin and its analogues in OSCC., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Merkel cell carcinoma (MCC) is an aggressive skin cancer, with a propensity for early metastasis. Therefore, early diagnosis and the identification of novel targets become fundamental. The enzyme nicotinamide N-methyltransferase (NNMT) catalyzes the reaction of N-methylation of nicotinamide and other analogous compounds. Although NNMT overexpression was reported in many malignancies, the significance of its dysregulation in cancer cell phenotype was partly clarified. Several works demonstrated that NNMT promotes cancer cell proliferation, migration, and chemoresistance. In this study, we investigated the possible involvement of this enzyme in MCC. Preliminary immunohistochemical analyses were performed to evaluate NNMT expression in MCC tissue specimens. To explore the enzyme function in tumor cell metabolism, MCC cell lines have been transfected with plasmids encoding for short hairpin RNAs (shRNAs) targeting NNMT mRNA. Preliminary immunohistochemical analyses showed elevated NNMT expression in MCC tissue specimens. The effect of enzyme downregulation on cell proliferation, migration, and chemosensitivity was then evaluated through MTT, trypan blue, and wound healing assays. Data obtained clearly demonstrated that NNMT knockdown is associated with a decrease of cell proliferation, viability, and migration, as well as with enhanced sensitivity to treatment with chemotherapeutic drugs. Taken together, these results suggest that NNMT could represent an interesting MCC biomarker and a promising target for targeted anti-cancer therapy., (© 2024. The Author(s).)

Paraoxonase-2 (PON2) is a ubiquitously expressed intracellular protein that is localized in the perinuclear region, the endoplasmic reticulum (ER), and mitochondria, and is also associated with the plasma membrane. PON2 functions as an antioxidant enzyme by reducing the levels of reactive oxygen species (ROS) in the mitochondria and ER through different mechanisms, thus having an anti-apoptotic effect and preventing the formation of atherosclerotic lesions. While the antiatherogenic role played by this enzyme has been extensively explored within endothelial cells in association with vascular disorders, in the last decade, great efforts have been made to clarify its potential involvement in both blood and solid tumors, where PON2 was reported to be overexpressed. This review aims to deeply and carefully examine the contribution of this enzyme to different aspects of tumor cells by promoting the initiation, progression, and spread of neoplasms.

Renal cell carcinoma (RCC) belongs to a heterogenous cancer group arising from renal tubular epithelial cells. Among RCC subtypes, clear cell renal cell carcinoma (ccRCC) is the most common variant, characterized by high aggressiveness, invasiveness and metastatic potential, features that lead to poor prognosis and high mortality rate. In addition, diagnosis of kidney cancer is incidental in the majority of cases, and this results in a late diagnosis, when the stage of the disease is advanced and the tumor has already metastasized. Furthermore, ccRCC treatment is complicated by its strong resistance to chemo- and radiotherapy. Therefore, there is active ongoing research focused on identifying novel biomarkers which could be useful for assessing a better prognosis, as well as new molecules which could be used for targeted therapy. In this light, several novel targeted therapies have been shown to be effective in prolonging the overall survival of ccRCC patients. Thus, the aim of this review is to analyze the actual state-of-the-art on ccRCC diagnosis, prognosis and therapeutic options, while also reporting the recent advances in novel biomarker discoveries, which could be exploited for a better prognosis or for targeted therapy.

Triple negative breast cancer (TNBC) displays a high aggressive behavior, tendency to relapse and early metastasize, leading to poor prognosis. The lack of estrogen receptors, and human epidermal growth factor receptor 2, prevents the use of endocrine or molecular targeted therapy, being therapeutical options for TNBC managements mostly limited to surgery, radiotherapy and mainly chemotherapy. While an important number of TNBCs initially responds to chemotherapy, they are prone to develop chemoresistance over the time. Thus, there is an urgent need to identify novel molecular targets to improve the outcome of chemotherapy in TNBC. In this work we focused on the enzyme paraoxonase-2 (PON2) which has been reported to be overexpressed in several tumors contributing to cancer aggressiveness and chemoresistance. Through a case-control study, we analyzed PON2 immunohistochemical expression in breast cancer molecular subtypes Luminal A, Luminal B, Luminal B HER2+, HER2 + and TNBC. Subsequently, we evaluated the in vitro effect of PON2 downregulation on cell proliferation and response to chemotherapeutics. Our results showed that the PON2 expression levels were significantly upregulated in the infiltrating tumors related to the subtypes Luminal A, HER2+ and TNBC compared to the healthy tissue. Furthermore, PON2 downregulation led to a decrease in cell proliferation of breast cancer cells, and significantly enhanced the cytotoxicity of chemotherapeutics on the TNBC cells. Although further analyses are necessary to deeply understand the mechanisms by which the enzyme could participate to breast cancer tumorigenesis, our results seem to demonstrate that PON2 could represent a promising molecular target for TNBC treatment., (© 2023. The Author(s) under exclusive licence to Japan Human Cell Society.)

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.

Background: The inter-individual differences in taste perception find a possible rationale in genetic variations. We verified whether the presence of four different single nucleotide polymorphisms (SNPs) in genes encoding for bitter (TAS2R38; 145G > C; 785T > C) and sweet (TAS1R3; −1572C > T; −1266C > T) taste receptors influenced the recognition of the basic tastes. Furthermore, we tested if the allelic distribution of such SNPs varied according to BMI and whether the associations between SNPs and taste recognition were influenced by the presence of overweight/obesity. Methods: DNA of 85 overweight/obese patients and 57 normal weight volunteers was used to investigate the SNPs. For the taste test, filter paper strips were applied. Each of the basic tastes (sweet, sour, salty, bitter) plus pure rapeseed oil, and water were tested. Results: Individuals carrying the AV/AV diplotype of the TAS2R38 gene (A49P G/G and V262 T/T) were less sensitive to sweet taste recognition. These alterations remained significant after adjustment for gender and BMI. Moreover, a significant decrease in overall taste recognition associated with BMI and age was found. There was no significant difference in allelic distribution for the investigated polymorphisms between normal and overweight/obese patients. Conclusions: Our findings suggest that overall taste recognition depends on age and BMI. In the total population, the inter-individual ability to identify the sweet taste at different concentrations was related to the presence of at least one genetic variant for the bitter receptor gene but not to the BMI.

The skin is constantly exposed to exogenous and endogenous sources of reactive oxygen species (ROS). An adequate balance between ROS levels and antioxidant defenses is necessary for the optimal cell and tissue functions, especially for the skin, since it must face additional ROS sources that do not affect other tissues, including UV radiation. Melanocytes are more exposed to oxidative stress than other cells, also due to the melanin production process, which itself contributes to generating ROS. There is an increasing amount of evidence that oxidative stress may play a role in many skin diseases, including melanoma, being the primary cause or being a cofactor that aggravates the primary condition. Indeed, oxidative stress is emerging as another major force involved in all the phases of melanoma development, not only in the arising of the malignancy but also in the progression toward the metastatic phenotype. Furthermore, oxidative stress seems to play a role also in chemoresistance and thus has become a target for therapy. In this review, we discuss the existing knowledge on oxidative stress in the skin, examining sources and defenses, giving particular consideration to melanocytes. Therefore, we focus on the significance of oxidative stress in melanoma, thus analyzing the possibility to exploit the induction of oxidative stress as a therapeutic strategy to improve the effectiveness of therapeutic management of melanoma.

Oral cancer is the most common tumor of the head and neck region. Its management is based on surgical and systemic therapies. Taste disorders represent the most common side effect of these treatments; indeed, dysgeusia is noted by 70% of oral cancer patients. Despite survival remaining the primary endpoint of cancer patients, taste impairments can cause psychological distress. This comprehensive review describes the last decade's knowledge from the literature regarding taste alterations in patients with oral and oropharyngeal squamous cell carcinoma. A total of 26 articles in English, including prospective, cross-sectional, and case-control studies, and clinical trials were evaluated. Literature analysis shows that anti-cancer treatments can destroy taste cells, decrease and alter their receptors, and interrupt nerve transmission. Furthermore, the tumour itself can destroy the oral mucosal lining, which encloses the taste buds. Dysgeusia typically occurs in 3-4 weeks of treatments, and usually taste sensation is recovered within 3-12 months. However, some patients exhibit incomplete or no recovery, even several years later. Thus, dysgeusia can become a chronic issue and negatively influence patients' quality of life, worsening their dysphagia and their nutritional status. Physicians should be focused on preventing oncological treatment-related symptoms, offering the most suitable personalized support during therapy.

Nicotinamide N -methyltransferase (NNMT) methylates nicotinamide (vitamin B3) to generate 1-methylnicotinamide (MNA). NNMT overexpression has been linked to a variety of diseases, most prominently human cancers, indicating its potential as a therapeutic target. The development of small-molecule NNMT inhibitors has gained interest in recent years, with the most potent inhibitors sharing structural features based on elements of the nicotinamide substrate and the S -adenosyl-l-methionine (SAM) cofactor. We here report the development of new bisubstrate inhibitors that include electron-deficient aromatic groups to mimic the nicotinamide moiety. In addition, a trans -alkene linker was found to be optimal for connecting the substrate and cofactor mimics in these inhibitors. The most potent NNMT inhibitor identified exhibits an IC 50 value of 3.7 nM, placing it among the most active NNMT inhibitors reported to date. Complementary analytical techniques, modeling studies, and cell-based assays provide insights into the binding mode, affinity, and selectivity of these inhibitors.

Nicotinamide N -methyltransferase (NNMT) methylates nicotinamide to form 1-methylnicotinamide (MNA) using S -adenosyl-l-methionine (SAM) as the methyl donor. The complexity of the role of NNMT in healthy and disease states is slowly being elucidated and provides an indication that NNMT may be an interesting therapeutic target for a variety of diseases including cancer, diabetes, and obesity. Most inhibitors of NNMT described to date are structurally related to one or both of its substrates. In the search for structurally diverse NNMT inhibitors, an mRNA display screening technique was used to identify macrocyclic peptides which bind to NNMT. Several of the cyclic peptides identified in this manner show potent inhibition of NNMT with IC 50 values as low as 229 nM. The peptides were also found to downregulate MNA production in cellular assays. Interestingly, substrate competition experiments reveal that these cyclic peptide inhibitors are noncompetitive with either SAM or NA indicating they may be the first allosteric inhibitors reported for NNMT., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Melanoma represents the most aggressive skin cancer, being responsible for the majority of deaths related with these neoplasms. Despite chemotherapy represents a frontline approach for management of the advanced stages of the disease, it displayed poor response rates and short-term efficacy due to melanoma cell resistance. Therefore, the discovery of molecules that can be used for effective targeted therapy of melanoma is crucial. In this study, we evaluated the impact of paraoxonase-2 (PON2) silencing on proliferation, viability, and resistance to treatment of the A375 melanoma cell line with chemotherapeutic drugs dacarbazine (DTIC) and cisplatin (CDDP). Due to the enzymes ability to counteract oxidative stress, we also evaluated the effect of enzyme knockdown on reactive oxygen species (ROS) production in cells treated with CDDP. The data reported clearly demonstrated that PON2 knockdown led to a significant reduction of cell proliferation and viability, as well as to an enhancement of A375 sensitivity to CDDP treatment. Moreover, enzyme downregulation was associated with an increase of ROS production in CDDP-treated cells. Although further analyses will be necessary to understand how PON2 could influence melanoma cell metabolism and phenotype, our results seem to suggest that the enzyme may serve as an interesting molecular target for effective melanoma treatment.

High-risk HPVs (HR-HPVs) are DNA viruses considered as primary etiologic factors in malignancies of the low female genital tract. Their presence has also been documented in oropharyngeal and laryngeal cancers. However, HPV infection is considered a necessary but not sufficient cause of tumoral development; meantime, increasing evidences on the tumorigenic role of cancer stem cells (CSCs) have been documented in the literature. CSCs represent a small subpopulation of neoplastic cells with self-renewal potential, capable of maintaining tumor growth and cell differentiation, also involved in metastatic process, recurrence, and resistance to chemotherapeutic agents. In the present study, performed on KB cell lines, we evaluated the tumor forming potential of CSCs, and their relationship with the HPV infection status. We started our study by identifying the most aggressive cell line on the minimal number of cells being able of growth in vivo in a model of athymic nude mice (BALB/c nu/nu). We used an oral-derived KB cell line separated in the KB-CD133+ and KB-CD133- populations, by using immunomagnetic beads and fluorescence-activated cell sorting (FACS). The separated populations were injected in athymic nude mice (BALB/c nu/nu). Xenograft tumors have been analyzed for tumor size, CD133 expression by immunohistochemistry (IHC) and for DNA HR-HPV integration by in situ hybridization (ISH), comparing CD133-enriched xenograft tumors versus the CD133 non-enriched ones. On standard conditions, the KB cell line has a poor population of glycosylated CD133 marker (<5.0%) when investigated with antibodies versus CD133, and more specifically its glycosylated epitope (AC133). Enriched CD133 KB cells possess a higher capacity of tumor growth in xenograft models of nude mice when compared to KB CD133-negative cells. We observed that the AC133 epitope, extensively used to purifying hematopoietic stem cells, is able to select an epithelial subpopulation of cancer stem cells with aggressive behavior. We retain that CD133 may be a useful target in anticancer strategies including pharmacological and immunological therapies., (© 2020 Scandinavian Societies for Medical Microbiology and Pathology.)

The goal of the current study was to identify potential roles of paraoxonase-2 in bladder carcinogenesis. T24 bladder cancer cells were transfected with plasmids inducing paraoxonase-2 silencing or overexpression. Upon the selection of clones stably down- or upregulating paraoxonase-2, cell proliferation, migration, and the production of reactive oxygen species were evaluated, before and after treatment with cisplatin and gemcitabine, used alone or in combination. The activity levels of both caspase-3 and caspase-8 were also analyzed. shRNA-mediated gene silencing and the overexpression of paraoxonase-2 revealed that the enzyme was able to promote both the proliferation and migration of T24 cells. Moreover, the knockdown of paraoxonase-2 was significantly associated with a reduced cell viability of T24 cells treated with chemotherapeutic drugs and led to both an increase of reactive oxygen species production and caspase-3 and caspase-8 activation. Conversely, under treatment with anti-neoplastic compounds, a higher proliferative capacity was found in T24 cells overexpressing paraoxonase-2 compared with controls. In addition, upon enzyme upregulation, both the production of reactive oxygen species and activation of caspase-3 and caspase-8 were reduced. Although further analyses will be required to fully understand the involvement of paraoxonase-2 in bladder tumorigenesis and in mechanisms leading to the development of chemoresistance, the data reported in this study seem to demonstrate that the enzyme could exert a great impact on tumor progression and susceptibility to chemotherapy, thus suggesting paraoxonase-2 as a novel and interesting molecular target for effective bladder cancer treatment., Competing Interests: The authors declare no conflicts of interest.

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.

Oral malignant melanoma (OMM) is an aggressive tumour, and shows deep tissue invasion at initial presentation. The prognosis is worse than that for cutaneous melanoma (CM), and the overall 5-year survival rate is 10-25%. A study of the molecular mechanisms involved in the development of OMM is necessary to identify new prognostic markers. In this study, we evaluated the possible role of nicotinamide N-methyltransferase (NNMT) in OMM. We carried out immunohistochemical analyses to evaluate the expression of NNMT in 15 OMM and 15 CM, measuring the percentage of positive cells and the value of NNMT expression intensity. Furthermore, we explored the relationship between NNMT levels and the prognostic parameters of patients with OMM. NNMT was significantly more expressed in CM compared with OMM, whereas higher staining intensity for NNMT was observed in OMM cases (P<0.05). In addition, a significant relationship was found between NNMT staining intensity and the presence of ulceration (P<0.05). Furthermore, univariate analysis showed a negative effect of NNMT expression on the disease-free survival rate (P<0.05). This study is the first to report the expression of NNMT in OMM and to compare OMM enzyme levels with those detected in CM. Data obtained seem to suggest the presence of potential molecular differences between these two tumours.

Oral squamous-cell carcinoma (OSCC) and most human tumors are characterized by an imbalance of regulatory mechanisms controlling cell processes such as apoptosis. Survivin, a member of the inhibitor of apoptosis family, is overexpressed in most solid and hematological malignancies and correlates with a reduced overall survival rate. Thus, the aim of this study was to find a correlation between nuclear Survivin expression and clinicopathologic data and the prognosis in OSCC patients. A total of 152 OSCC samples were investigated by immunohistochemistry for nuclear Survivin expression. Then, Survivin was scored semiquantitatively using an immunoreactivity score (IRS), calculated by multiplying the percentage of positive cells with the staining intensity. Using a digital image analysis software, OSCC patients were stratified into 4 groups. Results showed that patients with a lower IRS score displayed better survival rates than patients with a higher IRS score, reaching statistical significance. As the expression of Survivin at the nuclear level seems to suggest a poor prognosis in OSCC patients, the evaluation of nuclear Survivin IRS may be a useful tool to identify patients with more aggressive and disseminated disease, influencing follow-up and therapeutic protocols.

Nicotinamide N-methyltransferase (NNMT) is an enzyme that catalyses the methylation of nicotinamide to form N'-methylnicotinamide. Both NNMT and its methylated product have recently been linked to a variety of diseases, suggesting a role for the enzyme as a therapeutic target beyond its previously ascribed metabolic function in detoxification. We here describe the systematic development of NNMT inhibitors derived from the structures of the substrates involved in the methylation reaction. By covalently linking fragments of the NNMT substrates a diverse library of bisubstrate-like compounds was prepared. The ability of these compounds to inhibit NNMT was evaluated providing valuable insights into the structural tolerances of the enzyme active site. These studies led to the identification of new NNMT inhibitors that mimic the transition state of the methylation reaction and inhibit the enzyme with activity on par with established methyltransferase inhibitors.

Nicotinamide N-methyltransferase (NNMT) is one of the most abundant small molecule methyltransferases in the human body and is primarily responsible for the N-methylation of the nicotinamide (vitamin B3). Employing the cofactor S-adenosyl-l-methionine, NNMT transfers a methyl group to the pyridine nitrogen of nicotinamide to generate N-methylnicotinamide. Interestingly, NNMT is also able to N-methylate a variety of other pyridine-containing small molecules, suggesting a secondary role for the enzyme in the detoxification of xenobiotics. A number of recent studies have also revealed links between NNMT overexpression and a variety of diseases, including multiple cancers, Parkinson's disease, diabetes, and obesity. To facilitate further study of both the substrate scope and potential for inhibitor development, we here describe the development of a new NNMT activity assay. The assay makes use of ultra-high-performance hydrophilic interaction chromatography, allowing for rapid separation of the reaction products, coupled with quadrupole time-of-flight mass spectrometric detection, providing for enhanced sensitivity and enabling high-throughput sample analysis. We successfully demonstrated the general applicability of the method by performing kinetic analyses of NNMT-mediated methylation for a range of pyridine-based substrates. These findings also provide new insight into the diversity of substrate recognition by NNMT in a quantitative manner. In addition, we further established the suitability of the assay for the identification and characterization of small molecule inhibitors of NNMT. To do so, we investigated the inhibition of NNMT by the nonspecific methyltransferase inhibitors sinefungin and S-adenosyl-l-homocysteine, revealing IC50 values in the low micromolar range. The results of these inhibition studies are particularly noteworthy as they will permit future efforts toward the development of new NNMT-specific inhibitors.

The aim of this study was to analyze the placental expression and allele status of promoter region of Klotho in association with preeclampsia, which represents the most common hypertensive disease of pregnancy. Klotho mRNA and protein levels were determined using real-time PCR and Western blot, respectively, in placental tissue samples obtained from 34 patients affected with preeclampsia and 34 controls. A PCR-based genotyping analysis was carried out in the promoter region of Klotho gene. Moreover, expression levels of pluripotency markers, Nanog and Oct4, and telomere length were assessed using real-time PCR. Klotho mRNA and protein levels were reduced in preeclamptic placentas compared with controls. -744delA single-nucleotide polymorphism was significantly associated with preeclampsia. In pathological placentas, there was a downregulation of pluripotency markers and a reduced telomere length. This study is the first to evaluate the placental expression level of Klotho in association with preeclampsia. Further analyses will clarify its role in the pathogenesis of this pregnancy hypertensive disorder.

Oral squamous cell carcinoma (OSCC) is the most common malignancy of oral cavity. Human cancers are characterized by an imbalance of regulatory mechanisms controlling different cellular pathways, including apoptosis. Apoptosis occurs in a wide variety of physiological processes, such as embryonic development, tissue homeostasis or immune defense, and its role is to remove harmful, damaged, or unwanted cells. Defective apoptosis represents an important causative factor in the development/progression of cancer, and the ability of tumor cells to evade apoptosis can play a significant role in their resistance to conventional anticancer treatment. We investigated the expression profile of genes involved in the apoptotic mechanism in 21 paired tissue samples (OSCC and adjacent normal oral mucosa) by cDNA macroarray, in order to identify differentially expressed genes in oral cancer compared to normal tissue. To validate the results obtained by cDNA macroarray, quantitative real-time PCR, Western blot, and immunohistochemical analyses were performed. Results obtained by cDNA macroarray analysis showed different expression levels of CRADD, FADD, ATM, APAF1, and TP63 genes in OSCC compared to normal mucosa. Differential gene expression measurements (tumor vs. normal tissue) performed by real-time PCR showed an overexpression of FADD and a downregulation of ATM. Moreover, Western blot analysis confirmed that both CRADD and APAF-1 were decreased in OSCC compared to normal oral mucosa. As showed by immunohistochemistry, OSCC exhibited increased expression of p63 compared to normal tissue. Interestingly, a statistically significant positive correlation was found between p63 expression and the histological grade., (© 2012 Wiley Periodicals, Inc.)

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.

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.

Objective: To determine placental gene expression of endothelial and inducible nitric oxide synthases and measure nitric oxide levels in patients with hemolysis, elevated liver enzyme levels, and low platelet count syndrome., Study Design: Preterm placentas were obtained from 15 patients with hemolysis, elevated liver enzyme levels, and low platelet count syndrome and 30 controls matched for age, parity, and gestational age. mRNA levels were evaluated by real-time polymerase chain reaction, whereas nitric oxide and peroxynitrite production was measured by a commercially available kit., Results: Placental gene expression of inducible nitric oxide and endothelial nitric oxide synthases were significantly lower in the hemolysis, elevated liver enzyme levels, and low platelet count syndrome group than in controls, whereas nitric oxide and peroxynitrite production were significantly higher in hemolysis, elevated liver enzyme levels, and low platelet count syndrome compared with controls., Conclusion: The reduced endothelial nitric oxide and inducible nitric oxide synthases gene expression in women with hemolysis, elevated liver enzyme levels, and low platelet count syndrome may indicate extreme placental dysfunction that is unable to compensate the endothelial derangement and the related hypertension. The higher nitric oxide formation found in hemolysis, elevated liver enzyme levels, and low platelet count syndrome placentas could be explained as a counteraction to the impaired fetoplacental perfusion, typical of the syndrome., (Copyright © 2011 Mosby, Inc. All rights reserved.)

There is growing evidence that pregnancy complications such as preeclampsia, recurrent pregnancy loss (RPL), IUGR, and premature birth could be associated with abnormal immunologic interactions at the fetal-maternal interface. The restricted expression of HLA-G to the subpopulation of trophoblast cells which invade the uterus has generated much interest. The alternative splicing of HLA-G primary transcript, gives origin to seven isoforms, including both membrane-bound forms (HLA-G1, G2, G3, G4) and soluble forms (sHLA-G: sHLA-G5, G6, G7). sHLA-G consists predominantly of sHLA-G1 after its shedding by metalloproteinases, and secreted sHLA-G5 representing the quantitatively dominating and full-length isoforms. HLA-G expression and HLA-G genetic variations in both the mother and the embryo/fetus may be important for pregnancy outcome. It is also intuitively apparent that a gene with putative immunosuppressive and immunotolerant potential might be functional in both the mother and the embryo/fetus/placenta. Reduced or aberrant HLA-G expression seems to be associated with certain complications of pregnancy, among which preeclampsia and possibly the risk of miscarriage, and that this may be further linked to HLA-G polymorphisms. Most of the studies aimed at assessing the role of HLA-G in pregnant diseases have considered only the maternal genotype and ignored the contribution of the fetus. In this regard, the mother, placenta and the fetus form a synthesis. Therefore, studies on placental diseases should address HLA-G expression and genetic variations also to the fetus/placenta., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Breast cancer represents the most commonly diagnosed invasive malignancy in pre- and postmenopausal women in both developed and underdeveloped countries. Taking into account that treatment options, including surgery, have not been able to deal with the growing incidence of breast malignancy, it is required to develop mechanism-based novel agents for its prevention. Wide interest in some natural compounds as antiinflammatory agents and as alternative to the traditional medicines is increasing because they do not provoke any adverse effects and are effective in multiple organs, alpha-Bisabolol (BISA), a small oily sesquiterpene alcohol, was reported as chemopreventive agent in induced rat mammary carcinogenesis. The aim of the present study is to investigate the role played by two doses of BISA (via intramammary infusion) on the induction and development of mammary tumor in HER-2/neu transgenic mice as well as the BISA effect after tumor surgical resection. The main data show that (a) optimal dosage of BISA is 10 mg/mouse rather than 3.6 mg/mouse with no adverse effects (e.g., alopecia); (b) the number of the palpable tumor masses decreases in mice treated with 10 mg/mouse of BISA; (c) mice after surgical resection of the primary tumor and treatment with BISA (10 mg) are free from tumor for more weeks, after the surgical treatment; (d) using array analysis, some genes implicated in carcinogenesis mechanisms (NF-kappaBia, Map2k, Mapkl4, and HER2/ neu), angiogenesis process (Fgf), and inhibition of apoptosis (Birc5) are differently regulated after BISA treatment, with a downregulation of the HER2/neu as well as of Fgf and Birc5 genes; (e) the NK cell cytotoxicity increases in tumor-treated mice, especially after the removal of the first tumor mass. Such effectiveness could be important to achieve goals for a possible combination of BISA to conventional therapies in breast cancer and to tumor surgical removal (adjuvant therapy), as suggested for other sesquiterpene analogs.

Mitogen-activated protein kinase (MAPK) p38alpha was shown to be implicated in the organogenesis of the placenta, and such placental alteration is crucial for the development of hemolysis, elevated liver enzymes, and low platelets (HELLP) syndrome. We aimed to analyze for the first time human placental expression of MAPK p38alpha in pregnancies complicated by HELLP. The placental expression of MAPK p38alpha was investigated by semiquantitative polymerase chain reaction using cDNA extracted from placental tissue of 15 pregnancies with HELLP syndrome and 15 gestational age-matched controls. Seven patients with HELLP also had intrauterine fetal growth restriction (IUGR). In placenta from pregnancy complicated by HELLP, the expression of MAPK p38alpha is significantly decreased compared to the group with normal pregnancy (p < 0.001), while no difference was found between the HELLP and HELLP with IUGR subpopulations. Our study shows for the first time that MAPK p38alpha is expressed in the human placenta. Pregnancies with placental dysfunction and hypertensive complications are characterized by a significantly decreased expression of MAPK p38alpha. Our observations suggest that p38 MAPK signaling may be essential in placental angiogenesis and functioning.

We investigated expression levels of Nicotinamide N-Methyltransferase (NNMT), an enzyme involved in the biotransformation of many drugs and xenobiotic compounds, in oral squamous cell carcinoma (OSCC). Measurements were performed by immunohistochemistry and the relationship between tumour characteristics and NNMT levels in OSCC was studied to evaluate the effectiveness of NNMT as a prognostic marker in squamous cell carcinoma of the oral cavity. In conclusion, the present study suggests that NNMT may have potential as a biomarker and as a therapeutic target for OSCC.

AHSP inhibits cellular production of the reactive oxygen species. Reduced AHSP indicates reduced protection against oxidative stressors. Our objective was to investigate AHSP levels in recurrent miscarriage (RM). Trophoblast was collected from women of 10 weeks gestation: voluntary abortion controls (VA, n = 10); spontaneous first miscarriage with subsequent normal pregnancy (SMSN, n = 15) or with subsequent miscarriage (SMSM, n = 5); RM previously investigated (RMPS, n = 5) or not previously investigated (RM, n = 5). AHSP mRNA and protein were determined using real-time quantitative polymerase chain reaction (PCR) and Western blot, respectively. One-way ANOVA was performed to assess statistical significance (p < 0.05). ahsp mRNA levels were maximally reduced in RM and RMPS (8.0 x 10(-6) +/- 1.3 and 8.1 x 10(-6) +/- 0.7, respectively) compared with SMSN and VA (16.1 x 10(-6) +/- 2.3 and 26.1 x 10(-6) +/- 2.7, respectively). SMSM showed levels significantly reduced as well (9.0 x 10(-6) +/- 2.3). In RM, a reduced defense from oxidative stressors is evident at first miscarriage, identifying women at high risk for subsequent eventful pregnancy. Reduced AHSP may identify women at risk of experiencing further miscarriages.

Purpose: Survivin, an inhibitor of apoptosis protein and a cell cycle regulator, has been detected in the majority of human cancers. Five splice variants (survivin, survivin-2alpha, survivin-2B, survivin-3B, and survivin-DeltaEx3) have been identified; their expressions have been investigated here., Methods: By means of RT real-time PCR and immunohistochemistry, we have evaluated survivin isoform expressions at both mRNA and protein levels in human normal oral tissue, precancerous lesions, and oral squamous cell carcinoma (OSCC). Their correlations with the pathological findings have also been analyzed., Results: Expression levels of all survivin transcript variants were markedly elevated in OSCC when compared to normal tissues. One-way analysis of variance (ANOVA) revealed highly significant up-regulation of survivin (P = 0.001), survivin-DeltaEx3 (P = 0.001) and survivin-2B (P = 0.004), whereas survivin-3B showed a minor increase in OSCC compared to normal mucosa., Conclusions: Our findings suggest that survivin isoforms deregulation may have significant implications in tumor aggressiveness and prognosis.

Objective: Alpha hemoglobin-stabilizing protein (AHSP) inhibits the production of reactive oxygen species in various cells, including erythrocytes. Reduced AHSP can mean reduced protection from stressors. Our objective was to investigate whether AHSP is involved in the response to stress in pregnancy., Study Design: Placentas were collected from normal term pregnancies (n = 10) and pregnancies complicated by HELLP (n = 10), intrauterine growth restriction (IUGR; n = 10) or fetal death (IUFD; n = 6). AHSP messenger RNA (mRNA) and protein were determined using real time quantitative polymerase chain reaction (PCR) and Western blot, respectively. All statistical analyses were performed by using the GraphPad Prism Software. Differences were considered significant at p < 0.05., Results: Placental AHSP mRNA level in HELLP (4.16E10(-4) +/- 1.77) and IUFD (4.19E10(-4) +/- 3.37) were significantly decreased compared with controls (28.47E10(-4) +/- 14.86; p < 0.01), whereas levels in the IUGR group (7.55E10(-4) +/- 6.4) showed a trend toward being lower but the difference did not reach statistical significance. Western blot analysis results indicate a no significant increase of ASHP protein in the HELLP syndrome group and a significant decrease in the IUFD group compared with controls. There was no significant difference between the IUGR and control groups., Conclusion: ASHP mRNA expression in the placenta is decreased in complicated pregnancies, and it may be involved in the pathogenic mechanisms leading to the adverse pregnancy outcome.

alpha-Lipoic acid (LA) has been found previously to accelerate wound repair in patients affected by chronic wounds who underwent hyperbaric oxygen (HBO) therapy. Because proteinases are important in wound repair, we hypothesized that LA may regulate matrix metalloproteinase (MMP) expression in cells that are involved in wound repair. Patients undergoing HBO therapy were double-blind randomized into two groups: the LA group and the placebo group. Gene expression profiles for MMPs and for angiogenesis mediators were evaluated in biopsies collected at the first HBO session, at the seventh HBO session, and after 14 days of HBO treatment. ELISA tests were used to validate microarray expression of selected genes. LA supplementation in combination with HBO therapy downregulated the inflammatory cytokines and the growth factors which, in turn, affect MMPs expression. The disruption of the positive autocrine feedback loops that maintain the chronic wound state promotes progression of the healing process.

Thrombophilia is the increased tendency to thrombosis. Inherited and acquired factors may determine thrombophilia. Some physiologic conditions, such as pregnancy are themselves "thrombophilic". In pregnancy, in fact, there is a decrease of all natural anticoagulant systems, such as antithrombin, protein C and protein S that are partially compensated by an increased fibrinolysis. It has been well established that women with thrombophilic disorders are at greater risk of venous thromboembolism in pregnancy and puerperium. It has also been observed that those women have higher prevalence of those obstetric complications in which microplacental thrombosis may play a pathogenetic role, such as placental abruption, preeclampsia, intrauterine growth restriction, intrauterine fetal death, repeated spontaneous miscarriage. Given that those complications are not always associated with maternal thrombophilia, controversy still exists on the exact impact of the disorders with the adverse pregnancy outcomes. While we are convinced that thrombophilias are extensively implicated in pregnancy complications, we feel that there has not been completely elucidated the role of the different factors, the gestational age at which those factors may intervene, nor has been given enough relevance to the weight of fetal thrombophilias in the origin of some specific form of those obstetric complications. We should bear in mind that some thrombophilias may be inherited from the mother, the father or both. Our hypothesis is that maternal thrombophilias may be responsible for venous thromboembolism, preeclampsia HELLP and eclampsia, whereas fetal thrombophilia, may account for IUGR or stillbirth. This last would also explain some stillbirth or repeated late (>10 gestational weeks) miscarriage observed in non-thrombophilic mothers. The two sides of thrombophilia may, of course, concur, resulting in the more severe clinical presentations.