Overexpression and silencing of nicotinamide N-methyltransferase in human cancer cell lines

Il carcinoma polmonare rappresenta la principale causa di morte per cancro a livello mondiale. Il carcinoma orale rappresenta l’undicesima neoplasia più diffusa nel mondo. Nonostante i progressi effettuati in campo terapeutico, il tasso di sopravvivenza dei pazienti affetti da tali patologie rimane scarsa. Pertanto, risulta di fondamentale importanza l’identificazione di biomarcatori per una diagnosi precoce di tali neoplasie. Oggetto del presente lavoro di ricerca è l’enzima nicotinamide N-metiltrasferasi (NNMT). Al fine di esplorare il ruolo svolto dall’enzima nel metabolismo della cellula tumorale, i livelli di espressione dell’NNMT sono stati esaminati nella linea cellulare di carcinoma polmonare A549 ed è stato valutato l’effetto del silenziamento dell’enzima sulla tumorigenicità mediante il saggio MTT ed il saggio in soft-agar. Successivamente, è stato esaminato l’effetto dell’upregolazione dell’enzima sulla proliferazione cellulare nella linea cellulare di carcinoma orale HSC-2. Inoltre, è stato esplorato l’effetto dell’overespressione dell’NNMT sui livelli di espressione di geni coinvolti nel controllo del ciclo cellulare e dell’apoptosi, quali la β-catenina, Ki-67 e la survivina. Il silenziamento dell’NNMT nelle cellule A549 ha portato ad una significativa inibizione della proliferazione cellulare e della capacità di formare colonie in soft-agar, mentre l’overespressione dell’enzima nelle cellule HSC-2 ha determinato un significativo aumento della crescita cellulare. Inoltre, l’isoforma ΔEx3 della survivina è risultata significativamente upregolata nelle cellule HSC-2 overesprimenti l’NNMT. I risultati ottenuti hanno dimostrato il coinvolgimento dell’NNMT nella proliferazione della cellula tumorale, candidando l’enzima quale interessante bersaglio per una terapia antineoplastica. Inoltre, la correlazione osservata tra i livelli di espressione dell’NNMT e dell’isoforma ΔEx3 della survivina sembra suggerire un potenziale ruolo dell’NNMT nell’apoptosi.Lung cancer is the leading cause of tumor-related death worldwide. As the majority of patients are diagnosed at a late stage, current therapeutic strategies have limited effectiveness and the prognosis remains poor. Oral cancer is the 11th most common tumor in the world. Despite advances in cancer therapy, the 5-year survival rate has not improved in the last three decades. Therefore, the identification of biomarkers for early diagnosis and treatment of these neoplasms are needed. The present study is focused on the enzyme nicotinamide N-methyltransferase (NNMT). To explore the role of the enzyme in cancer cell metabolism, we first examined NNMT expression levels in the human lung cancer cell line A549 by Real-Time PCR, Western blot and catalytic activity assay, and evaluated the in vitro effect of NNMT knockdown on tumorigenesis by MTT and soft agar colony formation assays. Subsequently, the effect of enzyme upregulation on cell proliferation was explored in the human oral cancer cell line HSC-2. Moreover, apoptosis and cell proliferation were investigated at molecular level, by evaluating the effect of NNMT overexpression on β-catenin, survivin isoforms, and Ki-67 expression. NNMT knockdown in A549 cells led to a significant inhibition of cell proliferation and colony formation ability on soft agar, while upregulation of the enzyme in HSC-2 cells significantly increased cell growth. Moreover, a positive correlation between NNMT and survivin-ΔEx3 isoform expression levels was found in HSC-2 cells. Results obtained demonstrated a potential involvement of NNMT in proliferation and tumorigenic capacity of both lung and oral cancer cells, thus representing an interesting molecular target for an effective anti-cancer therapy. Moreover, the possible correlation observed between NNMT and survivin-ΔEx3 expression levels seems to suggest a potential role of the enzyme in the regulation of apoptosis

Il progetto Lab2Go per la diffusione della pratica laboratoriale nelle Scuole Secondarie di II grado

Even if laboratory practice is essential for all scientific branches of knowledge, it is often neglected at High School, due to lack of time and/or resources. To establish a closer contact between school and experimental sciences, Sapienza Università di Roma and the Istituto Nazionale di Fisica Nucleare (INFN) launched the Lab2Go project, with the goal of spreading laboratory practice among students and teachers in high schools

Overexpression and silencing of nicotinamide N-methyltransferase in human cancer cell lines

Il carcinoma polmonare rappresenta la principale causa di morte per cancro a livello mondiale. Il carcinoma orale rappresenta l’undicesima neoplasia più diffusa nel mondo. Nonostante i progressi effettuati in campo terapeutico, il tasso di sopravvivenza dei pazienti affetti da tali patologie rimane scarsa. Pertanto, risulta di fondamentale importanza l’identificazione di biomarcatori per una diagnosi precoce di tali neoplasie. Oggetto del presente lavoro di ricerca è l’enzima nicotinamide N-metiltrasferasi (NNMT). Al fine di esplorare il ruolo svolto dall’enzima nel metabolismo della cellula tumorale, i livelli di espressione dell’NNMT sono stati esaminati nella linea cellulare di carcinoma polmonare A549 ed è stato valutato l’effetto del silenziamento dell’enzima sulla tumorigenicità mediante il saggio MTT ed il saggio in soft-agar. Successivamente, è stato esaminato l’effetto dell’upregolazione dell’enzima sulla proliferazione cellulare nella linea cellulare di carcinoma orale HSC-2. Inoltre, è stato esplorato l’effetto dell’overespressione dell’NNMT sui livelli di espressione di geni coinvolti nel controllo del ciclo cellulare e dell’apoptosi, quali la β-catenina, Ki-67 e la survivina. Il silenziamento dell’NNMT nelle cellule A549 ha portato ad una significativa inibizione della proliferazione cellulare e della capacità di formare colonie in soft-agar, mentre l’overespressione dell’enzima nelle cellule HSC-2 ha determinato un significativo aumento della crescita cellulare. Inoltre, l’isoforma ΔEx3 della survivina è risultata significativamente upregolata nelle cellule HSC-2 overesprimenti l’NNMT. I risultati ottenuti hanno dimostrato il coinvolgimento dell’NNMT nella proliferazione della cellula tumorale, candidando l’enzima quale interessante bersaglio per una terapia antineoplastica. Inoltre, la correlazione osservata tra i livelli di espressione dell’NNMT e dell’isoforma ΔEx3 della survivina sembra suggerire un potenziale ruolo dell’NNMT nell’apoptosi.Lung cancer is the leading cause of tumor-related death worldwide. As the majority of patients are diagnosed at a late stage, current therapeutic strategies have limited effectiveness and the prognosis remains poor. Oral cancer is the 11th most common tumor in the world. Despite advances in cancer therapy, the 5-year survival rate has not improved in the last three decades. Therefore, the identification of biomarkers for early diagnosis and treatment of these neoplasms are needed. The present study is focused on the enzyme nicotinamide N-methyltransferase (NNMT). To explore the role of the enzyme in cancer cell metabolism, we first examined NNMT expression levels in the human lung cancer cell line A549 by Real-Time PCR, Western blot and catalytic activity assay, and evaluated the in vitro effect of NNMT knockdown on tumorigenesis by MTT and soft agar colony formation assays. Subsequently, the effect of enzyme upregulation on cell proliferation was explored in the human oral cancer cell line HSC-2. Moreover, apoptosis and cell proliferation were investigated at molecular level, by evaluating the effect of NNMT overexpression on β-catenin, survivin isoforms, and Ki-67 expression. NNMT knockdown in A549 cells led to a significant inhibition of cell proliferation and colony formation ability on soft agar, while upregulation of the enzyme in HSC-2 cells significantly increased cell growth. Moreover, a positive correlation between NNMT and survivin-ΔEx3 isoform expression levels was found in HSC-2 cells. Results obtained demonstrated a potential involvement of NNMT in proliferation and tumorigenic capacity of both lung and oral cancer cells, thus representing an interesting molecular target for an effective anti-cancer therapy. Moreover, the possible correlation observed between NNMT and survivin-ΔEx3 expression levels seems to suggest a potential role of the enzyme in the regulation of apoptosis

Overexpression of nicotinamide N-methyltransferase in HSC-2 OSCC cell line: effect on apoptosis and cell proliferation

Objectives: Oral squamous cell carcinoma (OSCC) is the most common malignancy of oral cavity. Despite advances in therapeutic approaches, the 5-year survival rate for oral cancer has not improved in the last three decades. Therefore, new molecular targets for early diagnosis and treatment of OSCC are needed. In the present study, we focused on the enzyme nicotinamide N-methyltransferase (NNMT). We have previously shown that enzyme expression is upregulated in OSCC and NNMT knockdown in PE/CA PJ-15 cells significantly decreased cell growth in vitro and tumorigenicity in vivo. Material and methods: To further explore the role of the enzyme in oral cancer cell metabolism, HSC-2 cells were transfected with the NNMT expression vector (pcDNA3-NNMT) and the effect of enzyme upregulation on cell proliferation was evaluated by MTT assay. Subsequently, we investigated at molecular level the role of NNMT on apoptosis and cell proliferation, by exploring the expression of β-catenin, survivin, and Ki-67 by real-time PCR. Moreover, we performed immunohistochemistry on 20 OSCC tissue samples to explore the expression level of NNMT and survivin ΔEx3 isoform. Results: Enzyme upregulation significantly increased cell growth in vitro. Moreover, a positive correlation between NNMT and survivin ΔEx3 isoform expression levels was found both in HSC-2 cells and in OSCC tissue samples. Conclusion: Taken together, our results indicate a possible involvement of NNMT in the proliferation and tumorigenic capacity of OSCC cells and seem to suggest that the enzyme could represent a potential target for the treatment of oral cancer. Clinical relevance: The involvement of NNMT in cell growth and anti-apoptotic mechanisms seems to suggest that this enzyme could be a new therapeutic target to improve the survival of OSCC patients