Study Of The Mechanisms Of Nicotinamide N-methyltransferase Overexpression Induced5-FU Resistance In Human Colorectal Cancer Cell Line SW480

Objective:To explore the effect of nicotinamide N-methyltransferase (NNMT) overexpression on colorectal cancer (CRC) cell’s sensitivity to5-FU and the related mechanisms and search for novel and potential targets for cancer therapy.Methods:The eukaryotic expression plasmids, pcDNA3.1and pcDNA3.1/NNMT was transferred to human CRC cell line SW480which has no endogenous NNMT expression and G418was used to screen the positive and stable cell strains. The expressions of NNMT mRNA and protein were verified by Real-Time QPCR and western blot assay. The sensitivity of SW480cells to5-FU was identified by MTT assay. The apoptosis rate was measured by flow cytometry analysis. Changes of activation levels of MAPK signaling pathways and apoptosis-related molecules were analyzed by western blot.Results:Two monoclonal cell models which stably overexpress NNMT, SW480/NNMT-29and SW480/NNMT-40, and one monoclonal blank control cell model named as SW480/Vector were successfully constructed and isolated. NNMT overexpression significantly decreased the sensitivity of SW480cells to5-FU, markedly reduced5-FU-induced apoptosis and both the extrinsic and intrinsic apoptosis pathways were involved in NNMT-induced5-FU resistance. Moreover, ERK, P38, JNK/MAPK signaling pathways were associated with the NNMT-induced5-FU resistance in SW480cells.Conclusions:NNMT enhances5-FU resistance in CRC cells associated with anti-apoptosis mechanism and MAPK signaling pathways, which suggest NNMT could be used as an indicator of the effect of5-FU in CRC and a potential target for cancer therapy.