title: [Suppression of NAMPT expression enhances the sensitivity of K562 cells to imatinib and its relative mechanism].
abstract: The aim of this study was to investigate the effect of suppression of nicotinamide phosphoribosyltransferase (NAMPT) expression on imatinib-sensitivity in chronic myelogenous leukemia (CML) cell line K562 and its mechanisms, NAMPT siRNA was synthesized and transfected into K562 cells. PI/Calcein staining technique was used to determine survival rate of transfected K562 cells at 48th hour after exposure to 1 µmol/L imatinib. MTS method was used to determine the proliferation changes of transfected K562 cell at 48th hour after exposure to different doses of imatinib, then half inhibitory concentration (IC(50)) was calculated. Expression of NAMPT at 3rd-48th hour after exposure to 1 µmol/L imatinib was determined by Western blot. To explore the effect of NAMPT-siRNA and imatinib on the expression of apoptosis-related genes, the microarray data from NCBI GEO Data-Sets was analyzed, then the results were confirmed by Western blot. The luciferase reporter assay was used to determine the effect of NAMPT and imatinib on transcriptional activity of NF-κB transcription factors. The results showed that after exposure to 1 µmol/L imatinib for 3 - 48 h, there was no significant change of NAMPT expression in K562 cells. The expression of NAMPT could be effectively inhibited by the NAMPT-siRNA. After exposure to 1 µmol/L of imatinib for 48 h, the survival rate of NAMPT-siRNA interference group was lower than that of negative control group (P < 0.05), indicating that suppression of NAMPT expression can increase the sensitivity of K562 cells to imatinib and enhance the killing effect of imatinib on K562 cells. The IC(50) of imatinib in NAMPT-siRNA interference group was the lowest compared with that of control group (P < 0.05) after exposure to different concentrations of imatinib for 48 h, the fitted survival curves showed that the slope of NAMPT-siRNA interference group was the largest ranging between 0.01 - 0.1 µmol/L of imatinib. Data mining of expression profiling indicated that the anti-apoptotic factor Bcl-2 decreased in K562 cells treated with either NAMPT-siRNA or imatinib, which was confirmed by Western blot. The inhibitory effect was much more significant when both NAMPT-siRNA and imatinib were used. The results of luciferase reporter assay showed that either NAMPT-siRNA or imatinib decreased transcriptional activity of NF-κB. The decreased effect was much more significant when both NAMPT-siRNA and imatinib were used. It is concluded that survival of K562 cells affected by imatinib may not be due to regulation of expression of NAMPT. When expression of NAMPT decreases, the K562 cells are more sensitive to imatinib, this may be related with the decreased transcriptional activity of NF-κB and its downstream effector Bcl-2.
authors.affiliation: Hematology Center, Beijing Children Hospital, Capital Medical University, Beijing 100045, China.
authors.forename: Ping-Ping
authors.initials: PP
authors.lastname: Lin
authors.forename: Xue-Jia
authors.initials: XJ
authors.lastname: Bai
authors.forename: Zhi-Xia
authors.initials: ZX
authors.lastname: Yue
authors.forename: Shao-Fei
authors.initials: SF
authors.lastname: Yan
authors.forename: Zong-Wen
authors.initials: ZW
authors.lastname: Li
authors.forename: Chao
authors.initials: C
authors.lastname: Gao
authors.forename: Yan-Yan
authors.initials: YY
authors.lastname: Mei
authors.forename: Kai-Ling
authors.initials: KL
authors.lastname: Wang
authors.forename: Wei-Jing
authors.initials: WJ
authors.lastname: Li
authors.forename: Wei
authors.initials: W
authors.lastname: Ding
authors.forename: Zhi-Gang
authors.initials: ZG
authors.lastname: Li
mesh_terms: D001549:Benzamides; D016207:Cytokines / Q000037:antagonists & inhibitors* / Q000378:metabolism; D016044:Fusion Proteins, bcr-abl / Q000378:metabolism; D006801:Humans; D000068877:Imatinib Mesylate; D020014:K562 Cells; D016328:NF-kappa B / Q000378:metabolism; D054409:Nicotinamide Phosphoribosyltransferase / Q000037:antagonists & inhibitors* / Q000378:metabolism; D010879:Piperazines / Q000494:pharmacology*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D011743:Pyrimidines / Q000494:pharmacology*
chemical_list: D001549:Benzamides; D016207:Cytokines; D016328:NF-kappa B; D010879:Piperazines; D019253:Proto-Oncogene Proteins c-bcl-2; D011743:Pyrimidines; D000068877:Imatinib Mesylate; D054409:Nicotinamide Phosphoribosyltransferase; C085455:nicotinamide phosphoribosyltransferase, human; D016044:Fusion Proteins, bcr-abl