Nicotinamide Increases The Sensitivity Of Chronic Myeloid Leukemia Cells To Doxorubicin

Objectives:Chronic myelogenous leukemia(CML)results from malignant transformation of a hematopoietic stem cell by the BCR-ABL oncogene.CML usually presents in a chronic phase but progresses to an accelerated phase and a terminal blast crisis.The resistance of leukemic cells to chemotherapy-induced apoptosis remains the most serious problem in the treatment of leukemia.Sirtuin 1(SIRT1)is a member of the sirtuin family that regulates numerous processes including aging,DNA repair,cell cycle,and cell survival under stress conditions,by deacetylating a number of factors including p53,FOXO,HSF1 and various DNA damage repair factors.Numerous reports have shown that SIRT1 may have a pathogenetic role in solid tumors and leukemias.Hence,there has been increased interest in investigating and designing molecular tools that might inhibit SIRT1.Nicotinamide,a main precursor of NAD+,has long been used in the treatment pellagra with minimal side effects,thus an effective inhibitor of SIRT1.In this study,we investigated the effects of nicotinamide in combination with doxorubicin(DOX)on CML cells.Methods:We analyzed the expression of SIRT1 in public available microarray datasets which included GSE47927,GSE4170 and GSE14671.JAVA program for GSEA was used to analyze the potential genes that are influenced by SIRT1 high expression.We also evaluated objective genes transcripts by real-time quantitative PCR.Protein expression was evaluated by western blotting and immunoprecipitation.Cell viability was determined by CCK8 assay.Cell proliferation was analyzed by flow cytometry by CFSE labeling.Apoptotic cells were detected by flow cytometry following Annexin V and propidium iodide staining.Morphologic changes and quantification of apoptotic cells were determined under fluorescent microscopy following Hoechst 33342 staining.The growth of xenograft tumors established by injection of K562/DOX cells subsutaneously into SCID mice was investigated to analyze the antitumor effects of nicotinamide and DOX.Results:Microarray datasets analysis indicated that SIRT1 expression level was increased in CML and was significantly associated with the TKI response.SIRT1 was over expressed in the DOX-resistant K562/DOX cell line compared with DOX-sensitive K562 cell line.Exogenous nicotinamide efficiently inhibited the deacetylation activity of SIRT1,inhibited proliferation and induced apoptosis in a dose-dependent way in K562 and K562/DOX cells.The effects of nicotinamide were more evident in K562/DOX cells,in line with their high constitutive levels of SIRT1.Nicotinamide enhanced DOX-induced cell proliferation inhibition and apoptosis in K562 and K562/DOX cells.Further analysis revealed that the combination of DOX and nicotinamide increased FOXO3a acetylation,and also increased cleavage of caspase-3 and PARP in K562/DOX cells.In vivo experiments,using SCID mice model showed that the combination of DOX and nicotinamide significantly inhibited the tumor growth,and the weight of the mice was not significantly affected by drug treatment.Conclusions:In summary,nicotinamide efficiently increases the sensitivity of CML cell lines to DOX.Our results suggest that SIRT1 could be used as a potential prognosis marker and therapeutic target,which could provide new clues for SIRT1 inhibition in CML treatment.