Nontoxic-dose Of Deguelin Selectively Target The Axis Of MtNPM1/SIRT1

Background:Nucleophosmin1(NPM1) is an oligomeric nucleolar phosphoprotein located on the nucleolus, shuttles between the nucleolus and cytoplasm, and plays important roles in maintaining the structure of the nucleus and nucleoli, DNA repair, transcriptional regulation, and histone chaperoning. NPM1was higher expressed in proliferation activity of cells such as stem cell and tumor cell. NPM1mutations consistently result in nucleotide gain at the C-terminus of one NPM1allele, which reduces nuclear localization signal, causes aberrant cytoplasmic localization of NPM1, drives NPM1interacting protein or transcription factor in the nucleus also located in cytoplasm that leads to the disorder in biological function of cell, then causing the occurrence and progression of cancer. NPM1gene mutations that occur in nearly60%of adult AML cases of which have a normal karyotype, is the most common molecular abnormalities in the AML, become an important factor in the pathogenesis of NPMc+AML. Despite the improvements of hematopoietic stem cell transplantation combined with chemotherapy has greatly promoted the treatment of AML, but AML still can not be cured. The strategy of induction differentiation therapy have changed the outcome of some AML subtype, such as arsenite/retinoic acid for the treatment of AML-M3(PML-RAR alpha fusion gene), significantly improving the disease remission rate and survival time. At present, induction differentiation therapy of leukemia has become a hotspot. Therefore, high efficiency and little side effect of drug development is another effective way of acute myeloid leukemia treatment. Deguelin, as a natural monomer medicine, has been screened out by our group basic on two projects of the National Natural Science Fund Project which could regulate expression and function of nuclear pore protein and nuclear phosphoprotein in leukemia. We first found that high doses of deguelin could activate apoptosis signal pathway and induce apoptosis, while inducing differentiation of NPMc+AML cells at low doses, but the differentiation mechanism is not clear. The present experiments will be related to this research.Methods:Human NPMl mutant leukemia cell lines (OCI-AML3) and NPM1wild leukemia cell lines (OCIM2) were used for in vitro studies. Cells viability and proliferation were evaluated by MTT assay, LDH release assay and Trypan blue exclusion assay. Cell apoptosis and differentiation assays were assessed by flow cytometric analysis, Wright-Giemsa staining assay and Nitroblue Tetrazolium (NBT) reduction assay. Differentiation associated molecular events assessed by Western Blot, Real-Time Quantitative PCR and Immunofluorescence analysis. Gene silencing was done by si-RNA and gene overexpression was achieved by transfection with overexpression vector mediated by lentivirus.Results:Deguelin inhibited the proliferation and induced apoptosis of OCI-AML3cells expressing Mt NPMl in a concentration-dependent manner, while OCIM2cells harboring Wt NPMl showed low sensitivity to deguelin. Deguelin treatment at the differentiation-induced concentration of2μM for5days significantly induced differentiation of OCI-AML3cells, the phenomena were not presented in the OCIM2cells. Immunoblot analysis revealed deguelin significantly reduced the levels of Mt NPM1and HDACs (HDAC1/HDAC3/SIRT1), but not Wt NPM1, accompanied by increasing the expression of proteins and mRNAs of CEBPβ and G-CSFR. In addition, immunoprecipitation and immunoblot analysis confirmed deguelin significantly down-regulated Mt NPM1via the ubiquitin-proteasome pathway, while the regulation of SIRTl protein at post-translationally. si-NPM Mut treatment induced differentiation of OCI-AML3, reduced the expression of SIRT1, and upregulated the levels of proteins and mRNAs of CEBPβ and G-CSFR while did not change the levels of HDAC1/3. After si-SIRT1treatment which did not alter the levels of Mt NPM1, its pro-differentiation effect is similar to the action of si-NPM Mut treatment, suggesting that the decline of SIRTl was partially accountable for differentiation of NPMc+cells induced by down-regulation of Mt NPM1. At last, overexpression of Mt NPM1appreciably hampered deguelin-induced differentiation of NPMc+OCI-AML3cells compared with GFP-expressing cells.Conclusion:Nontoxic-dose of deguelin inhibited the proliferation and induced differentiation of NPMlc+OCI-AML3cells. The effect of deguelin-induced differentiation is associated with the down-regulation of Mt NPM1/SIRT1. Meanwhile, the decline of HDAC1/HDAC3was partially responsible for deguelin-induced differentiation.