Deguelin And Oridonin Target NPM1Mutant Protein

Part â… . Deguelin, selective silencing of the NPM1mutant, potentiates apoptosis and induces differentiation In AML cells carrying NPM1mutation.Background: Nucleophosmin (NPM1) is a multifunctional protein that functions as molecular chaperone, shutting between the nucleolus and the cytoplasm. In up to one-third of patients with acute myeloid leukemia, mutation of NPM1results in the aberrant cytoplasmic accumulation of mutant protein and is thought to be responsible for leukemogenesis. Deguelin, a rotenoid isolated from several plant species, has been shown to be a strong anti-tumor agent.Methods; Human leukemia cell lines were used for in vitro studies. Drug efficacy was evaluated by apoptosis and differentiation assays and associated molecular events assessed by Western Blot. Gene silencing was done by small interference RNA.Results:Deguelin exhibited strongly cytotoxic activity in the cell line of OCI-AML3, and selectively down-regulated NPM mutant protein, accompanied by up-regulation of the activity of caspase-6and caspase-8in relatively high concentration, while oridonin, triptolide and betulinic acid did not alter the protein level of both NPM mutant and NPM wild type (NPM-wt). Deguelin induced differentiation of OCI-AML3in nontoxic concentration whereas no effects were found in OCIM2cells expressing NPM-wt, which was associated with activated caspase-8, p53, p21and30kD band of C/EBPa decreasing. Moreover, treatment with siRNA to NPM mutant decreased p53, p21, pro-caspase-8and30kD band of C/EBPa, as well as inhibited proliferation and induced differention of the OCI-AML3cells.Conclusion:Deguelin is a potent in vitro inhibitor of NPM1mutation protein which provides the molecular basis for its anti-leukemia activities in NPMlc+acute myeloid leukemia cells. Part â…¡. Oridonin relocates NPM1mutation protein and induces apoptosis in acute myeloid leukemia cells bearing NPM1mutationBackground:Oridonin is a natural product described to have potential as an inhibitor of carcinogenesis and inflammation in recently, and AML with NPM1mutations were recognized as a provisional entity in the new2008WHO classification. We investigated whether oridonin was able to induce apoptosis of NPM1c+cells and its ability to interfere with the NPMlc+protein traffic in vitro using OCI-AML3cell line.Methods:The growth inhibitory effect was detected by MTT assay; Cell apoptosis were evaluated by AV/PI using flowcytometry and Hoechst33258staining; Processing of caspases, cleavage of PARP, expression and subcellular localization of NPM-mut and NPM-wt were detected by western blot and Immunofluorescence staining; Nup98mRNA level was detected by RT-PCR; The expression and subcellular localization of Crm1and Nucleoporin98(Nup98) were evaluated by western blot and Immunofluorescence staining; Results:Oridonin induced apoptosis of oCI-AML3cells characterized by activation of caspase-3, cleavage of PARP and relocation of NPM-mut. Exposure of OCI-AML3cells to oridonin results in upregulation of pl4arf and p53. The amount of Nup98mRNA and protein was increased, accompanied by accumulation in nucleus after treatment with oridonin. In addition, without change the expression of Crml, oridonin dislocated Crm-1from cytoplasm into nucleus, suggesting that re-location of NPM-mut might cause by the accumulation of Crm-1in nucleus.Conclusion:Apart from an effect on translocation of NPM mutation back into the nucleus, oridonin significantly increase the expression of p53and pl4arf, which may contribute to the apoptosis of the leukemia cells. Oridonin may be an effective therapeutic agent in the treatment of NPM1c+leukemia cells.