The Anti-leukemia Effect Of Atorvastatin And The Molecular Mechanism

Objective: The aim of our study is to investigate the anti-leukemia activity and molecular mechanism of Atorvastatin in human chronic myeloid leukemia(CML)cell line K562 and acute myeloid leukemia(AML)cell line HL60,thus providing the theoretical basis for expanding the novel application of AT for therapy of CML and AML.Methods: 1.MTT assay was used to determine the anti-proliferative activity of Atorvastatin on the growth of K562,HL60 and normal peripheral blood mononuclear cells(PBMCs).2.Flow cytometry was used to analyze the effect of Atorvastatin on the cell cycle,apoptosis,intracellular ROS level as well as mitochondrial membrane potential(MMP,Δψm)of K562 and HL60 cells.3.Immunofluorescence method was used to monitor the expression and localization of Yes-associated protein(YAP)after treatment with Atorvastatin alone or co-treatment with 20 μM Atorvastatin and 50 μM mevalonate acid(MVA)in K562 and HL60 cells.4.Western-blot was used to evaluate the effect of different concentration of Atorvastatin on cell cycle related protein phosphorylation and expression of cyclin D1,p27,p-p Rb,cyclin B1 and cdc2,the expression of apoptosis regulators of caspase-9,caspase-3,PARP,Bcl-2,Bax and cytochrome c(cyt c),as well as the expression of YAP(nuclear)and phosphorylation of p-YAP(cytosolic)of K562 and HL60 cells.5.Western-blot was used to detect the effect of Atorvastatin alone or co-treatment with Atorvastatin and MVA,Atorvastatin and Geranylgeranyl pyrophosphate(GGPP),Atorvastatin and Farnesyl pyrophosphate(FPP)on the expression of YAP(nuclear),as well as the phosphorylation of p-YAP(cytosolic)of K562 and HL60 cells.Results: 1.Growth inhibitory effect of Atorvastatin on human leukemia cells.MTT assay showed that Atorvastatin inhibited K562 and HL60 cell proliferation in a dose-dependent manner with the IC50 values of 10.55 μM and 10.26 μM,respectively.Also,Atorvastatin showed weak inhibition against normal cell PBMC with IC50 of 102.70 μM2.Atorvastatin affected cell cycle distribution of CML and AML.PI staining combined with flow cytometry and Western-blot showed that Atorvastatin induced cell cycle arrest in G2/M phase by down-regulating the expression levels of cyclin B1 and cdc2 in K562,as well arrested G0/G1 phase in HL60 through the down-regulation of cyclin D1 expression and p-p Rb phosphorylation,up-regulation of p27 expression level.3.Atorvastatin induced apoptosis in CML and AML,which might be mediated by ROS-involved mitochondrial apoptotic signaling pathway.Flow cytometry and Western-blot showed the apoptotic cell populations of K562 and HL60 were increased to 34.67% and 39.16% after treatment with Atorvastatin(20 μΜ).Also,with increase of Bax,decrease of Bcl-2,cyt c was released from mitochondria to cytoplasm,as well increased cleavage of caspase-9,caspase-3 and PARP.After treatment with different concentration of Atorvastatin,the amount of ROS increased and the relative MMP reduced dose-dependently.4.Atorvastatin inhibited YAP nuclear localization of CML and AML.Immunofluorescence showed that Atorvastatin caused the inhibitory effect of YAP nuclear localization.Western-blot showed that the expression of YAP(nuclear)down-regulated while the phosphorylation of p-YAP(cytosolic)up-regulated in a dose-dependent action after treatment with Atorvastatin of K562 and HL60 cells.5.The inhibitory effect of Atorvastatin on YAP nuclear localization was dependent on the MVA pathway.Immunofluorescence and Western-blot showed that re-exposure MVA and GGPP,but not to FPP,rescued the Atorvastatin-induced inhibition of YAP nuclear localization.Conclusions: 1.Atorvastatin inhibited K562 and HL60 cell proliferation activity,affected cell cycle distribution,as well induced apoptosis in vitro.2.The inhibitory effect of Atorvastatin on YAP nuclear localization was dependent on the MVA pathway,which may be mediated by inhibiting synthesis of intermediate metabolites.