Effect Of Homoharringtonine On Induction Of Apoptosis In T-cell Leukemia With Involvement Of Gene Regulation And Its Signaling Pathway

OmECTrvEHomoharringtonine (HHT) is a cephalotaxine alkaloid isolated from genusCephalotaxus. HHT was first introduced to clinic for treatment of leukemia inl970' in China and has been proven to have strong ami-leukemia activity It isused as an effective anti-leukemia drug clinica1ly in treatment of acute myeloidleukemia as well as chronic myeloid leukemia. In the last few decades, studieshave shown that the inhibitory effects of HHT on leukemia cell groWth includeinhibition of protein synthesis, decrease of clonogenicity and induction ofleukemia cell differentiation. As evidenced by 'H-Thymidine incopofationassnys, HHT also demonstrated the capacity to inhibit DNA synthesis inleukemia cells. Recent studies indicated that HHT is caPable of uP-regulating ofc-myc and c-fos, consequently promoting aPoptosis in myeloid leukemia cellssuch as K562 cells. Although the HHT's effect on inducing aPoptosis in cellline models such as HL-60, K562 and Jurkat is obvious, the detailed mechanismof HHT in regulation of aPoptosis in leukemia is not yet clear To date, little isknOwn aboul the role that HHT plays in regUlation of aPoptosis in lymphocyticleukemia. The study on the mechanisms of HHT in regulation of aPoptosis inleukemia cells will give us insigh into further understanding of the role HHTmay play in regulation of aPoptosis in leukemia cel1s and to underiine thesignaling pNay that HHT may interact with duing aPoPt()sis induced by thechemotheraPelltic drug. The data generated from this Stlldy will also providevaluable information for designing chemotheraPeutical regimens in clinicaltreatment of lymphocytic leukemia and to improve the clinical olltcome ofpatients receiving anii-leukemic chemotheraPyMATERIALS AND METHODS1 In this study, four T-cell leukemia cell 1ines Mo1t-3, Molt~l6,Ichikawa,Jurkat and one myeloid cell line HL-60 were used in the experimentsto detect the effects of HHT on induction of aPoptosis and to study the possiblemechanisms of HHT in regulation of aPoptosis of the cells. Wth the excePtionof Molt-3 and Moltl6 cells, which expressed a wild type of p53, the other twolines expressed mutant-type p53 in Ichikawa and Jurkat, null p53 expressed inHL-60. Cell cycle was analyzed by fluorescence-activated Flow Cytometry(FACS); DNA breakage resulting from HHT treatment was detected by TdTtransferase labeling. The morphologic observation using Giemsa staining wasmade on various cells treated by HHT.2 FACS analysis was also used fOr detection of FAS protein expressed oncell surface, for evaluation of aPoptosis in the cells after treatment with ani-Fasand anti-Fas ligand (Fas-L ) aniibodies, for detCction of mitochondrialmembrane potential (MMP ) and the generation of reactive oxygen species(ROS ) in the cells, as well as the effect of ami-oxidants on genefation of ROSin the cells tfCated with HHT Caspase-3 activity as well as the effects of theinhibitors of CasPase-3, 8, 9 on MMP and on aPoPtosis in the cells induced byHHT was also evaluated.3 Semi-quanitative RT-PCR was used to evaluate the mRNA expression ofBax and Bcl-2 in the cells treated with HHT4 By labelling the cells with Mito Tracker Red CM-H,XROS and stainingsubsequently with ani-cytochrome C and anti-Bax anibody the subcellularlocalization and re-distribution of Bax and cytochrome C in cells after treatmentWth HHT was analyzed by Confocal Laser Scan MicroscopyussUrrsl The results demonstfated that HHT was caPable of inducing aPoptosis in theT-cell leukemia cells in time- and dose-dependam manners. The statlls of p53has an effect on cell cycles of the cells as Gl arrest was observed in the two T-cell lines with wild-type p53 and Ichikawa cells with mutant p53 aftertreatmellt with HHT Twniy-four hours after tfCattheflt of Molt-3, Molt-l6,Ichikawa, Jurkat and HL-60 cells with HHT at different doses of 2ng/ml,10ng/ml, 25ng/ml and 50ng/ml, significan aPoptotic cells were detected in theMol...