In Vitro Study Of Effect Of 2-methoxyestradiol In Human Leukemia Cell Lines CEM And Its Related Mechanism

【Objective】To study the effects of 2-methoxyestradiol (2-ME2) on proliferation inhibition and cell cycle and apoptosis induction in human T lymphocyte leukemia cell line CEM cells and on related mRNA and protein expressions.【Methods】CEM cells were treated with different concentractions. MTT assay was used to examine the effect of growth inhibition.DNA fragmentation assay and Annexin V-FITC/PI staining were used to detect the effect of apoptosis.The loss of mitochondrial transmembrane potential and the distribution of cell cycle were analyzed by flow cytometry.The expression of c-myc,bcl-2,p21,p53 and h-TERT mRNA were detected by RT-PCR; and the protein expressions of c-myc,bcl-2,cyto-c,pro-caspase-3,pro-caspase-9,PARP,p21,p53 and Akt signal pathway molecules Akt and p-Akt were detected by Western-blotting.【Results】(1)2-ME2 inhibited proliferation of CEM cells in a time- and dose-dependent manner and the concentration of 50% growth inhibition(IC50) was 2.0μmol/L.2-ME2 significantly induced apoptosis in CEM cells as measured by detection of DNA Ladder and staining with Annexin-V/PI; The FCM analysis showed that the loss of mitochondrial transmembrane potential and cell cycle arrest at G2/M phase;These showed that 2-ME2 induced apoptosis and cell cycle arrest in CEM cells a dose-dependent manner between certain range of concentrations of 2-ME2. 2-ME2 decreased the expression of h-TERT mRNA and c-myc,bcl-2,p53 mRNA and protein,increased the expression of p21 mRNA and protein,down-regulated proteins level of procaspase-3,procaspase-9,PARP(116Kda),p-Akt,up-regulated expression of cytoplasmic Cyto-c and PARP 85Kda apoptosis-related cleavage fragment protein ,while had no effect of total Akt protein in CEM cells after treated with 2.0μmol/L of 2-ME2 for 0,24,48 and 72h.【Conclusions】(1)2-ME2 could efficiently induce proliferation inhibition in time- and dose-dependent manners and apoptosis in a dose-dependent manner in CEM cells, the mechanisms may invovle in reduce the mitochondrial transmembrane potential, releasing Cyto-c to cytoplasmic,down-regulation of h-TERT,bcl-2,c-myc,pro-caspase-3 and pro-caspase-9 mRNA and/or protein expressions may involve.Akt signal pathway may be involved in apoptosis of CEM cells after treatment with 2-ME2.(2)2-ME2 also can induce the cell cycle arrest at G2/M phase in a dose-dependent manner,the mechanism may invovle in upregulate the expression of p21 mRNA and protein in a p53-independent manner in CEM cells,and then inhibit the growth of CEM cells.