Egcg Inducing The Apoptosis In Human Leukemia HL-60Cells Through AKT1Passageway

Objective: Leukemia is one of common malignant tumours. Leukemia patientsgained good curative effect because they were treated by chemotherapy. But lots ofchemotherapy drugs have serious side effects, which, because of part of patients giveup treatment. The aim of this study is to investigate mechanism and effect ofEpigallocatechin-3-gallate （EGCG） inducing the apoptosis of human leukemia HL-60cells and provid experimental information for development and search for new drugs.Methods: By MTT method we have detected50%of inhibiting concentration （IC50）by which EGCG inhibted HL-60cellular growth. The proliferation of HL-60cellinhibited by EGCG was observed through the cellular growth curve and soft agarformation. The cell apoptosis of EGCG affecting HL-60cells was checked byacridine orange （AO）, flow cytometry （FCM） and DNA gel electrophoresis. Theexpression of AKT1gene and protein was respectively analyzed by real timefluorescent quantitation RT-PCR and Wester blotting. Meantime, the phosphorylationlevel of AKT1protein and activated Caspase-3protein in HL-60cells treated byEGCG were detected by Western-blotting.Results:1. IC50that EGCG inhibted HL-60cells proliferation was58.70mg·L^-1.2.The results of the cellular growth curve showed to increase inhibitory effct of HL-60cellular growth with EGCG concentration increasing and treatment time extended（n=3, P＜0.05）.3. The results of the soft agar formation indicaded that the volume ofHL-60cellular colony was gradually small and the number of HL-60cellular colonywas decreased and HL-60cellular colony formation was inhibited by EGCG with itsconcentration increasing （n=3, P＜0.05）.4. The result of acridine orange （AO）showed that after EGCG treated HL-60cell48hours, the typical morphologycharacteristic of cell apoptosis exemple orange-red or red karyopyknosis, nuclear edge-collection and nuclear fragmentation occurred in HL-60cell. The number ofcellular apoptosis increasing with EGCG concentration increased （n=3, P＜0.05）.5.The result of flow cytometry （FCM） displayed that the apoptosis rate of HL-60cellgradually raises with EGCG concentration increasing and EGCG induced HL-60cellapoptosis for concentration depence on.6. The result of DNA gel electrophoresisshowed that DNA ladder strap was detect in HL-60cell treated by EGCG and it ismore obviously with concentration increased.7. The results of real time fluorescentquantitation RT-PCR found that the expression of AKT1gene in HL-60cellsobviously degraded with EGCG concentration increasing （P＜0.05）.8. Thewestern-blot results of AKT1protein exhibited that expression of in HL-60cellsobviously decreased with EGCG concentration increasing （P＜0.05） and EGCGinhibited expression of AKT1protein for concentration dependence on.9.Thephosphorylation level of AKT1protein in HL-60cells was depressed by EGCG anddepended on its concentration （P＜0.05）. Moreover, the activated Caspase-3proteinwas advanced by EGCG and depended on its concentration （P＜0.05）.Conclusion:EGCG induced the apoptosis of HL-60cells through inhibiting the expression ofAKT1gene and phosphorylation AKT1.