Influence Of Liushenwan On Inducing Apoptosis In Leukemia Cells And Apoptosis-related Genes

PURPOSE:Leukemia is a primary disease of hematopoietic tissues with pathologic characters of wide abnormal hyerplasia of leukemia cell in hematopoietic tissues such as bone marrow and infiltration to other organs. Apoptosis is a phyisoprocess adjusted by many factors to keep stabilzation of body which plays a important role in keeping normal hematopoiesis. Once apoptosis is inhibited, excessive, damaged and cancerated cells can not be cleared so tumor will occur. Apoptosis is one of the mechanisms related to the happeniss of leukemia and inducing cancerated leukemia cells to apoptosis is a important way to treat leukemia.After many years' clinical practice, it turns out that Liushenwan is effective in treating leukemia. Our study will observe the action of Liushenwan in inducing apoptosis in leukemia cells and expression of genes correlated to apoptosis by cell culture in vitro and the methods of cells form inspecting , TUNEL, DNA electrophoresis and flow cytometer thus argue its mechanism of treating leukemia. METHODS:1 Cells culture: using RPM1-1640 culture medium and routine methods to culture NB4 and HL-60 cells in vitro.2 Drug extraction: extracting the utility components into achromatic liquid through reflux distillation.3 Observing the inhibition on cells proliferation by MTT reduction assay.4 Apoptosis detecting:4.1 Morpholog: observe cells smears stained with Giemsa staining with light microscope.4.2 DNA electrophoresis: extract DNA with chlotoform/isoarmyl alcohol and do agarose gel electrophoresis.4.3 Terminal-deoxynucleoitidyl transferase mediated nick end labeling.4.4 Flow cytometer: analyse cell cycle and detect apoptosis cells ratro byPI/Annexin V-FITC.5 Influence on expression of genes correlated to apoptosis: test the expression of mRNA of genes bcl-2, c-myc and p53 by methods of RT-PCR. RESULTS:1 Liushenwan inhibit cells proliferation with a obvious correlation with action time and the inhibitive ratio gets to more than 60% after 48h with common dose-effect correlation.2 Apoptosis characters were seen after induced by Liushenwan . DNA electrophoresis showed typical step strap.Liushenwan showed obvious effect to induce apoptosis tested by methed of TUNEL. The apoptosis ratio related with action time and drug concentration. The highest apoptosis ratio was seen in cells induced by Liushenwan of 100ug/ml after 32h which is 64.3% (NB4 cell) and 68.6% (HL-60 cell) .The results tested by flow cytometer revealed that the cells induced by Liushenwan of 50ug/ml after 32h showed the highest apoptosis ratio which is 37.7%(NB4 cell)and 23.2%(HL-60 cell)and the apoptosis ratio decended after induced by Liushenwan of lOOug/ml.3 Liushenwan rised the level of bcl-2 mRNA expression while up regulate the mRNA expression of c-myc and p53 in NB4 cell.CONCLUSION:Extrinsic experiments show that Liushenwan can inhibit proliferation of acute myeloblastic leukemia cell, HL-60 and acute promyelocytic leukemia cell, NB4 with a high inhibition ratio and time dependency. Liushenwan can induced apoptosis and the apoptosis characters of cells form and biochemistry were seen. mRNA level of gene bcl-2 which inhibit apoptosis descended, while mRNA level of p53 and c-myc increased which was likely to be one of the mechanisms of the effect of Liushenwan to induced apoptosis.