Inhibitory Effects Of Oleanolic Acid On Human Leukemic Cells And Its Mechanisms

Apoptosis is the most common mechanism by which the multicellular organisms eliminate damaged cells. It is essential to embryonic development and the maintenance of homeostasis. Apoptosis is activated through two major signaling pathways—the 'intrinsic' and the 'extrinsic' pathways. The extrinsic and intrinsic pathways converge via activation of intracellular enzymes called 'caspases'. Bcl-2 family and ROS involve in apoptosis and they are the important members in the signal transduction. Multiple pathways interplay sophisticatedly at the same time and form complicated net work to regulate this process when cell apoptosis happen. So it will be helpful to identify new proper candidate targets for anti-tumor drugs by studying the apoptosis signal transduction pathway. Many drugs can induce apoptosis via Bax/mitochondria/Caspase-9 pathway. But in the events of oncologic cell's apoptosis induced by OA (oleanolic acid), which mitochondria apoptotic pathways are involved, the relations of the bcl-2 gene family with other apoptosis related gene and the action of death receptor pathway are not known.The objective of this experiment is to study the effects of OA on the signal transduction pathways in HL-60 cell apoptosis and the human leukemia model by transplanting HL-60 cells into SCID mice. The concrete contents are as follows:1. The study on apoptosis of HL-60 cells induced by OA in vitroThe human acute leukemic cell line HL-60 cells were incubated in RPMI-1640 medium containing 10% fetal bovine serum. OA was dissolved in DMSO at a concentration of 80mmol/L as storage liquid. When storage liquid was used, it was diluted with RPMI-1640 medium containing 10% fetal bovine serum to the wanted concentrations.HL-60 cells were incubated with oleanolic acid at the final concentrations of 40μmol/L,60μmol/L,80μmol/L,100μmol/L for 12, 24, 48 or 72h, respectively. Blank group, control group (both containing 0.05%DMSO) and the positive group of As2O3 were set up. The results of MTT assay showed that OA inhibited the cells proliferation in a time-dependent and dose-dependent manner. All the groups treated with 80μmol/L OA showed statistics significance except the 48 h group and 72 h group. Compared these groups with the corresponding groups treated with 100μmol/L OA, there were no statistics significance except the 12 h groups(p<0.05). So we adopted the concentration of 80μmol/L and the duration of 48h in our successive experiments. After staining with DAPI, we examined the morphologic change of karyon of HL-60 cells that treated with OA with fluorescent microscope, we found that living cells have the even fluorescence, but apoptosis cells have the condensed nubby or granular fluorescence and apoptotic bodies. In order to ascertain that this phenomenon is apoptosis but not necrosis, we tested the genome DNA with gel electrophoresis. In electrophoresis analysis we found the typical ladder pattern due to multiple DNA fragments. We also detected the activity of apoptosis associated protein caspase-3, the results indicated the caspase-3 was activated after HL-60 cells were exposed 24h and the activity of caspase-3 was the highest at 48h. However, when the Caspase-3 inhibitor (z-DEVD-fmk) was used, we found the activity of Caspase-3 was the same as the control group. The flow cytometry (FCM) showed that a marked G1 arrest occurred when HL-60 cells were exposed to OA. All these results show that OA can induce the apoptosis of HL-60 by activating the caspase-3 and block HL-60 cells into G1 phase.2. The signal transduction pathways in HL-60 cells apoptosis induced by OA in vitroIn order to make sure via which pathway OA induces apoptosis, we first detected the mRNA of Fas, FasL and FADD by RT-PCR and the protein of Caspase-8 by western blot. These results indicate that OA induces HL-60 cells apoptosis via Fas signal transduction pathway.We tested the change of reactive oxygen species (ROS) with FCM, the expression of Caspase-9, Cyt-c, Smac/DIBLO and the volume of tBid in the mitochondria by western blot, the mRNA of bcl-2,bid, bax and bcl-xL which belong to the bcl-2 family by RT-PCR. The results show that the content of ROS ascends; Caspase-9 which the final executor in the mitochondria pathway is activated; the volume of Cyt-c and Smac/DIABLO increase in the cytoplasm; the content of tBid ascends in the mitochondria; the mRNA expression of bid, bax is up-regulated in a time-dependent manner, but the mRNA expression of bcl-2, bcl-xL is down-regulated. All the results show that OA can up-regulate the expression of bid, bax and down-regulate that of bcl-2, bcl-xL to induce HL-60 cells apoptosis; the mitochondria pathway possibly involves in the mechanism of the HL-60 cells apoptosis induced by OA.3. Effect of OA on human leukemia SCID mice modelWe made severe combined Immunodeficiency (SCID) mice our object of study to observe the effect of OA on the leukemic cells in the body. 4 to 6-week old SCID mice were inoculated 1×107 HL-60 cells via the abdominal cavity injection. Every week after inoculation the peripheral blood white cell count and the positive rates of promyelocytes on smears were examined. Histological assay confirmed that all mice developed myeloid leukemia after 28 days. 200mg/kg OA were injected into leukemia mice model via hypodermic injection for 14 days. Evaluated the normal physiological conditions of the mice, tested the percentage of leukemic cells in the blood and marrow to ascertain the curative effect of OA and the effect on the differentiation of leukemic cells. The results show that the drug group can apparently decrease the percentage of white cell in the peripheral blood, the positive rates of promyelocytes on blood smears, and the percentage of the immature cells on bone marrow smears(p<0.05).OA can prolong the survival time of the leukemia mice model, inhibit the leukemic liver and spleen infiltration. OA can down-regulate the proteins expression of Bcl-2, CyclinD1, CyclinA in liver and spleen, and the expression of CD11b is up-regulated in spleen. As a result the proliferation inhibition and differentiation enhancement of the leukemic cells occur.All these results indicate that OA can induce the apoptosis of HL-60 cells and block these cells into G1 phase. The Fas signal transduction pathway and the mitochondria pathway possibly involve in the mechanism of the HL-60 cells apoptosis induced by OA. Our experiments also indicate that OA have an effect on the human leukemia SCID mice model, inhibit proliferation of HL-60 cells and make HL-60 cells differentiate.