| Background:Leukemia is a malignant clonal disease of hematopoietic stem cell, along with the constant research of leukemia, now we can diagnosis and assessment leukemia from type of the morphology, cell chemistry, cellular immunology, cytogenetics, molecular biology, In recently, the clinical treatment of leukemia are still chemotherapy, chemotherapy has the advantages of killing leukemia cells of fast proliferation rapidly, reducing the malignant cells at short-term, achieving the purposes of rapid relief. However, chemotherapy is difficult to reach remission, relapse and related complications after chemotherapy appear to be resolved, because of the drug itself and the other reasons such as untolerance, chemotherapy drugs can not kill all the leukemia cells in vivo, the presence of residual leukemic cells are able to the reasons of remission failure or relapse; In addition, leukemia cells can loss sensitivity to cytotoxic drugs because of anti-drugs, leading to chemotherapy could not induce apoptosis of leukemia cells, which is another important reasons of relapse and chemotherapy failure. Chemotherapy drugs not only kill leukemia cells, but also kill large numbers of human normal cells, resulting in decreased immune function, and produce gastrointestinal side effects, liver and kidney dysfunction and other system complications, an increase of chemotherapy-related mortality, influenced quality of life of patients. Clinical using of All-trans retinoic acid have create a new method for the treatment of leukemia. Indirubin is the main treatment component of chronic myeloid leukemia, in addition to acute myeloid leukemia. Traditional Chinese medicine can help chemotherapy drugs killing leukemia cells and promote apoptosis, increase refractory and relapsed acute leukemia remission rate;The study showed that the role of traditional Chinese medicine is from one of the major alkaloids, purified alkaloid combined with chemotherapy drug to treat leukemia maybe a feasible way of remission and reversaling multidrug resistance. The method can reduce complications mortality, enhance long-term survival and quality of life.Evodiamine is dry and nearly ripe fruit of Rutaceae Evodia Evodia Evodia rutaecarpa (Juss.) Benth. Tigers E.rutaecarpa (Juss.) Benth.var.officinalis (Dode) Huang and sparse hair Evodia E.rutaecarpa (Juss.) Benth.var. bodinieri (Dode) Huang. Evodia contains a variety of chemical constituents, among of these constituents the separation of evodiamine have some biological action such as anti-inflammatory analgesic, antioxidant, weight reduction, relaxation of bronchial and so on. There are a variety of evodiamine anti-tumor effect, but the normal role of peripheral blood mononuclear cells are very less. In recent years, anti-tumor effect of Evodia founded are: lung cancer, colorectal cancer cells, thyroid cancer, melanoma, prostate cancer, cervical cancer, fiber tumor cells, breast cancer cells (including multi-drug resistant tumor), human acute myeloid leukemia cells U937 cells. Also founded that evodiamine can inhibit tumor cell migration, inhibit tumor cell metastasis.The choice of the Chinese medicine evodiamine because of the advantages of as monomer,against multiple tumor cells, less effect on normal peripheral blood cells, the large number of studies found that caspase-3, NF-KB, MAPK, AKT, AIF, Bcl-2, Bax and so on involved in evodiamine induced apoptosis of tumor cells, but the mechanism of apoptosis in malignants has not been fully elucidated, In this study, different origin leukemia cells were selected to explore apoptosis inducted by evodiamine, further investigate the apoptosis mechanisms of leukemia cells induced by evodiamine, provide experimental basis for clinical application.MethodsPart I:Evodiamine effect on leukemia cells inducing appotosis.1. CCK8 was used to detect 24,48,72 h growth inhibition effect of K562,Jurkat,Raji cells by different concentrations of evodiamine(2.5,5,10,20,40,80μmol/L);2. Inverted microscope was used to detect the clone formation rate changes of the three cells induced by evodiamin cultured by methylcellulose semisolid culture assay;3. Apoptosis morphological changes of cells by 15μmol/L evdiamine induced for 24h were observed through inverted microscope and Fluorescence microscope;4. Flow cytometry was used to detect apoptosis rate of three kinds of cells induced by different concentrations of evodiamine;5. Flow cytometry was used to detect intracellular reactive oxygen species (ROS) level of three kinds of cells induced by different concentrations of evodiamine;6. Flow cytometry was used to detect cell cycle changes of three kinds of cells induced by different concentrations of evodiamine.Part II:Evodiamine induces apoptosis of cells and explores its possible mechanism Western blotting was used to detect the protein levels changes of three kinds of cells induced by different concentrations of evodiamine,which was related protein in Mitochondrial apoptosis pathway: Procapase-3, cleaved-caspase-3, Procapase-9, cleaved-caspase-9, PARP, cleaved-PARP, Bax, Bcl-2. PCR are used to detect the mRNA level of Bcl-2, Bax of K562 cell induced by different concentrations of evodiamine.Statistical methods:The results of experimental data with mean±standard deviation (x±s) that significant for the 0.05 standard, using SPSS 13.0 statistical packages for statistical analysis. Group compared with one-way ANOVA (One-Way ANOVA); three cells differences analysis of multi-way classification ANOVA. inter-group comparison was significant using SNK method;the relationship between concentration and inhibition rate was using correlation analysis,the relationship between protein expression level and inhibition rate was also using correlation analysis.ResultsPart I:Evodiamine effect on leukemia cells inducing appotosis.1. The growth inhibition effect of different kinds of leukemia cells induced by evodiamineCCK-8 showed that 2.5,5,10,20,40,80μmol/L evodiamine reduced proliferation of three kinds of cells respectively, the inhibition rate induded by different concentrations of Evo on the K562,Jurkat,Raji cells was different (F=206.691, P<0.001; F=219.711, P<0.001; F=287.341, P<0.001), with the concentration increasing, inhibition rate of three kinds of cells increased Inhibition rate between the three time points include 24h,48h and 72h were different (P<0.05). At 24h,48h,72h, there was different between the inhibition rate of three kinds of cells (F=198.311, P<0.001),and SNK inter-group comparison method showed that inhibition rate of K562 was higher than that of Jurkat, inhibition rate of Jurkat cells was higher than Raji cells(P<0.05).Inhibition rate between the three time points include 24h,48h and 72h were different (F=164.902, P<0.001), and with the time increasing, inhibition rate increased (P<0.05).2.Evodiamine effect on the colony formation of three leukemia cells in vitro7,14 Days later, the inverted microscope was used to observe the cloning formation ratio after induced by Evodiamine using the concentration of 15umol/L for 24 hours. Cloning was formated at the seventh day, clonal growth is almost even into piece at the fourteenth day,2.5,5,10 umol/L of evodiamine, respectively affect on K562, Jurkat, Raji cells, no cloning.3.Evodiamine induced apoptosis of different kinds of leukemia cells(1)Morphological changes of cells after induced by evodiamineThe inverted microscope was used to observe the morphology changes of three cells before and after drug induced by 15umol/L,the cell bodies were round, translucent, uniform size, nucleus were round and located in the central of cell; after effect of drug, we can see more visible cellular debris in the flask, cell bodies survival became moreslender,and the tail was raised, can be seen the small apoptotic bodies near round of cell bodies, cell transmittance were poor.(2) Fluorescence microscopy15umol/L evodiamine role in three types of cells after 24 hours, fluorescence microscopy was used to observe cell morphology before and after drug inducing, compared with the control group, three types of cells, after 15umol/L evodiamine inducing, appeared the nuclear pyknosis, margination, and apoptotic bodies.(3) Apoptosis rate changes of the three cells after evodiamine inducedFlow cytometry was used to detect the apoptosis rates of the three cells induced by evodiamine, the results showed 5,10,25 umol/L concentrations of evodiamine were stimulated in K562,Jurkat,Raji cells, apoptosis was different.The apoptosis rate was also different with different concentration, with statistical significance (early apoptosis F=25.672, P< 0.001; late apoptosis F=10.611, P<0.001); single cells, early apoptosis and late apoptosis rate showed concentration-dependent (P<0.05); compared with apoptosis rate of three cells, SNK method showed that the early apoptosis rate displayd no significant difference (5umol/L, F=4.686, P= 0.059; 10 umol/L, F=1.393, P=0.318; 25 umol/L, F=1.373,P=0.323), the late apoptotic K562 cells was higher than the other cells, with statistical significance (P <0.05).(4) Evodiamine effects on cell cycleFlow cytometry was used to detect cell cycle, the results showed that G0/G1,G2/M arrest between three kinds of cells was no statistical significance (F=1.029;0.574, P=0.431;0.747).Three kinds of cells in the control group more than 50% of cells was in S phase, G0/G1 of the second, G2/M phase minimum; Evo of different concentration role in K562 cells,the ratio of G0/G1> G2/M phase cells was different (F=17.877, P<0.001; F=738.451, P<0.001), SNK method showed that there was no difference between control group and 5,10μmol/L Evo groups,significantly difference in 25μmol/L group (P<0.05); Evo of different concentration role in Jurkat cells,the ratio of G0/G1> G2/M phase cells was different (F=1893.323, P<0.001; F=32.902, P<0.001), SNK method showed that there was no difference between control group and 5μmol/L group,significantly difference in 10,25μmol/L groups (P<0.05); Evo of different concentration role in Raji cells, the ratio of G0/G1,G2/M phase cells was different (F=2065.452, P <0.001; F=8.149, P<0.001), SNK method showed that there was no difference between control group and 5,10μmol/L groups,significantly difference in 25μmol/L groups (P<0.05)(5) Evodiamine of intracellular reactive oxygen species (ROS) levelsThe results showed:the levels of ROS in three kinds of cells were different, ROS levels was different with the different concentrations (F=20.223, P<0.001). Compaired with control group of three kinds of cells, ROS levels were different (F=38.113, P<0.001),SNK inter-group comparison discoveried that ROS levels of Jurkat cells was higher than K562 cells in the control group, ROS levels of K562 cells was higher than Raji cells in the control group (P<0.05), but ROS levels of Jurkat cells after treatment was no significantly increased, intracellular ROS levels of K562 was more significantly increased than others; On 10 umol/L, ROS level appeared differences among three types of cells (F=34.722, P=0.001), ROS levels of K562 cells detected were higher than Jurkat and Raji cells at this time, and with the concentration increasing,ROS level increased (P<0.05),PartⅡ: Evodiamine induces apoptosis of cells and explores its possible mechanismApoptosis protein change levels induced by Evodiamine1. Caspase family(1) K562 cells were induced by evodiamine,with the concentrations of Evo increased, caspase-3 protein precursor expression gradually decreased (r=-0.834, P <0.001), activated caspase-3 expression increased (r=0.806, P<0.001); caspase-9 precursor protein expression was decreased (r=-0.623, P=0.006), expression levels of activated caspase-9 was increased (r=0.641, P=0.004).(2) Jurkat cells were induced by evodiamine,with the concentration of Evo increased,precursor caspase-3 protein expression was decreased(r=-0.738, P <0.001), activated caspase-3 was increased (r=0.822, P<0.001); caspase-9 precursor protein expression was decreased(r=-0.835, P<0.001), the activated caspase-9 expression was gradually increased (r=0.897, P<0.001).(3) Raji cells were induced by evodiamine,with the concentration of Evo increased,precursor caspase-3 protein expression was decreased (r=-0.913, P <0.001), activated caspase-3 was increased (r=0.767, P<0.001); caspase -9 precursor protein expression was decreased (r=-0.948, P=0.006),activate caspase-9, there was gradually increased (r=0.855, P<0.001).2. Bcl-2 family(1) Evodiamine affected on K562 cells,with the increase of drug concentration, the expression of pro-apoptotic protein Bax increased gradually (r=0.929, P< 0.001); anti-apoptotic protein Bcl-2 expression gradually decreased (r=-0.902, P< 0.001); substrate of activated PARP levels was gradually increased (r=0.719, P< 0.001).(2) Evodiamine affected on Jurkat cells, with the increase of drug concentration, the expression of pro-apoptotic protein Bax increased gradually (r=0.666, P= 0.003); anti-apoptotic protein Bcl-2 expression gradually decreased (r=-0.675, P= 0.002); substrate of activated PARP level was gradually increased (r=0.802,P<0.001).(3) Evodiamine affected on Raji cells, with the increase of drug concentration, the expression of pro-apoptotic protein Bax increased gradually(r=0.932,P<0.001); anti-apoptotic protein expression of Bcl-2 gradually decreased (r=-0.924, P<0.001); substrate of activated PARP levels was gradually increased, (r=0.809, P<0.001).Conclusion1. Evodiamine inhibited the proliferation of different kinds of leukemia cells with time - dose dependent manner. Roling on K562 cells was stronger than the other two cells.2. There was cloning forming of control group,which was no cloning forming in drug group.3. The cells of control group, the cell bodies were round, translucent, uniform size, nucleus were round and located in the central of cell; after 15umol/L evodiamine inducing, appeared the nuclear pyknosis, margination, and apoptotic bodies by Hochest 33342 staining.4. Three kinds of leukemia cells can be induced apoptosis by Evo, apoptosis rate of K562 cell was higher than others.5.Evodiamine can block the cell cycle of leukemia cells in G2/M phase,there was no significant different between three kinds of cells.6.Evodiamine can impact intracellular reactive oxygen species (ROS) levels of leukemia cells,the ROS level of K562 cell was higher than others.7.Evodiamine increased caspase-3, caspase-9, Bax protein expression levels of K562,Jurkat and Raji cells, decreased Bcl-2 protein expression. |
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