| Leukemia is one of the most common malignant tumor in beings,which is a heterogeneous group of clonal origins of malignant disease. The incidence of leukemia is reported to be 3-4/100,000 per year. The main treatment method for leukemia is dependent on chemotherapy. With the development of chemotherapy plans and the apperence of new medicines, supportive care and stem cell transplantation to enhance the application of the treatment of leukemia has been significantly improved. However, the emergence of mulyidrug resistance is not become part of the leukemia patients in remission, or the main reason for relapse after remission. Therefore, the searching for new anticancer drugs, and exploring new treatment options is of great significant.In recent years, some of traditional Chinese medicine and its active ingredient reversal of multidrug resistance of leukemia caused people's attentions. Celastrol or Tripterygium, one of traditional Chinese medicines, is belonging to Wilfordil hook and celastrol is one of the monomers of tripterygium. Chemically, Celastrol belongs to triterpene constituents with molecular weight 450 and its molecular formula is C29H3gO4.It reported that tripterine inhibite immune; inhibition of vascular endothelial cells in vitro value;inhibition of HMC-1 cells adhesion and adhesion molecule expression; Celastrol also induced apoptosis of tumor such as non-small cell lung cancer line H1299,human acute myeloid leukemia cell line HL-60,malignant glioma lines and retinoblastoma.In this study, we use human chronic myelogenous leukemia blast crisis cell lines K562 and K562/A02 cell line resistance to adrimycin as the target cell to determine the reverse effect of Tripterine by CCK-8 and flow cytometry,and detected the concentration of intracellular ADM and P-gp expression by Tripterine.Materials and Method1,Culture of K562 and K562/A02K562 cells were cultured with 10% newborn calf serum RPMI-1640 medium, which include in 100U/mL,100μg/mL penicillin and streptomycin per liter.K562/A02 cells were cultured with the same medium, but this medium add adriamycin(ADM1μmol/L).The two kinds of cells were cultured at 37℃,5%CO2 incubator in noemal culture. Every 2 to 3 days passage 1 times. K562/A02 cells changed with adriamycin-free medium two weeks before experiment. Logarithmic growth phase cells were used for experiments.2,The sensitivity of cells to ADM or Celastrol and the reversal of drug resistance were determined with CCK-8method.Logarithmic growth phase cells were used for experiments. Living cell counting methed based on Trypan bluedye staining staining was used for tumor cell killing test. Take 20μL cell suspension with the examinated under microscope to count 100 cells for calculating living cell persatages. Trypan bluedye stained cells deal and non-stained cells means living. Take activity greater than 95% of cells for experimental cell. K562 or K562/A02 cells of 2～3×109cells per well were inoculated into 96- well. High to low concentrations of ADM were added(such as 160,80,40,20,10,5,2.5,1.25μmol/L).The same method with Celastrol (such as 320,160,80,40,20,10,5,2.5μmol/L).Each bore hole had three repetitive wells. The volume of every wells was 150μl. The cells cultured in 37℃,5%CO2 incubator after 72 hours, each well by adding 10μl CCK-8 reagent. Continued to train 2 hours. Zero with an empty hole, put the 96-well in automatic microplate reader at absorbance measured at 450nm,and the reference wavelength of 650nm. The measured data using statistical software to calculate SPSS16.0.The SPSS 16.0 software could calculate the growth inhibition rate of 50% of the drug concentration that is half inhibition(IC50).The inhibition was calculated as follows:Inhibition=(1-Experimental group's OD)/Control group's OD);Resistance index=IC50 of K562/A02/IC50 of K562.The experiment was repeated three times and averaged the datas.3,The drug resistance of Celastrol were determined with CCK-8.Logarithmic growth phase K562/A02 cells, which activity greater than 95%. K562/A02 cells of 2～3×109cells per well were inoculated into 96- well. The experiment groups divided into the following groups:①K562/A02;②K562/A02+ Celastrol;③K562/A02+VLP.The concentration of ADM and other drugs as discussed earlier. Each bore hole had three repetitive wells. The volume of every wells was 150μL. The VLP(5μg/L) as positive control. The cells cultured in 37℃,5%CO2 incubator after 72 hours, each well by adding 10μl CCK-8 reagent. Continued to train 2 hours. Zero with an empty hole, and the detection wavelength as discussed earlier. The experiment was repeated three times. The reversal fold was calculated as follows: Reversal fold= The IC50 of before reversal of K562/A02/The IC50 of after reversal of K562/A02.4,The intracellular of ADM concentration of K562/A02 and K562 by Flow cytometry.Logarithmic growth phase K562/A02 and K562 cells inoculated into 24- well, which activity greater than 95%. The experiment groups divided into four groups:①K562/A02;②K562/A02+ADM(IC50);②K562/A02+ Celastrol (IC50);④K562/A02+ADM(IC50)+Celastrol (IC50). Following 48 hours in incubator, collected cells,washed with ice-cold PBS (4℃,0.01mol/L, Ph7.4) twice. Resuspended in ice-cold PBS.The samples were stored at 4℃first detected by flow cytometry.(Ex=488nm,Em=575nm)5,P-gp expression detected by flow cytometry Logarithmic growth phase K562/A02 cells inoculated into 24- well, which activity greater than 95%. The experiment groups divided into the following groups:①K562/A02;②K562/A02+ADM(IC50);③K562/A02+ Celastrol (IC50)④K562/A02+ADM(IC50)+ Celastrol (IC50). Following 48 hours in incubator, collected cells,washed with ice-cold PBS (4℃,0.01mol/L,Ph7.4) twice. Added the P-gp antibody following with the instruction. Used flow cytometry to detect the P-gp expression after incubated at room temperature away from light 30 min.Result1,The results of K562 and K562/A02 cells inhibition by CelastrolThe IC50 of Celastrol to K562/A02 and K562 cells were 295.58±23.288μmol/L,411.59±26.5511μmol/L.The results of two cells has a significant difference (P<0.05). This result explain that Celastrol has non-drug resistance in K562/A02 cells. The IC50 of ADM to K562/A02 and K562 cells were 59.755±6.883μmol/L,0.749±0.741μmol/L. The resistance of K562/A02 cells to ADM was 79.78 times of K562 cells. It explained that ADM has evident drug resistance in K562/A02. The cytotoxic does of Celastrol could reduce the IC50 of ADM in K562/A02. This cytotoxic does could increase the ADM's sensitivity. After incubating with cytotoxic does of Celastrol, the IC50 of ADM was 0.507±0.070μmol/L,which decreased significantly than only used ADM (P<0.05), and the resistance was reversed by 117.860 times. And the IC50 of VLP(5μg/L)was 17.196±6.303μmol/L,which decreased significantly than only used ADM too(P<0.05).The resistance was reversed by 3.745 times.2,The results from concentration of intracellular ADM and P-gp expression were tested with flow cytometry (FCM)Tested with Celastrol, the concentration of intracellular ADM included obviously. After incubation of cytotoxic and non-cytotoxic does of Celastrol, the intracellular concentration of ADM were73.727±8.626,102.86±4.518.It increased 1.537 and 1.102 times in K562/A02 and K562 cells(P<0.05).When used ADM alone, the crest of P-gp expression has not obvious change. When used Celastrol alone, the crest of P-gp expression has obviously lower. And when Celastrol with ADM, the crest of P-gp expression lower obviously than ADM.Conclusion1. The IC50 of Tripterine to K562/A02 and K562 cells had a significant difference. The resistance of K562/A02 cells to ADM was 79.78 times of K562 cells.2. After incubating with cytotoxic does of Celastrol, the IC50 of ADM was obviously lower.3. After incubation of cytotoxic and non-cytotoxic does of Celastrol, the intracellular concentration of ADM increased obviously.4. Celastrol could down-regulate P-gp expression obviously. |
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