Effects Of Celastrol On The Proliferation And Apoptosis Of Acute Myelogenous Leukemia Primary Cells

Background and objectiveAcute leukemia is one of the most common malignant tumors in human beings. The incidence of leukemia is reported to be 3～4/100,000 per year.Approximately 70%is AML,and it occurs mostly to adults.The main treatment method for AML is dependent on chemotherapy.With the development of chemotherapy plans and the appearance of new medicines,the prognosis of patients suffering from AML has being greatly improved.However,the multi-drug resistance and recurrence of leukemia remain to be serious and popular problem.So it is great importance to look for new anti-tumor drugs and establish the associated curative strategy.In the recent years,some traditional Chinese medicines have been confirmed to possess anti-tumor effects and this causes great attentions.Tripterygium,one of traditional Chinese medicines,is belonging to Wilfordil hook,and it possesses many pharmacological actions,such as anti-inflammation,immunological suppression,bacteriostasis and anti-tumor.Celastrol is one of the monomers extracted from the root of tripterygium.Chemically,Celastrol belongs to triterpene constituents with molecular weight 450 and its molecular formula is C₂₉H₃₈O₄.It is reported that Celastrol either inhibit immune or inflammatory responses vitro,it is found that celastrol show a function of growth inhibiting in many kinds of tumor cells.In overseas and domestic,the research of celastrol in treatment of leukemia is still in the stage of fundamental researches.And there are some reports about celastrol acting on some leukemia cell lines,but the researches about cells from AML patients in vitro are rare at home and abroad,So it remains to be explored further.In this study,we used cells from acute myelogenous leukemia patients as the target cells to determine the effect of Celastrol on growth depression and apoptosis in vitro,we attempted to explore the possibility that celastrol in treatment leukemia preliminary,and to provide the theory basis for celastrol in clinical application.Materials and methods1.Separate and culture of AML primary cellsBone marrow mononuclear cells were separated from bone marrow of untreated AML patients by Ficoll.The AML cells of 1×10⁶/ml were inoculated into suspension culture flask and then cultured at 37℃for 2～3 generation under the atmosphere of 5%CO₂.To experiment used the cells that cytoactive above 95% in exponential phase.2.Trypan bluedye staining methodLiving cell counting method based on Trypan bluedye staining was used. Brietly,40μl of each the cell suspensions was stained,and then detected under microscope to count 100 cells for calculating living cell percentages.Trypan bluedye stained cells were dead and non-stained cells means living.3.MTT assayThe AML primary cells grew at exponential phase of growth,whose viability were more than 95%were used in the MTT assay.The AML cells(1×10⁶/mL) were incubated in a 96-well plate and 180μl per well.There were 4 experiment groups and the final concentration were 1μmol/L,2μmol/L,5μmol/L,10μmol/L, respectively.Meanwhile the control groups without celastrol and the blank groups were set.experiments were repeated for four times.Add 20μl MTT was added to each wells after the cells were cultured for 3h,6h,12h and 24 h at the atmosphere of 37℃,5%CO2.centrifugated the cell suspensions,Discarded the supernatant.And then added 150μl DMSO to each well.Shaked lightly to solution the formazan.Optical density(OD) was detected by an ELISA at 490nm wavelength. Then the growth inhibitory rates were counted,and the optimal concentration and time was set from the curve which the abscissa was the concentrations and the ordinate was proliferation inhibited rates.4.Annexin V-FITC/PI法Flow cytometry using Annexin V-FITC/PI was performed to detect tumor cells apoptosis.Briefly,10μl Annexin V-FITC was added into 100μl AML primary cell suspensions which treated with 1μmol/L,2μmol/L,5μmol/L,10μmol/L Celastrol or treated with 2μmol/L celastrol for 3h,6h,12h,24h,and then incubated at 37℃for 10 min.The cells were washed with PBS and then resuspended.5μl of PI solution was added to stop the reaction,and flow cytometry was applied to detect the apoptotic cells as soon as possible,experiments were repeated for three times. Meanwhile the negative controls that without Annexin V-FITC and PI were set.5.PI单染法Flow cytometry using PI staining was performed to analyze quantitatively the Gl sub-peak(apoptotic peak) of AML primary cells.Treated the AML primary cells with 1μmol/L,2μmol/L,5μmol/L,10μmol/L celastrol for 6h,And then fixed the treated cells with 70%ethanl for about 24h.Washed by PBS for twice.Added RNAase at 37℃for 30min,and then added PI,mixed and stained for 15min at 4℃. Flow cytometry was performed to analyze quantitatively the apoptotic peak of AML primary cells.6.Statistical analysisAll the data were showed in the form of mean±standard deviation,The multi-samples mean values were compared with variance analysis by SPSS13.0 software.Takingα=0.05 as the significant standard of test.Results1.The proliferation inhibited rates of AML primary cells treated by Celastrol with different concentrations and different durations.After the AML primary cells were treated with different concentrations of celastrol,the results of MTT were analyzed,cells growth were significantly inhibited compared with blank control group(P＜0.01).There was no statistical difference among 2μmol/L,5μmol/L and 10μmol/L celastrol(P＞0.05) except at the concentration of 1μmol/L(P＜0.01).The optimal concentration was 2μmol/L of celastrol.After AML primary cells were treated with celastrol for 3h,6h,12h, 24h,the result showed that the difference among 6h,12h,24h groups had no statistical significance(P＞0.05).But compared with 3h group,the difference had statistical significance(P＜0.01),The optimal time was 6h.Conclusively,celastrol was capable of inhibiting the AML primary cells proliferation with optimal concentration at 2μmol/L and optimal time at 6h.2.correlation among the concentration and duration of Celastrol and the changes of apoptotic ratesThe detection results by flow cytometry indicated that the apoptosis was seen after treated with 1μmol/L,2μmol/L,5μmol/L,10μmol/L Celastrol for 6h, apoptotic rates were significantly higher with apoptotic rates at(34.03±17.07)%,(49.53±19.84)%,(50.56±15.80)%,(28.28±21.21)%,respectively.Compared with the control group at(4.95±1.85)%,the difference had statistical significance (P＜0.01).there was no statistically significant difference between 2μmol/L and 5μmol/L groups(P＞0.05).while the two groups were significantly higher than others(P＜0.05).The apoptotic rate decreased significantly at the concentration of 10μmol/L than the at the concentration of 5μmol/L(P＜0.05).After the cells were treated with 2μmol/L Celastrol for 3h,6h,12h,24h,the apoptotic rates were (19.67±0.16)%,(37.44±1.01)%,(40.10±1.42)%,(46.53±0.37)%,respectively, and there was statistical difference among apoptotic rates in different groups.3.The quantitative analysis of apoptotic peak by flow cytometryFlow cytometry was used to analyze quantitatively the G1 sub-peak(apoptotic peak) of treated-cells.The apoptotic peak was seen after treated with 1μmol/L,2μmol/L,5μmol/L,10μmol/L Celastrol for 6h,respectively.The apoptotic rates were(19.34±5.28)%,(39.84±9.99)%,(46.40±2.67)%,(32.32±8.42)%, respectively,and the difference was significant compared with the control(P＜0.01). The apoptotic rates had no significant difference between the 2μmol/L celastrol group and 5μmol/L,10μmol/L groups(P＞0.05),but the difference had statistical significance compared with 1μmol/L celastrol group(P＜0.05).And the apoptotic rate was lower obviously in 10μmol/L group than in 5μmol/L group(P＜0.05).Conclusions1.The proliferation of AML primary cells can be inhibited at the concentration of (1～10μmol/L) Celastrol,And the optimal concentration is 2μmol/L celastrol and the optimal time is 6h.2.When the concentrations of Celastrol is within 1～10μmol/L,apoptosis of the AML primary cells can be induced by celastrol.Prompting that inducing apoptosis may be revolved in the anti-leukemia mechanism of Celastrol.