Effect And Possible Mechanism Of Tripterine On Inducing Apoptosis Of Human Acute Myelocytic Leukemia HL-60 Cells

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. The main treatment method for leukemia is dependent on chemotherapy that induces apoptosis of the tumor cells. With the development of chemotherapy plans and the apperence of new medicines, the prognosis of patients suufering from leukemia has being greatly improved. However, the multidrug resistance and recurrence of leukemia remain to be serous and popular problems. For example, many kinds of tumor cells show the resistance to antitumor drugs such as VCR, MMC and DAM. So it is great importance to look for new antitumor drugs and establish the associated cuarative strategy.In the recent years, some of traditional Chinese medicines have been confirmed to possess antitumor effects and this causes great attentions. Tripterygium, one of traditional Chinese medicines, is belonging to Wilfordil hook and Tripterine is one of the monomers of tripterygium. Chemically, Tripterine belongs to triterpene constituents with molecular weight 450 and its molecular formula is C29H38O4. It is reported that tripterine either inhibite immune and inflammatory responses or show a function of antitumor in vitro. But so far the mechanism of antitumor has not been exactly clear yet.In this study, we use human acute myelocytic leukemia HL-60 cells as the target cell to determine the effect of Tripterine on inducing apoptosis of the tumor cells and the associated quatuative and duration patterns by using flow cytometry and transmission electron microscopy and so on. Furthermore, for understanding to the possible inducing mutor cell poptic mechanisms of Tripterine, the effects of the monomer on the expression levels of Fas, FasL and NF-KBP65 in the HL-60 cells before or after treatment with TripterineMATERIALS AND METHODS1. Culture and treatment of HL-60 cellsHL-60 cells of 1×10⁶/ml per well were inoculated into 24 well plates and then pre-cultured at 37℃ for 24 h under the atmosphere of 5%CO₂. In the tested group the final concentrations of 0.5, 1.0, 1.5 and 2.0 μmol/L Tripterine were given respectively while another group with no tripterine was used as control. All the HL-60 cells with or without treatment of Tripterine were cultured for 24 h under the sam conditions as above and then the cells were collected. On the other hand, HL-60 cells teated with 1.5 μmol/L Tripterine was cultured for 0, 4, 8, 12, 16 and 24 h, respectively, and then the cells was collected. All the collected cells were washed with PBS twice and then resuspened. The concentration of the cell suspensions was regulated to 1×10⁶/ml.2. Tumor cell killing testLiving cell counting method based on Trypan bluedye staining was used for tumor cell killing test. Briefly, 30 μl of each the cell suspensions which treated with different dosages of Tripterine or non-treated with the monomer was stained and then examinated under microscope to count 100 cells for calculating living cell persatages. Trypan bluedye stained cells were dead and non-stained cells means living.3. Examination of tumor cell apoptosisFlow cytometry using FITC-annexin V/PI was performed to detect tumor cell apoptosis. Briefly, 10 μl FITC-annexin V was added in 100 μl of each the cellsuspensions which treated with different dosages of Tripterine or non-treated with the monomer and then incubated at 37℃ for 10 min. The cells were washed with PBS and then resuspended. 50 μl of PI solution was added to stop the reaction and flow cytometry was applied to examinate the apoptic cells.4. Observation with transmission electron microscopyThe cell cultures treated with 1.5 μmol/L Tripterine for 0 or 24 h were centrifuged and then the supernatants were discarded. The precipitates were washed twice with pre-cold 0.01 mol/L PBS (pH 7.4). The cells were colleceted and then fixed with phosphate buffer containing 2.5% glutaraldehyde. The ultrasections of fixed cells were prepared and then the morphological alterations of the cells were observed by transmission electron microscopy.5. Detection of Fas and FasL100μl of each the cell suspensions respectively treated with 1.5 μmol/L Tripterine for 0, 12 and 24 h was added with 10 μl of FITC-labelled monoclonal antibody against Fas or FITC-labelled monoclonal antibody against FasL. After incubation at 37℃ for 30 min, the percentages of Fas positive rate or FasL positive rate was decided by flow cytometry. In these tests, FITC-labelled monoclonal antibody IgG1 was used as the negative control.6. Detection of NF-κBP65 activity100μl of each the cell suspensions respectively treated with 1.5 μmol/h Tripterine for 0, 12 and 24 h was added with 100 μl of FITC-labelled rabbit-anti-human monoclonal antibody against NF-kBP65 and then incubated at room temperature for 30 min. The cells were washed twice with .PBS and then added with FITC-labelled mouse-anti-rabbit monoclonal antibody. The cells were incubated under the protection from light at room temperature for 30 min and then re-washed twince. Flow cytometry was performed to quantitatively measure NF-κBP65 positve percentages in the cells after fixed with 1% para-aldehyde.RESULTS 1. Correlations among the concentration and duration of Tripterine andthe changes of apoptic ratesThe results of Trypan bluedye staining showed that the living cell percentages were 93.1±1.4, 92.1±1.1, 86.4±1.4, 84.1±1.7 and 80.2±1.5 after treated with 0.5, 1.0, 1.5, 2.0 and 2.5 μmol/L of Tripterines, respectively, while the living cell percentage without Tripterine treatment was 98.3±0.6. The detection results by flow cytometry indicated that the apoptic rates were 6.13±0.83,14.31±1.35, 36.71±2.28, 31.85±2.06 and 20.17±2.19, after treated with 0.5, 1.0, 1.5, 2.0 and 2.5 μmol/L of Tripterines, respectively. When compared these rates to the apoptic rate from the control, there was a statistically significant differences between them (P<0.05). Among the results, the poptic rates were risen in pace with the concentrations of 0.5～1.5 μmol/L Tripterine. When Tripterine concentration was as high or more than 2.0 μmol/L, the poptic rate was decreased in pace with the increase of dosages. After the cells were treated with 1.5 μmol/h tripterine for 4, 8, 12, 16 or 24 h, the apoptic rates were 2.67±0.26, 7.44±0.66, 12.68±1.15, 22.10±0.95 and 38.53±1.83, respectively, which presented statistically significant differences compared to apoptic rate1.92±0.26 from the control (P<0.05).2. Morphological features of the poptic cellsThe normal HL-60 cells had similar round or oval shapes. After being treated by 1.5 μmol/L Tripterine for 16 h and 24 h, respectively, the cells displayed the typical morphological changes of apoptosis such as cellular atrophy, many vacuoles in plasmid, chromosome rim aggregation and crescent nucleus. Part of the cells appearred apoptic bodies in their nucleuses and this pathogic change become more obvious in pace with duration time.3. Percentages of Fas, FasL and NF-κBP65 positive cellsAfter the cells treated with 1.5 μmol/L Tripterine for 12 and 24 h, respectively, the expressions of both the Fas and FasL were remarkably increased. The Fas-positive cell percentages were 47.91±1.21 and 65.74±1.20, respectively, while the positive percentage in the control was 14.23±1.42. There was a statistically significant difference between the positive percentages from the tested and control groups (P<0.05). The FasL-positive cell percentages were 37.92±1.73 and 43.50±2.11, respectively, while a percentage of 15.61±1.10 for the control. Asignificant difference between the positive percentages from the tested and control groups (P<0.05) could also be found. After the cells were treated with 1.5 μmol/L Tripterine for 24 h, the NF-κBP65 positive percentage was 4.78±0.30, which obviously lower than that from the control (8.34±1.52) (P<0.05).CONCLUSION1. When the concentrations of Tripterine were between 0.5 μmol/L to 1.5 μmol/L, apoptosis of the HL-60 cells could be happened with a dosage-dependent pattern. And 1.5 μmol/L Tripterine could cause the highest poptic rate. When the concentration of Tripterine is equal to or higher than 2.0 μmol/L, the apoptic rates were decreased, suggesting the exsistance of a possible threshold value of effective concentration. When the concentration of Tripterine was 1.5 μmol/L, the apoptic rates of HL-60 cells showed a duration time-dependent pattern.2. The HL-60 cells without Tripterine treatment presented low expressions of Fas and FasL. When the cells treated with Tripterine, the positive rate of the cells expressing Fas and FasL remarkably increased, indicating that the inducing the expression Fas and FasL in the target cells and then initiating the associated intracellular signaling pathways may be revolved in the anti-tumor mechanism of Tripterine.3. The down regulation of the NF-κBP65 expression levels in the HL-60 cells that treated with 1.5 μmol/L Tripterine for 24 h implied another possible anti-tumor mechanism of Tripterine by inhibiting NF-κB signaling pathway.