Effect Of Andrographolide On Apoptosis Induction And Proliferation Inhibition Of Human Leukemia Cell Lines U937 And HL60 Cells And Its Mechanism

Objective: Malignant tumors are the most serious diseases of mankind.There are about 7 million people die of cancer each year all over the world and over a million people die of cancer in China. Leukemias are malignant hematopoietic disorders. They are account for 5% of all malignant tumors. Although there are many methods for the treatment of leukemia, such as Bone Marrow Transplantation, Immunotherapy, Gene Therapy, chemotherapy is the most important regimen for treating leukemia. However, the most severe side-effect of chemotherapy is the suppression of normal tissues and cells even causing severe fatal complications. So we have to look for new effective therapeutic drugs without or with very slight side-effect. Recent studies showed the treatment of leukemia with chemical drugs and traditional Chinese herbal medicine had better effect than chemotherapy alone.Apoptosis, a form of programmed cell death, has attracted the focus of many researchers in recent years. Several studies showed that some natural Chinese herbal medicine could inhibit the growth of tumor cells and induce apoptosis of tumor cells. It is hopeful that Chinese herbal medicine could be used in clinical anti-tumor therapy.Andrographolide (Andro) is a chemical compond extracted and purified from Andrographis paniculata. Andro is the most effective component of Andrographis paniculata and has been used extensively as an antipyretic, anti-inflammatory and antivirus medicine for a long time. Recently, it's found that Andro has very important pharmacological activities other than what mentioned above, such as immune activity regulation effect, anti-tumor effect, and hepatoprotective effect.However, there is no report about whether Andro can inhibit the growth of leukemia cell lines of U937 and HL60 cells. Therefore, the present study is designed to explore the effects of Andro, at different concentrations, on proliferation inhibition and aopopotosis induction of in leukemia cells.Methods:1 Cell culture: HL60 cells and U937 cells were cultured in RPMI-1640 medium which was supplemented with 10% newborn bovine serum, at 37℃, 5% CO₂, and fully humidified atmosphere. Cells at logarithmic growth phase were treated with Andro (at different final concentrations, 10μmol/L, 20μmol/L, 40μmol/L, 80μmol/L) as different treatment groups or without Andro treatment as control group for 48 hours, then used for the following experiments.Cell proliferation assay: A cell counting kit-8 (CCK-8) was used to measure the growth inhibition effect of Andro. After the cells were treated with or without Andro for 48 hours, 10μL of CCK-8 solution were added to each group of cells, then the cells were incubated at 37℃for 2 hours. The absorbance of each sample was measured at 450 nm by using an enzyme-labeling measuring instrument. The inhibition rates were calculated based on the following formula: the inhibition rate (%) = (the absorbance value of control group- the absorbance value of experiment group)/ the absorbance value of control group×100% . The 50% inhibiting concentration (IC50) were also calculated from the result of this experiment.2 Hoechst33342 staining and Giemsa staining were performed to observe the index of cell apoptosis. The cell morphology changes of apoptosis were observed under an optical microscope and a fluorescence microscope.3 Flow cytometry (FCM) was used to measure cell apoptosis rate and cell cycle distributions after U937 and HL60 cells were treated with Andro (20,40,80μmol/L) for 48h.4 Expression of bcl-2 mRNA and NF-κB mRNA in U937 and HL60 cells were detected by semi-quantitative RT-PCR after these cells were treated with Andro (20, 40, 80μmol/L) for 48h.5 Enzyme activities of caspase3/7 were measured by enzyme-labeling measuring instrument and Caspase-Glo kit after U937 and HL60 cells were treated with Andro (20,40,80μmol/L) for 48h.Results:1 Andro (1¹00μmol/L) inhibited significantly the proliferation of U937 and HL60 cells in vitro (P<0.05), the growth inhibition effect was in a dose-dependent manner. The highest inhibition rate was 83.97%, observed in U937 cells treated with Andro at a final concentration of 100μmol/L for 48 hours. The highest inhibition rate of 71.64% was observed in HL60 cells treated with Andro at a final concentration of 100μmol/L for 48 hours.The IC50 of Andro for U937 was 21.86μmol/L and 46.41μmol/Lfor HL60 cells.2 After the treatment with Andro, U937 and HL60 cells showed some typical morphologic features and changes of apoptosis under an optical microscope, such as the reduced cell volume, the condensation of chromatin, the pyknosis and fragmentations of nuclear. The percentage of cells with bright blue fluorescence increased, under a fluorescence microscope, in Andro treated groups compared with that of control group.3 The results of FCM showed that Andro arrested U937 cells at G₀/G₁ phase, and the percentage of G₀/G₁ phase cells increased from 51.30% to 94.10% after the cells were treated with Andro. Andro also induced apoptosis of U937 cells. The apoptosis rate of cells treated with 80μmol/L andro for 48h increased to 52.48%. While Andro arrested HL60 cells at S phase, the percentage of HL60 cell increased from 39.48% to 67.93% after Andro treatment. However, only 17.75% of U937 cells underwent aopoptosis after the cells were treated with Andro.4 The results by semi-quantitative RT-PCR showed that bcl–2 mRNA and NF–κB mRNA in U937 and HL60 cells decreased significantly after these cells were treated with different concentrations of Andro for 48h, compared to that of control group (P<0.01).5 The enzyme activity of caspase3/7 increased significantly in U937 and HL60 cells, after the cells treated with Andro (P<0.05).Conclusion:1 Andro could significantly inhibit the proliferation of human Leukemia cell line cells of U937 and HL60 in vitro and in a dose -dependent manner; its growth inhibition effect on U937 and HL60 cells may be related to its inducing cell apoptosis and cell cycle arrest.2 The cell cycle of U937 and HL60 cells was arrested by Andro, maybe through downing-regulating the expressing of bcl-2 and NF-κB genes. .