An Experimental Study On Apoptosis Of Leukemic Cells Induced By Brucea Javanica Oil Emulsion And Its Regulation Of Apoptosis-Related Genes

Objective: Apoptosis or programmed cell death(PCD) is a process of active cell death controlled by genes, by which the organism maintains self-stabilization and regulates the equilibrium between proliferation and death of body cells. It has specific morphologic & biochemical features and no evident inflammation. The prevention of apoptosis is an important characteristic of malignant tumors. To induce tumor cell apoptosis with drugs has become an important way in tumor therapy. In recent years, it has always been a hopeful way in the treatment of tumors by inducing apoptosis of tumor cells with Chinese traditional medicine. So study on apoptosis of tumor cells induced by Chinese traditional medicine would be a hot topic in oncology in the following years. Brucea javanica oil emulsion is oil-in-water emulsion made from extractions from the fruit of Brucea javanica (L)Merr, and with refined soybean phospholipid as emulsifying age. In recent years, experiments in vivo and in vitro have proved the suppressive effect of brucea javanica oil emulsion to solid tumor cells. It has aroused great attentions that brucea javanica oil emulsion has remarkable antitumor effects in clinical treatment for carcinomas of esophagus and rectum. But no research had been reported about brucea javanica oil emulsion inducing apoptosis of leukemic cell lines, K562 and U937, as well as apoptosis-related genes at home and abroad. Inorder to explore the effect and mechanism of brucea javanica oil emulsion to leukemic cells in vitro, we systematically observed the apoptotic process of leukemic cells induced by brucea javanica oil emulsion with morphologic and molecular biologic methods.Materials and Methods: A human chronic myelogenous leukemia (CML) cell line ( K562 cell) and a human acute monocytic leukemia cell line(U937 cell) were selected. MTT assay was used to test the proliferation of K562 and U937 cells pretreated by brucea javanica oil emulsion. Morphological changes of K562 cells and U937 cells pretreated by brucea javanica oil emulsion were observed by light microscope and fluorescence microscope. Ultrastructural changes were observed by transmission electron microscope (TEM). The apoptotic changes were verified by morphology, DNA fragmentation electrophoresis and flow cytometry (FCM). FCM and RT-PCR were used to detect the expressions of Bcl-2 and c-myc genes in U937 cells pretreated by brucea javanica oil emulsion . Furthermore, Flow cytometry analysis was used to test the expression changes of Fas/FasL and c-fos genes in K562 cell pretreated by brucea javanica oil emulsion. The activity of caspase-3 was detected by colorimetric assay. The intracellular free calcium was detected by laser scanning confocal microscopy during the apoptosis process in U937 cells. Results:1. After treatments with different doses of brucea javanica oil emulsion for 48hs, the proliferation of K562 cells was inhibited, and showed a dose-dependent relationship. The value of IC50 was 187 U g/ml. After 24hs, 48hs and 72hs treatments with 500 u g/ml brucea javanica oil emulsion, the proliferation of K562 cells was inhibited as a time-dependent relationship. The proliferation of U937 cells was significantly inhibited by brucea javanica oil emulsion and showed a dose & time-dependent relationship. The IC50 value was 214 u g/ml;2. Typical morphological changes of apoptotic cells were observed in K562 and U937 cells treated by brucea javanica oil emulsion. By Wright-Giemsa staining, they showed morphological features such as chromatin compaction, condensation of cytoplasm,nuclear fragmentation and formation of apoptotic bodies. After fluorescence staining, compact fluorescent granules could be observed in nucleus or cytoplasm. K562 and U937 cells treated with brucea javanica oil emulsion ,The ultrastructural features of apoptotic cells characterized by cell volume reduction,cytoplasm shrinkage, condensation, fragmentation of chromatin,and presence of apoptotic bodies under electron microscope.3. Apoptotic apex was observed in K562 and U937 cells by FCM after incubation with brucea javanica oil emulsion. The apoptotic ratios of K562 cells pretreated with 500 u g/ml brucea javanica oil emulsion forl2hs, 24hs, 48hs, 72hs were 6.15%, 7.36%, 9.11%, 20.13%, respectively, showed significant differences comparing with the control cells(P<0.01). The results showed that brucea javanica oil emulsion induced apoptosis of K562 cells in a time-dependent relationship. The apoptotic ratios of K562 cells pretreated with 125, 250, 500 U g/ml brucea javanica oil emulsion for 72hs were 1.41%, 7.57%, 9.11%, respectively, showed significant differences comparing with the control cells(P<0.01). The results showed that brucea javanica oil emulsion induced apoptosis of K562 cells in a dose-dependent relationship. Treated with 125, 250 and 500 u g/ml brucea javanica oil emulsion for 24hs, 48hs and 72hs, the apoptotic ratios of U937 cells detected by FCM showed significant differences comparing respectively with the control cells (?<0.01).The percentage of sub-Gi cells in U937 cells was increased in a time-and concentration-dependent manner.4. Pretreated with brucea javanica oil emulsion in 500 u g/ml for 72hs, typical DNA ladder were observed.5. 500 u g/ml brucea javanica oil emulsion treated with U937 cells ,the ratioof U937 cells on G0/Gi phase were increased ,which indicated that transition from G0/Gi phase to S phase was blocked.6.The positive ratio of Fas expression in K562 cells pretreated by 500 u g/ml brucea javanica oil emulsion for 48hs was higher than that in the control (P<0.0l). Both FasL and c-fos oncoprotein expression levels decreased after treatment with brucea javanica oil emulsion, but no significant difference in FasL expression level showed (P>0.05).7. Both expression levels of Bcl-2 and c-myc decreased and showed significant differences comparing with the control (P<0.01) .8. The Caspase-3 activity was significantly higher in the apoptotic cells than in the control one in U937 cells.9. In U937 cells pretreated with brucea javanica oil emulsion for 30 minutes, the concentration of intracellular free calcium increased greatly. Conclusions: Brucea javanica oil emulsion could inhibit the proliferation of K562 and U937 cells in vitro in time and dose-dependent manners. 500 u g/ml brucea javanica oil emulsion arrested U937 cells at Go-Gi phase.Brucea javanica oil emulsion induced apoptosis of K562 and U937 cells in vitro, which might be one of its anticancer mechanisms. Brucea javanica oil emulsion could up-regulate the expression of Fas gene and down-regulate the expression of c-fos gene in the apoptotic process of K562 cells, which might be one of the mechanisms of brucea javanica oil emulsion inducing apoptosis. Down-regulation the expression of Bcl-2 and c-myc genes might be one of mechanisms of inducing U937 cells apoptosis. Caspase-3 activation participated in the apoptotic process of U937 cells. Intracellular free calcium was an important signal molecule in signal transduction of apoptosis in U937 cells.Significances: In this study, distinct growth suppressions of leukemic K562 and U937 cells pretreated with brucea javanica oil emulsion was observed by MTT method. We not only demonstrated in morphology and biochemistry that...