The Effect Of Reactive Oxygen Species On Glaucocalyxin A-induced Apoptosis In Human Leukemia HL-60 Cells

Background and Objective: Rabdosia japonica(Burm. f.)Hara var. glaucocalyx (Maxim.) Hara is a herbal plant of the Labiatae family, and used as a folk medicine in China for the treatment of hepatitis, gastricism, mastitis and coughing. Recently, many literatures reported that Rabdosia plants generally have anti-tumor activity, and ent-kaurane diterpenoids are their active ingredients. However, few reports on the anti-tumor activity of R. japonica are presented. In this thesis, the anti-tumor effect of glaucocalyxin A (GLA) and glaucocalyxin C (GLC), two main diterpenes isolated from the whole plant of R. japonica, were evaluated in vitro. The mechanism of apoptosis and the effect of ROS on GLA-induced apoptosis were investigated. Nowadays, it has become the trend to explore the pharmacological effects of TCM by molecular biological methods to reiterate the underlying pharmacological mechanism and to find new anti-tumor drugs. In such a circumference, a deeper study on the molecular mechanism of GLA in anti-tumor effect is promising and valuable for TCM development.Methods: Firstly we compared the cytotoxicity of GLA and GLC as determined by MTT assay. Then we evaluated the binding energy between GLA/GLC and Bcl-2 by autodock technology. Morphological and biochemical changes of apoptosis were investigated using AO/EB double staining, DAPI staining, TUNEL assay, DNA ladder and Annexin V-FITC/PI double staining and the ratio of apoptotic cells was also measured by flow cytometry. Spectrophotometric method was used to detect the activities of caspase-3, -8 and -9. The mitochondrial membrane potential and ROS burst were measured by flow cytometry and fluorescence microscope. Oxygen consumption was measured using the Clark-type oxygen electrode, and the content of MDA and the activities of antioxidase were also detected. The subcellular localization of cytochrome c was determined by immunofluorescent labeling. Western blotting was used to detect the protein level of Bcl-2 and Bax. RT-PCR to detect the gene changes of Fas. Meanwhile, MTT assay, flow cytometry and fluorescence microscope were used to detect the effect of NAC on apoptosis induced by GLA.Results: GLA displayed potent cytotoxic activity against all 15 different types of tumor cells, but GLC have no cytotoxicity on all 15 cancer cell lines with our selective concentrations. Moreover, the autodock result showed that the binding energy and Ki between Bcl-2 and GLA were lower than Bcl-2 and GLC, this means that GLA bind Bcl-2 is easier than GLC. GLA induced apoptosis in HL-60 cells, and the ratio of apoptotic cells increased from 7.8% to 40.6% in a dose-dependent manner, but no obvious hypodiploid cells were detected in GLC treatment group. HL-60 cells treatment with GLA resulted in MMP reduction, ROS burst, cytochrome c release from mitochondrias, Bax/Bcl-2 expression increased, Fas transcription increased and caspase activation. NAC, a general free radical scavenger, was used to block ROS generation, then cell viability, MMP of HL-60 cells increased, and the ratio of apoptotic cells decreased.Conclusion: GLA, but not GLC, inhibited HL-60 cell proliferation and induced cell apoptosis. GLA induced apoptosis in human HL-60 cells through both mitochondrial pathway and death receptor pathway. GLA induced apoptosis in human HL-60 cells through a novel mechanism of action that involves ROS generation.