| Hepatoma is one of the most common malignant tumors, and at least 120 thousand people die from hepatoma each year in China. The current therapies are not good at curative effects on hepatoma, so research and development of anti-hepatoma drugs is an important mission for us. In recent years, the orientation in research of anti-tumor drugs has changed from single cytotoxic drugs to diversified drugs. Especially, more and more evidences show that the dysequilibrium among cell proliferation, differentiation, and apoptosis is related to generation and evolution of cancer, and many cancers are involved in apoptosis blocking. And thus, it will be a new tactics for curing hepatoma and other cancers, if we hold a profound research on apoptotic mechanism and seek effective and distinctive anti-tumor drugs initiating apoptosis.Since the first affiliated hospital of Harbin Medical University attempted to cure acute promyelocytic leukemia (APL) with arsenic compound (sodium arsenite) and achieved a good curative effect early in 1970s, researchers have paid an increasing attention to the effect of arsenic compounds on cancer, and made considerable, systematic, and worldwide studies. Moreover, it has also been reported that paeonol is effective against cancer in vivo and in vitro, induces apoptosis of tumor cells, and affects the express of apoptotic genes. Based on these advances, we synthesized the unreported azo compound, 2-methoxy-4-hydroxy-5-acetyl-azobenzene-4'-arsenic acid (R2), from paeonol by coupling reaction with arsanilic acid, and conducted experiments of growth inhibition, apoptosis analysis, and mechanism study of HepG2 cell line in order to find out a new organic arsenic drugs which could induce tumor cell's apoptosis in vivo and in vitro at a lower concentration while have little effect on normal cells.The main research methods, contents, and conclusions are as follow:1.MTT assay to observe the growth inhibition of human hepatoma cell line HepG2 by various concentration of R2 at different times showed that R2 caused the significant inhibition of HepG2 cell growth, and R2-mediated reduction of cell viability was dose- and time-dependent. It was most effective in the first 24 hours and at a higher concentration.2.We observed morphologically with inverted microscope, HE dyeing, transmission electron microscope, and fluorescence microscope, and found that R2 induced apoptosis of HepG2 cell at concentration of 5×10-7 and 10-6mol.L-1. 3.Further morphological and biochemical analysis were made by FCM coupled with fluorescent dyes. PI dyeing showed that cells in G0/G1 phase reduced obviously while cells in S phase increased visibly. Annexin-V/PI double dyeing showed apoptotic cells were much more than necrotic cells. Rhodamine 123 dyeing showed that mitochiondrial transmembrane potential of HepG2 treated with R2 declined, which was dose- and time-dependent.4. Agarose gel electrophoresis result showed that HepG2 treated with R2 for 24h produced a DNA ladder band.5.Proteins related to apoptosis were analyzed by immunohistochemistry dyeing, and the result showed that the express of Bcl-2 reduced,which was dose-dependent. The express of p53 reduced more remarkably than Bcl-2.6. Proteinases related to apoptosis of HepG2 were analyzed by colorimetry. The result showed three Caspase proteinases increased during HepG2 cell apoptosis induced by R2, which was dose- and time-dependent. Among them, Caspase-9 and Caspase-3 increased more remarkably than Caspase-8.7.LD50 obtained from acute toxicologic experiment was 580.12mg/kg. It implied R2 had less acute toxicity.
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