The Isolation Of Several Antitumor Principals And Studies On Their Mechanisms Of Apoptosis Induction On HepG2 Cells

Today,traditional Chinese medicine has been widely used in biomedical science and clinics for its potential biological activities,such as used to treat diabetes, angiocardiopathy and cancer.It is one of the most important resources for new drug discovery and development.In this study,we isolated several anti-tumor principals from Chinese medecine Angelica dahurica and Suillus placidus.The effects of the isolated materials on antitumor were investigated.At first,bioassay-guided fractionation procedure was used to get the furanocoumarin,Imperatorin(IM),from the roots of Angelica dahurica.The anti-cancer effect of IM was examined in 9 hunman cancer cell lines by using MTT assay.Human hepatoma cells HepG2 were preferentially killed by IM with an IC50 of 60.5μM after 48h treatment.It was also found that IM was effective against HepG2 cells in animal study.The tumor volume was significant decreased by 31.93% and 63.18%after 50mg/kg and 100mg/kg IM treated 14 days.No significant weight loss and damage towards the liver and heart was found in IM-treated mice.Further study on the mechanism of its anti-tumor activity indicates that IM inhibits proliferation of HepG2 cells through apoptosis in a time and dose dependent manner, as shown by externalization of phosphatidyl serine,morphological changes,DNA fragmentation,loss of mitochondrial membrane potential,release of cytochrome c into cytosol and activation of caspase-3,8,9.Western blot analysis revealed significant increase in the protein levels of Fas death receptor,adaptor FADD,protein p21 and p53,proapoptotic protein tBid,Bax,Bad,and a decline of Bcl-2.These results suggest that the induction of apoptosis by IM is through both the death receptor and mitochondrial pathway.Taken together,IM might be an effective agent to treat liver cancer.Suillin,a bioactive component,was isolated from the ethyl acetate extract of the mushroom Suillus placidus.The anti-cancer effect of suillin was examined in 8 human cancer cell lines by using MTT assay.It is of interest to note that human liver cancer cells(HepG2 cells,Hep3B cells,Huh 7 cells) were preferentially killed by suillin with an IC₅₀ value of 2μM for 48h treatment.Machanistically,suillin induces apoptosis in HepG2 cells as characterized by DNA fragmentation,phosphatidyl-serine (PS) externalization,activation of caspase-3,-8,-9,depolarization of mitochondrial membrane potential,as well as release of cytochrome c into cytosol in a dosedependent manner.Western blot analysis revealed that the protein level of the pro-apoptotic protein Bad was significantly increased while Bid was steadily declined. The expressions of Fas death receptor and its adaptor FADD protein were also strongly increased with increase in the concentration of suillin.These results suggest that suillin activates apoptosis through both death receptor and mitochondrial pathways.Accordingly,suillin might have a potential to be an effective agent against liver cancer.