Isolation Of Anticancer Compounds From Toad Skin And The Molecular Mechanism Study

Part I Isolation of Anticancer Compounds from Toad Skin and the Molecular Mechanism StudyCancer has long been the number one killer in many parts of the world. Conventional cancer therapies such as surgery, chemotherapy and radiotherapy have a number of limitations such as relatively high cost, development of resistance and are often accompanied with many adverse side effects. Therefore, there is a pressing need to search for new drugs that are safe and effective for cancer treatments despite significant progress in oncology therapeutics in the last decades. Traditional Chinese medicines are sometimes used as an adjunct to radiotherapy or chemotherapy for cancer. Toad has been found to be a source of agents with anticancer properties for thousands of years in Chinese medicine. Actually, several compounds, such as bufalin and cinobufagin, had been successfully isolated from toad skin and always used in treating cardiovascular diseases. Clinical and laboratorial materials also showed that some of them had the effects of anticancer, but up to now, the exact constituents of toad skin that responsible for this inhibition of tumor have not been identified and its anticancer mechanism has not been fully understood.In the present study, we fractionated spray-dried toad skin and used SMMC-7721 (human hepatoma cell line) and A549 (human lung adenocarcinoma epithelial cell line) to assess the inhibitory effects of the fractions. The proliferation of cells was detected by CCK-8 assay. The cell cycle distribution was analized by Flow Cytometry.The spray-dried toad skin was fractionated by process-scale chromatography to give 4 fractions,Ⅰ,Ⅱ,ⅢandⅣ.CCK-8 assay demonstrated fractionⅣposses anti-tumor activity, while fractionsⅠ,ⅡandⅢnot. FractionⅣwere then fractionated by HPLC and 11 subfractions were yielded. Further cell proliferation assay showed that HCS411 has inhibitory effect on cancer cell growth and HCS408 has marginal effect, while other subfractions have not inhibitory effect. Further study demonstrated HCS411 significantly inhibited SMMC-7721 cell growth in a dose- and time-dependent manner. And we also found HCS411 triggered a notable G2/M cell cycle arrest in SMMC-7721 cells indicating that the growth inhibition of endothelial cells by HCS411 can be induced by the mitosis inhibition through the arresting at the G2/M phase of the cell cycle. Given the important role of p53 in cell cycle arrest, our result showed P53 was not involved in G2/M arrest in SMMC-7721 cells exposed to HCS411. Further HPLC analysis yielded 60 subfractions from HCS411, and 7 of which were verified to be effective to inhibit cancer cell growth. More importantly, we found that the subfraction 2-SX048429 was extremely potent to inhibit the xenograft tumor growth in vivo.A strength of this study was our approach to active constituent identification, which was based on an unbiased, bioassaydirected fractionation of spray-dried toad skin, and yielded consistent and convincing results using relatively small amounts of material. The results of this study demonstrate that some subfractions in spray-dried toad skin may contain bioactive compounds with potential chemopreventive activity. These results lay a solid foundation for the generation of antitumor active microcompound from toad skin and are meanful to the development of new antitumor drug. Part II Effects of Nickel on Cyclin Expression, Cell Cycle Progression and Cell Proliferation in Human Pulmonary CellsThe exposure to nickel has largely increased in industrial societies due to the environmental pollution by heavy metals at all stages of production, use, and disposal. Frequent exposure to high-concentrated nickel compounds has been considered as one of the potential causes of respiratory cancers. Previous studies have demonstrated that the cell signaling activation leading to cell cycle redistribution plays a critical role in carcinogenesis. In current study, notable increase of G1/S cell cycle transition was observed in human bronchial epithelial cells with 12 hours nickel exposure. The inductions of G1/S phase related cyclin D1 and cyclin E by nickel, which were demonstrated for the first time, may be responsible for the nickel induced G1/S transition. In addition, we verified that hypoxia-inducible factor-1alpha (HIF-1α), as an important transcription factor of nickel response, was not required for the cyclin D1 induction. On the other hand, Beas-2B cells treated with nickel compounds for 24 and 48 hours present obvious G2/M cell cycle arrest as well as significant cell proliferation inhibition. Given that the role of p53 in G2/M arrest has been widely proved, its contribution in nickel-induced G2/M arrest was excluded, respecting that its protein level, ser15 phosphorylation and transcriptional activity were not changed in nickel response. Further study revealed that cyclin B1, an important cyclin which prevents cells from M-phase exit and is frequently involved in the promotion of many kinds of tumor, was remarkably up-regulated by nickel on a HIF-independent manner, and the essential role of cyclin B1 in M-phase arrest and cell proliferation inhibition was further verified by knocking down its expression using specific small interference RNA. Taken together, our results demonstrate that nickel exposure can induce G1/S cell cycle transition by facilitating cyclin D1 and/or cyclin E expression, until the M-phase blockage caused by up-regulated cyclin B1 expression (after a latent phase) veiled the effect of cyclin D1 and/or cyclin E and lead to the overall cell cycle blockage and cell proliferation inhibition.