| Cancer is a severe health menace to human being. About 5 million people died of cancer each year in the world according to the statistic report of World Health Organization. Therefore the prevention and treatment of cancer is a very challenging task. Chemotherapy is one of three common ways to treat cancer while surgery and radiotherapy are the two others. Anticancer drugs have been constantly studied for many years. Drug screening proves to be a very important step in the study. Reasonable design of screening method and system is especially indispensable during the screening process. Governments all over the world have attached great importance to the screening of anticancer drugs, and have devoted a lot of resources to the drug development. On the other hand, there are a large number of compounds including synthetic drugs, natural compounds and fermentation products waiting for screening. At the meantime finding a safer, more effective and less side effect anticancer drug has always been the object of researches. In recent years, with the rapid development of life science and technology, new anticancer drugs continually appear.Results of study of the molecular mechanisms of tumorigensis showed that malignant tumor cells invariably lose the control of various basic processes. These processes include: regulation of cell cycle, malfunction of cellular signal transduction pathways, apoptosis, stability of telomere, angiogenesis and complex interactions between cell and its extracellular matrix, etc.To develop anticancer drugs with high specificity, researcher making use of the recent development in the cancer biology, have begin to pay more attention to looking for special molecule and drug target which effect in the etiology and pathology of cancer, such as apoptosis inducers,signal transduction blockers and angiogenesis inhibitors, etc.Apoptosis is a tightly regulated cellular suicide process. Apoptosis can be triggered by various stimuli, e.g. by DNA damage as a cause of defect in DNA repair mechanisms, treatment with cytotoxic drug or irradiation, by lack of survival signals, etc. During apoptosis, various changes in gene expression and biochemical reactions occurred. Some proteases activity will be upregulated. Increasingly evidences have shown that apoptosis is closely related with cancer genesis, development, treatment and prognosis. Accordingly many anticancer drugs are being used in cancer treatment mainly by inducing apoptosis. The efficiency of these anticancer drugs is correlated with their ability to induce apoptosis. Anticancer research using regulators of apoptosis as potential targets to selectively induce tumor cell apoptosis have made many significant advances.Curcuma longa Linn is Chinese traditional medicine. According to modern medicine research, it exhibits many activities including anti-inflammation, anticancer, antioxidant, anti-bacterial, anti-HIV etc. Although its chemical composition is very complex, a lot of effective compounds have been isolated. At present research of curcuma longa is focused on its anticancer, antioxidant, anti-bacterial, anti-HIV activities. A sesquiterpene, ar-turmerone is also an effective component of curcuma longa. It possesses anti-bacterial, fungicidal, and antivenom activity, etc. In our present research, we found that ar-turmerone showed cytotoxic activity, and could induced apoptosis in various cancer cell lines.This study investigated the mechanisms of apoptosis induced by ar-turmerone isolated from Chinese traditional medicine turmeric (Curcuma longa L) via pharmaceutical chemistry, analytical chemistry, cell biology and biochemistry techniques. Ar-turmerone was first isolated and purified and clear evidences have been showing that ar-turmerone can induce apoptosis in various cancer cells. I. Isolation, purification and structural analysis of ar-turmerone Curcuma longa was extracted with methanol. The methanol extracts were loaded on a silica gel column and eluted, then were separated by HPLC. Three fractions were obtained. Structure of fraction 1 was identified to be ar-turmerone by *H- and I3C-NMR.II. Study on cytotoxic activity of ar-turmerone The cytotocicity was measured using the microculture tetrazolium (MTT) method in the K-562, U-937, RBL-2H3, L-1210, SNU, Jurkat cell lines. Ar-turmerone exhibited potent and dose-dependent cytotoxicity on all these cancer cell lines. The IC50 of ar-turmerone on these cell lines were 20-60 jj.g/ml. Ar-turmerone also showed an increased inhibition ratio on cell viability at a concentration < 80 ng/ml in these cell lines. These results showed that ar-turmerone exhibit potent cytotoxicity, and inhibit cancer cell proliferation.III. Study on induction of apoptosis by ar-turmerone Using agarose gel electrophoresis, flow cytometry, Acridine orange staining, laser scanning confocal microscopy, the effect of ar-turmerone on apoptosis were studied. The K-562, U-937, L-1210, RBL-2H3 cell line exhibited some typical morphologic and biochemical features of apoptosis, including cell shrinkage, nuclear condensation, DNA fragmentation, and formation of apoptosis bodies, Apoptosis induced by ar-turmerone was in a proper dose- and time-dependent manner. The SNU and Jurkat cells didn't exhibit DNA fragmentation and apoptotic body. The results primarily showed that ar-turmerone induces selective apoptosis on various cell lines, especially on leukemia cell.IV. Study on the apoptosis mechanisms induced by ar-turmerone Cell cycle analysis was performed via PI staining and flowcytometry (FCM). Cells arrest at G2/M phase accumulated when cells were incubated with 20 ng/ml, 40 ng/ml ar-turmerone for 12h and 24 h. Again ar-turmerone displayed a time- and dose-dependent effect. G2/M phase is an important checkpoint in cell cycle. These results showed that ar-turmerone blocks the cell cycle at G2/M phase, and thus inhibits cancer cell proliferation, which primarily accounts for its cytotoxicity obserced proviously. Caspases play a direct role in proteolytic cleavage of cellular proteins responsible for progression to apoptosis. The activities of caspase-3, caspase-8 were thus further assayed in U-937 cell with cell being incubated with 20 ug/ml, 40 jjg/ml ar-turmerone for 24 h. Caspase-3 and caspase-8 were activated when treated with ar-turmerone. This result suggested that ar-turmerone might induced apoptosis in U-937 cell by extrinsic apoptosis signal pathway. The activities ofcaspase-3, caspase-8, caspase-9 were also assayed in K562 cell with cells being incubated with 40 |J.g/ml, 80 ug/ml ar-turmerone for 24 h. In comparison with U-937 cell, while Caspase-3 and caspase-9 were activated, caspase-8 was not activated in K-562 cell line. This result suggested that ar-turmerone might induced apoptosis in K-562 cell by intrinsic apoptosis signal pathway.
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