| Angiogenesis correlate to the growth and metastasis of many kinds of tumours intimately. Tumor angiogenesis is a so complicated process that many factors are involved in, including adhesion,matrix degradation and migration. Therefore, the development of angiogenesis inhibitors is a new very active research field. Chinese medicine has drawn simple and low toxicity characteristics. Therefore,mang investigators are absorb in the natural medicine research. natural medicine as the research object, has become today's hot spots for exploring and studying a new type of anti-cancer drugs.This test is to study and prove that traditional Chinese medicine Scutellaria barbatae D.DON flavonoids have the activity of anti-tumor.Followed by the results of the preliminary research team,that is, the flavonoid of Scutellaria barbatae D.DON can inhibit the expression of VEGF in gastric cancer cells. Through the researches of tumor cell proliferation, apoptosis, Survivin and PTEN, we want to look into antitumor mechanism of Scutellaria barbatae D.DON flavonoids, which lays a theoretical foundation for the further development of high efficiency and low toxicity of anticancer drugs.Systematic solvent separation method and column separation method was employed to extract different parts of Scutellaria barbatae D.DON. The effects of different parts of Scutellaria barbatae D.DON on proliferation of tumor cell were tested by MTT method. At last,we selected the most effective part and investigated the mechanism of inhibiting tumor growth, invasion and metastasis through apoptosis,flow cytometry techniques,immunocytochemistry and Western blot.The main results are showed as below:1.The extraction and separation of scutellaria barbatae D.DON flavonoids: we found eight different polarity parts which were A-H through silica gel column chromatography using mixed solvent CHC13-MeOH and thin-layer chromatography(TLC).Finally we selected the part of D which was active through the MTT assay. The different concentrations of scutellaria barbatae D.DON flavonoids D(10μg/ml,20μg/ml,40μg/ml,80μg/ml,160μg/ml) can effectively inhibit the proliferation of SGC-7901 cell lines and BGC-823 cell lines,which was showed concentration- dependent .2.Scutellaria barbatae D.DON flavonoids induced apoptosis of tumor cells: With the increase of drug concentration,the apoptosis rate of tumor cells gradually increased. The apoptosis rate was increased in SGC-7901 cells and BGC-823 cells treated with 30μg/ml .60μg/ml or 120μg/ml Scutellaria barbatae D.DON flavonoids after 24 h as compared with the control group(p<0.01).3.Scutellaria barbatae D.DON flavonoids inhibited the protein expression of Survivin from SGC-7901 cells and BGC-823 cells: Through immunocytochemical method, we can see that with the increase of drug concentration and the extension of time, brown-stained gradually became shallow, indicating that the protein expression of Survivin gradually reduced. The protein expressions of Survivin both declined in SGC-7901 cells and BGC-823 cells treated with 60μg/ml and 120μg/ml Scutellaria barbatae D.DON flavonoids after 24h as compared with the control group(p<0.01). The protein expressions of Survivin both declined in SGC-7901 cells and BGC-823 cells treated with 30μg/ml. 60μg/ml and 120μg/ml Scutellaria barbatae D.DON flavonoids after 48h as compared with the control group(p<0.01).The protein expression of Survivin declined in SGC-7901 cells treated with 60μg/ml Scutellaria barbatae D.DON flavonoids after 48h as compared with 24h(p<0.05). The protein expression of Survivin declined in BGC-823 cells treated with 60μg/ml and 120μg/ml Scutellaria barbatae D.DON flavonoids after 48h as compared with 24h(p<0.01).We can see that by Western blot, with the increase of drug concentration and time,the color of the strips on Survivin expression faded which was consistent with the results by immunocytochemistry.The protein expression of Survivin declined in SGC-7901 cells treated with 40μg/ml and 80μg/ml Scutellaria barbatae D.DON flavonoids after 24h as compared with the control group(p<0.05). The protein expression of Survivin declined in SGC-7901 treated with 160μg/ml Scutellaria barbatae D.DON flavonoids after 24h as compared with the control group(p<0.01). The protein expressions of Survivin both declined in SGC-7901 treated with 80μg/ml and 160μg/ml Scutellaria barbatae D.DON flavonoids after 24h as compared with the control group(p<0.01).The protein expression of Survivin declined in SGC-7901 cells treated with 160μg/ml Scutellaria barbatae D.DON flavonoids after 48h as compared with 24h(p<0.05).4.Scutellaria barbatae D.DON flavonoids promoted the protein expression of PTEN from SGC-7901 cells and BGC-823 cells: Through immunocytochemical method, we can see that with the increase of drug concentration and the extension of time, brown-stained gradually became deep, indicating that the protein expression of PTEN gradually increased.The protein expression of PTEN increased in SGC-7901 cells treated with 120μg/ml Scutellaria barbatae D.DON flavonoids after 24h as compared with the control group(p<0.05). The protein expression of PTEN increased in SGC-7901 cells treated with 120μg/ml Scutellaria barbatae D.DON flavonoids after 48h as compared with 24h(p<0.05).The protein expression of PTEN increased in BGC-823 cells treated with 60μg/ml Scutellaria barbatae D.DON flavonoids after 24h as compared with the control group(p<0.05). The expression of PTEN increased in BGC-823 cells treated with 120μg/ml Scutellaria barbatae D.DON flavonoids after 24h as compared with the control group(p<0.01). The expression of PTEN increased in BGC-823 cells treated with 60μg/ml and120μg/ml Scutellaria barbatae D.DON flavonoids after 48h as compared with the control group(p<0.01). The protein expression of PTEN increased in BGC-823 cells treated with 120μg/ml Scutellaria barbatae D.DON flavonoids after 48h as compared with 24h(p<0.05).We can see that by Western blot, with the increase of drug concentration and time,the color of the strips on PTEN expression gradually darkened which was consistent with the results by immunocytochemistry.Conclusion: Scutellaria barbatae D.DON flavonoids can inhibit the expression of Survivin and promote the expression of PTEN in vitro, Its mechanism may be that Scutellaria barbatae D.DON flavonoids can inhibit expression of VEGF through inhibiting Survivin and promoting PTEN , inhibit the proliferation of tumor cells, induce tumor cell apoptosis in vitro which finally can inhibit the tumor cell migration and block tumor angiogenesis. |
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