Isolation And Purification Of Anti-tumor Ingredients From The Gekko And Study Of Its Anti-tumor Mechanism

Malignant tumor is one of the most serious diseases which threaten the health of people.Besides inhibiting the growth of cancer cells,most of anti-tumor agents can kill normal cells and lead severe side effects and drug resistance.So,it becomes an important target to find a kind of anti-tumor new drugs with efficiency and low side effects.In recent years,many inspiring achievement had been made by scientists.They isolate a lot of anti-tumor ingredients from traditional Chinese medicine.So far,it have been reported that many natural products from traditional C hinese medicie has good therapy effects,low cytotoxity and less drug resistance.Growing evidences have demonstrated that the Gekko effectively suppressed the development of tumors and improved cancer patients’clinical symptoms and survival rates.However complex composition from the Gekko limited its clinical efficiency.The aim of the study is to gain a kind of anti-tumor ingredients from the Gekko and explore its anti-tumor mechanisms.This study will provide experimental and theoretical basis for the clinical application of Gekko ingredients.Chapter 1 Separation and Purification of the Gekko Anti-tumor IngredientsObjective:The aim of this study was to isolate and purify a kind of small molecule anti-tumor ingredients from Gekko,and screening for in vitro anti-tumor activities.Methods:The dried Gekko was selected and ground to a fine powder.The anti-tumor ingredients were isolated and purified by the following procedure which consisted colloid milling,water precipitation,ethanol supernatant（55%）,ultra-filtration intercept,Sep Hadex G-25,Sep Hadex LH-20,High Performance Liquid Chromatography.CCK8 assay was used to detect the anti-tumor activity of the ingredients from each step of separation in four cancer cells（human esophageal cancer Ec 9706 cells,human prostate cancer DU145 cells,human hepatoma SMMC 7721cel s,human cervical cancer SiHa cel s）.Results:1.Less 3000Da Ultrafiltration ingredients from the Gekko ethanol extract significantly inhibited the proliferation of DU145,Ec 9706,SMMC 7721 and SiHa cells at 24h and 48 h respectively.Ec 9706 cells were the most sensitive among four cancer cell.The 50%inhibitory concentration(IC₅₀)of ultrafiltration ingredients on Ec9706 cells were 1.21 mg/mL and 1.04 mg/mL at 24 h and 48 h.2.Three active peaks were obtained by Sephadex G-25.The ingredients from peak 3 showed the best anti-tumor activity by CCK8 screening.The IC₅₀ of ingredients in peak 3 on Ec 9706 cells were 0.92 mg/mL and 0.36 mg/mL respectively at 24 h and48 h.3.After LH-20 separation and purification,we gained 17 ingredients.The No.15ingredient had the best anti-tumor activity and it IC₅₀ on Ec 9706 cells were 70.2μg/mL and 54.6μg/mL respectively at 24 h and 48 h.Conclusion:1.In this experiment,a kind of less than 3000 Da active ingredients（LH-20-15）from the Gekko was gained by separation and purification by the procedure of alcohol precipitation,ultra-filtration and gel chromatography.The main ingredients were identified as polypeptides by protein purification system and high performance liquid phase.2.The products of each step have obvious anti-tumor activity on four tumor cells and the human esophageal cancer Ec 9706 cells was the most sensitive to each product.Chapter 2 LH-20-15 regulates the proliferation and apoptosis of Ec 9706 cellsby targeting the MACC1/c-Met signaling pathwayObjective:The aim of this study was to investigate the effects of LH-20-15 on the proliferation and apoptosis of Ec 9706 cells and its regulation on MACC1/c-Met/Akt signaling pathway.Methods:Ec 9706 cells were treated with LH-20-15 for 48h in vitro.The CCK8 and Ed U click reaction assay were used to assess cell proliferation.Cell apoptosis was determined by Hoechst 33258 staining,Annexin V/PI staining and Caspase-3 assay.MACC1 protein was detected by Western blotting and Immunocytochemistry.The protein expressions of Caspase 3,Bcl-2,Bax,p-c-Met,c-MET,p-Ak,and p-BAD were measured by Western blotting.Results:1.The proliferation of Ec 9706 cells was inhibited by LH-20-15 in a dose-dependent manner.The inhibitory rate was 13.8%,20.5%,28.2%,34.1%,42.6%,58.9%,89.9%respectively.2.The results of Ed U experiments showed that the control cells showed deep brown staining.Compared with the control group,the brown staining of the Ec 9706cells gradually decreased in dose dependence after LH-20-15 treatment.This result suggested that LH-20-15 inhibited DNA synthesis of Ec 9706 cells.3.Annexin V/PI staining results showed that different doses LH-20-15 induced the apoptosis of Ec 9706 cells significantly.The apoptosis rate of Ec 9706 cells were respectively 28.4%,35.7%,50.6%,54.6%,61.4%and 84.3%at 0.3μg/mL,1μg/mL,3μg/mL,10μg/m,30μg/mL and 100μg/mL.4.Hoechst 33258 results showed markly increasing apoptotic cells with typical fragmented nuclei in LH-20-15 treatment group.The apoptotic cells nuclei showed karyopyknosis,fragmentation and highlight blue fluorescence.5.Compared with the control group,after administration of 0.1μg/mL,0.3μg/mL,1μg/mL,3μg/mL,10μg/mL,30μg/mL,100μg/mL LH-20-15 for 48 h,the Caspase 3 enzyme activity of Ec 9706 cells were increased significantly.6.Western blot results showed that LH-20-15 low,medium and high dose groups（15,30,60μg/mL）significantly increased the protein expressions of Bax,p-Bad and Caspase 3 and reduced protein levels of MACC1,p-c-Met,p-Akt and Bcl-2.7.The immunocytochemistry results showed that the MACC1 expression decreased significantly in Ec 9706 cells treated with LH-20-15 at 15,30,60μg/mL.Conslusions:LH-20-15 inhibited the proliferation and induced the apoptosis o f Ec 9706 cells by targeting the mitochondrial pathway and MACC1/c-Met signaling pathway.