Isolation, Purification And Experimental Study Of Hepatocelluar Carcinoma Treatment By Extract Of Centipedes

Hepatocellular carcinoma (HCC) was one of the most common malignancy tumor in worldwide. It was the third leading cause of death attributable to cancer. The incidence of 50-150 HCC cases per 100,000 population and year in parts of Africa and Asia where HCC was responsible for a large proportion of cancer deaths. In recent 20 years, despite the emergence of various therapeutic modalities such as diagnosis at an early stage, hepatic resection and radiofrequency ablation were therapy and chemotherapy. In China, HCC was the second the incidence of and mortality of cancer.The treatment for the patient mainly depended on the stage of the tumor and the liver function. As we all known, although surgery (partial hepatectomy or total hepatectomy with orthotopic liver transplantation) could be curative for localized small liver tumors, therapeutic options for patients with advanced or metastatic HCC were limited. The patients who had no chance to undergo hepatic resection or could not bear the surgery may be cured by combination of region treatment with integration treatment. There were many methods for it, But all treatments had their own respective limitations; and the therapeutic effect for the patients with advanced stage of HCC was still poor. Further elucidation of the molecular pathogenesis of HCC and exploration of an effective drug may facilitate the development of more effective therapeutic interventions for HCC.Centipede as a drug applied to prophylaxis and treatment of diseases had a history of more than two thousand years. The extract of whole centipede was proved to have many effects in disease treatment in recent years. But the reports about its effect in carcinomas' treatment were in odds and ends, and the method for experiment of tumor therapy only limits to MTT. Up to now, little information of its detailed mechanism of action had been known to us. In order to investigate the sensibility and mechanisms of HCC cell to extract of centipede (ECP),we took HCC Bel-7402 cell line and model of heterotopic grafting carcinoma for HCC Bel-7402 to research treatment with ECP by a series of methods.What we did aim to give its theory to clinic treatment with ECP for HCC. ChapterⅠAssay sensibility of HCC Bel-7402 cell line to ECP in vitroObjective:To investigate the inhibitory effect of ECP, supernatant and protein from ECP on human HCC Bel-7402 cell line.Method: Bel-7402 cell line were cultured in vitro; ECP,the supernatant from ECP and its protein were applied to the interference of the growth of Bel-7402 with different drug concentration; MTT method and cell amounts were employed to investigate the sensibility to them. We observe cell morpheus changes through inversion light microscope. LO2 cell line was cultured in vitro. ECP were applied to the interference of the growth of LO2 with different drug concentration; MTT method and cell amounts were employed to investigate the sensibility to them.Result:①According to light microscope and cell amounts after 48 hours, we found out that these were viable counts of Bel-7402 decrease at the ECP,the protein of ECP and 5-Fu, no evident depressant effect for cells Bel-7402 at the supernatant from ECP and for cells LO2 at the ECP.②The result of MTT method showed that the inhibition ratio to Bel-7402 of ECP,the protein of ECP,the supernatant from ECP and 5-Fu was 0.788,0.453,0.198 and 0.944, respectively. The inhibition ratio to LO2of ECP was 0.095.③The result of MTT method showed that the inhibition ratio of various concentration of ECP at 12mg/ml,1.2mg/ml,0.12mg/ml,0.012mg/ml and 0.0012mg/ml was 0.788,0.608,0.308,0.207 and 0.099, respectively, and IC50 of ECP to Bel-7402 was 9.597mg/ml. The inhibition ratio of various concentration at 2.9mg/ml,0.29mg/ml,.029mg/ml,.0029mg/ml and 0.00029mg/ml was 0.453,0.208,0.145,0.077 and 0.063, respectively, and IC50 to Bel-7402 was 1.372mg/ml.④Drug concentration survival curve showed that the viable counts of Bel-7402 had direct relations with ECP and the protein of ECP concentration. There existed distinct discrepancies for different groups (P<0.05)⑤The result of MTT method showed that the inhibition ratio of various concentration of ECP at 12mg/ml,1.2mg/ml,0.12mg/ml,0.012mg/ml and 0.0012mg/ml was 0.095,0.040,0.037,0.022 and 0.005, respectively.Conclusion:①ECP and the protein of ECP could inhibit the growth of Bel-7402 cells. The inhibitory effect was concentration dependent. The supernatant from ECP had no transparent inhibiting on Bel-7402.②ECP had no transparent inhibition on LO2. ChapterⅡIsolation, Purification the anti-tumor protein extracted from ECPObjective:To investigate the sensibility of HCC Bel-7402 cell line by treatment of the protein of ECP was isolated and purified by gel filtration, ion-exchange chromatography. The molecular weights of proteins were identified by SDS-PAGE electrophoresis method.Method:①Bel-7402 cell line were cultured in vitro. The protein of ECP was isolated and purified by gel filtration on CL-6B, DEAE-Sepharose-FF anion-exchange chromatography and gel filtration on Sephadex G-75, applied to the interference of the growth of Bel-7402 with different drug concentration; MTT method and cell amounts were employed to investigate the sensibility to them.②We observe cell morpheus changes through inversion light microscope.③The molecular weights of effective components were identified by SDS-PAGE.Result:①A,B,C,D,E were separated from the protein of ECP by gel filtration on CL-6B column. According to light microscope,the cell amounts and MTT after 48 hours, we found out that the proteins compound C and E had remarkable suppressive on the proliferation of Bel-7402 in vitro. The protein molecular weights of C and E were mainly distributed between 1.6×106～2.2X 106,1×104～4×105, respectively. The result of MTT method and drug concentration survival curve showed that the viable counts of Bel-7402 had direct relations with C and E concentration. The cell proliferation was slower than that of contrast group (P＜0.05)②F,G,H,I,J,K,L were separated from the compound C and E by DEAE-Sepharose-FF anion-exchange chromatography. According to light microscope,the cell amounts and MTT after 48 hours, we found out that the proteins compound G and I had remarkable suppressive on the proliferation of Bel-7402 in vitro. The pI of G and I less than 6.8. The result of MTT method and drug concentration survival curve showed that the viable counts of Bel-7402 had direct relations with G and I concentration. The cell proliferation was slower than that of contrast group (P＜0.05)③M,N,O,P were separated from the compound G and I by gel filtration on Sephadex G-75 column. T According to light microscope,the cell amounts and MTT after 48 hours, we found out that the proteins compound O and P had remarkable suppressive on the proliferation of Bel-7402 in vitro. The protein molecular weights of O and P were mainly distributed between 4×104～6×104,2×104～4×104 respectively. he result of MTT method and drug concentration survival curve showed that the viable counts of Bel-7402 had direct relations with G and I concentration. The cell proliferation was slower than that of contrast group (P<0.05).IC50 of O and P to Bel-7402 was 1.132mg/ml and 1.129mg/ml, respectively.④In SDS-PAGE,7 major bands were showed in O (molecular weights: 120Kd(1=103),60 kD,48 kD,35 kD,33 kD,25 kD,20 kD.6 major bands were showed in P (molecular weights: 120kD,80 kD,35 kD,33 kD,25 kD,20 kD).⑤A,B,D,F,H,J,L,M,N from ECP could inhibit the growth of HCC Bel-7402 cell line at different concentration, but the growth inhibiting rate were low.Conclusion:①O,P were separated from the protein of ECP by gel filtration on CL-6B column,DEAE-Sepharose-FF anion-exchange chromatography,gel filtration on Sephadex G-75 column, which were compound of acidic protein. The protein molecular weights of O and P were mainly distributed between 2×104～6×104.②O,P could inhibit the growth of the Bel-7402. The inhibitory effect was concentration dependent. And its suppressive effect on the proliferation of Bel-7402 was nearly 8.5 times more than the crude proteins of ECP. ChapterⅢTo explore the mechanisms in human Bel-7402 cell line treated with ECPObjective: To explore the effect of antitumor molecular mechanisms, the HCC Bel-7402 cell line were treated with ECP.Method:Bel-7402 cells line were treated with ECP (10mg/ml) in different time (0,3,6,12,24,48h). The expression of Galectin-7 and p-JNK1 protein expression and mRNA were examined by Western Blot, Real Time-PCR and immunofluo-rescence staining. We treated Bel-7402 cells line with Galectin-7-siRNA, then Bel-7402 cells line were treated with ECP (10mg/ml) in 48h. The expression of Galectin-7 protein expression and mRNA, and p-JNK1 protein expression were examined by Western Blot, Real Time-PCR and immunofluorescence staining.Result:①Western Blot, Real Time-PCR and Immunofluorescence staining showed that expression of Galectin-7, p-JNK1 protein and mRNA were up regulated in a time-dependent manner in Bel-7402 stimulated with ECP. There was significantly difference among ECP groups and contrast group (P<0.01)②Western Blot, Real Time-PCR and Immunofluorescence staining showed that expression of Galectin-7, p-JNK1 protein and mRNA were decreased in Bel-7402 stimulated with ECP in the presence of Galectin-7-siRNAs compared with Bel-7402 stimulated with ECP (P<0.05)③Western blot showed that induction of p-JNK1 protein was decreased in Bel-7402 stimulated with ECP in the presence of Galectin-7-siRNAs compared with Bel-7402 stimulated with ECP (P<0.05). There was significantly difference amongs Galectin-7-siRNAs groups and contrast group (P<0.05)Conclusion:Galectin-7, p-JNK1 protein and mRNA were up regulated in a time-dependent manner in Bel-7402 stimulated with ECP. ECP maybe activate Galectin-7-JNK pathway to induce apoptosis of Bel-7402.