Experimental Study Of Hepatocelluar Carcinoma Treatment By Extract Of Centipedes

Hepatocellular carcinoma(HCC)is one of the most commonmalignancy in China. In recent 20 years, despite the emergence of varioustherapeutic modalities such as hepatic resection, radiofrequency ablationtherapy and chemotherapy, the prognosis of HCC remains poor, and theaverage life time is very short.The treatment for the patient mainly depends on the stage of thetumor and the liver function. As we all known, hepatic resection includeliver transplantation is the most effective treatment and the first choice tocure the HCC radically. However, due to different stages of the tumor,the exist of cirrhosis and different functions of liver, the resection rateof the patients who undergo radical hepatic resection is often low (10％～30ï¼…).The patients who has no chance to undergo hepatic resection orcannot bear the surgery may be cured by combination of region treatmentwith integration treatment. There are many methods for it, But alltreatments have their own respective limitations; and their therapeuticeffects for the patient with advanced stage of HCC are still poor. Further elucidation of the molecular pathogenesis of HCC and exploration of aneffective drug may facilitate the development of more effectivetherapeutic interventions for HCC.Centipede as a drug applied to prophylaxis and treatment of diseaseshas a history of more than two thousand years. The extract of wholecentipede was proved to have many effects in disease treatment in recentyears. But the reports about its effect in carcinomas' treatment are in oddsand ends, and the method for experiment of tumor therapy limits only toMTT. Up to now, little information of its detailed mechanism of actionhas been known to us. In order to investigate the sensibility andmechanisms of HCC cell to ECP, we take HCC Bel-7404 cell line andmodel of hefterotopic grafting carcinoma for HCC Bel-7404 in nudemouse as a object to research treatment with extract of centipede (ECP)by a series of methods. What we do aim to give its theory to clinictreatment with ECP for HCC.Chapterâ… Assay sensibility of HCC Bel-7404cell line to ECP in vitroObjective: To investigate the sensibility, cell cycle and mechanismof HCC Bel-7404 cell line by treatment of ECP, and to provideevidences of clinic therapeutic theory for HCC. Method:â‘ Bel-7404 cell line were cultured in vitro; ECP wasapplied to the interference of the growth of Bel-7404 with different drugconcentration; MTT method and different drug concentration-timesurvival curve are employed to investigate the sensibility of HCC cell(Bel-7404) to ECP.â‘¡We observe cell morphous and it's Ultrastructuralchanges through inversion light microscope and electron microscope.â‘¢We detect cell cycles and apoptosis ratio to ECP group by flowcytometry(FCM).â‘£To investigate expression degrees ofrelated-apoptosis genes XIAP and Bax, we also detect HCC Bel-7404 cellline after 48 hours treated with ECP by immunohistochemical method.Result:â‘ ECP can inhibit the growth of HCC Bel-7404 cell lineobviously at different concentration with time-concentration dependent.The result of MTT method showed that the inhibition ratio of variousconcentration of ECP at 80mg/ml,40mg/ml,20mg/ml,10mg/ml,5mg/ml,2.5mg/ml and 1.25mg/ml is (76.58±3.58)%,(71.06±3.43)%, (63.98±4.70)%, (56.74±11.30)%, (36.89±8.13)%,(31.26±3.42)% and (17.93±4.34)% respectively, and IC₅₀of ECP to Bel-7404 is 9.5mg/ml. Inhibitionratio increases when ECP concentration adds with direct correlation tendency.â‘¡Drug concentration-time survival curve shows that the viable counts ofBel-7404 has direct relations with ECP concentration and administrativetime. The survival curve for the contrastive group show viable counts ofBel-7404 elevate firstly, and then descend gradually until to equation to cell numbers of the onset at the seventh day. As for the treatment group ofthe concentration at 5mg/ml, the curve also show viable counts ofBel-7404 elevate firstly, and then descend gradually after 48 hour. For thegroups of 10mg/ml and 20mg/ml, viable counts of Bel-7404 sharplydecrease after 24 hours treated at the concentration of 10mg/ml and20mg/ml. There exist distinct discrepancies for different groups (P＜0.01).â‘¢Observed through light microscope after 48 hours, we fred out that these isno evident depressant effect for cells Bel-7404 at the ECP concentrationof 5mg/ml,but over 10mg/ml, viable counts of Bel-7404 decrease, andcyto-morph transform to round and shrink. At the ECP concentration of80mg/ml, dead cells Bel-7404 increase, many irregular cells debris can beseen, and many dead cell cast-off from culture flask. When observedthrough electron microscope for treatment groups we see the increase of cellsdensity, chromatic agglutination, chromatin margination, nuclear pyknosis,nuclear fragmentation and apoptotic body formation.â‘£The result ofFCM display for treatment groups that ECP mainly retard cell cycle phasein G0/G1 after 48 hours.(A)The ratio of G0/G1 is different at the differentconcentration. It's (50.8±3.5)% for the contrast After 48 hours', whentreatement with concentration of 5mg/ml, 10mg/ml and 20mg/ml,G0/G1phase account to (58.24±2.4) %,(65.7±4.1)% and (72.1±5.3)%,respectively.(B) Proliferation index(PI) for cells Bel-7404 for thecontrast is (49.2±1.9)%. For the treatment groups at the concentation of 5mg/ml,10mg/ml and 20mg/ml,PI is (41.8±4.1)%, (35.3±3.7)% and(27.9±2.0)%.(C)Apoptosis ratio:The apoptosis ratio for the contrast is(1.56±0.17) % after 24 hours for cells Bel-7404. For the treatmentgroups at the concentation of 5mg/ml, 10mg/ml and 20mg/ml,It is (4.99±0.59)%, (7.71±0.92)% and (12.1±0.63)% respectively. It is significantdifference from treatment group and control group (P＜0.05). After 48hours,the apoptosis ratio for the contrast is(1.754±0.145) %, For thetreatment groups at the concentation of 5mg/ml, 10mg/ml and 20mg/ml,Itis (5.90±0.35)%, (9.80±0.95)% and (17.3±1.71)% respectively. It issignificant difference from treatment group and control group,and also fordifferent time when in the same concentration of ECP.â‘£It shows thatXIAP gene express gradually to weaken but Bax gene to enhance in HCCcells with the increase of ECP concentration, these are statisticalsignificance in contrast to the control (P＜0.05).Conclusion:â‘ ECP can inhibit the growth of the Bel-7404. Theinhibitory effect is time-concentration dependent.â‘¡ECP can induceBel-7404 cell line to apoptosis in low concentration, And directly kill it ifin middle-high concentration.â‘¢ECP mainly retard cell cycle phase ofBel-7404 in G0/G1, suppress it to proliferation and can induce it toapoptosis, the inhibitory effect is time-centration dependent.â‘£One of themechanisms of treatment with ECP is to promote Bax gene and inhibitXIAP gene expression for HCC Bel-7404. Chapterâ…¡Investigation to the effect and mechanisms ofHCC BEL-7404 treatment with ECP in vivoObjective: To investigate the effects and mechanisms of HCCBel-7404 treatment with ECP.Method:â‘ To set up model of hefterotopic grafting carcinoma forHCC Bel-7404 in nude mouse, and observe its tumor growth andmetastasis by intragastric administration of ECP, including its weight ofthymus gland and spleen.â‘¡To detect XIAP,Bax,VEGF and Ang-2 intumor tissue with immunohistochemical methodsResult:â‘ The achievement ratio for hefterotopic graftingcarcinoma for HCC Bel-7404 in nude mouse is 100%.â‘¡It has distinctiveinhibitive effect for ECP to hefterotopic grafting carcinoma for HCCBel-7404 in nude mouse. At the 31st day postgraft of nude mouse, theaverage grafting carcinoma volume of the contrast is (1187.5±117.4)mm³, and for the treatment group is (506.1±65.7)mm³; The averagegrafting carcinoma weight of the contrast is (1059±125.2)mg, but forthe treatment group is (647±122.89)mg, The inhibitive ratio reaches to 38.91%. thymus gland and spleen index for the treatment group are(2.15±0.43)% and (10.63±3.7)% respectively, for the contrast group,it's(0.81±0.27)% and (7.90±2.58) % respectively, there are significantdifference for them(P＜0.05).â‘¢The staining cells population, stainingintensity and the optical density (OD) of XIAP, VEGF and Ang-2 for thecontrast group are higher than the treatment group, but they are lower forBax. there are significant difference for them (P＜0.05)Conclusion:â‘ ECP can inhibit hefterotopic grafting carcinoma forHCC Bel-7404 in nude mouse, the mechanisms relate to inhibit tumorAngiogenesis and to promote HCC Bel-7404 cells to apoptosis.â‘¡ECPcan't inhibit immunological function of nude mouse but can increase it.Chapterâ…¢: Application of the techniques and methods ofProteomics into separating differential expression proteinsin HCC cells treatment with ECPObjective: To screen the relevant differential expression proteinsafter treatment with ECP, and to investigate the molecule mechanism ofECP for HCC Bel-7404.Method: we use proteomic Techniques and methods to analysis the mechanism of treatment with ECP for HCC Bel-7404. Firstly,comparative two-dimensional gel electrophoresis(2-DE) technology wasapplied to separate the total protein of HCC Bel-7404 treatment group byECP and its control group respectively. The well-resolved, reproducible2-DE patterns of treatment group of ECP and control group wereestablished. Then, PDQuest software was used to analyze 2-DE images,and the differential expression proteins between the two groups wereidentified by both peptide mass fingerprint (PMF) and peptide sequencetag (PST) based on MALDI-TOF-MS (Matrix-assisted laserdesorption/ionization time of flight mass spectrometry).Result: We select the differential expression proteins for betweentreatment group and control group which spots variance is over double toanalyse with MALDI-TOF-MS after spots enzymolyed, and found out76 protein spots at difference level of expression (p＜0.05), including 41spots decreasing in treatment group, and 35 spots increasing in controlgroups. 14 of the total pieces of PMF were be matched searching inSWISS-PROT/TREMBL database by Mascot software. Among theidentified 14 protein spots, the expression level of proteins whichup-regulates in treatment group is 8 in total, including: Peptidyl-prolylcis-trans isomerase A (PPIA),Lamin-β1,Flavin reductase (FRase),Transferrin-binding protein 2 (Tbp2),Sialic acid synthase (SAS),Glyceraldehyde-3-phosphate dehydrogenase (GAPDH),Cyclin dependent kinase proflin 1(P²¹)and Galectin-7(Gal-7). But 6 proteins intreatment group is down-regulation, which include Keratin (CK-19),Actin,Transitional endoplasmic reticulum ATPase (TERATPase),Glutathione transferase omega-1(GSTO1-1),Profilin-1(PFN1) andAlkaline phosphatase (ALP).Conclusion:â‘ Through analysis of two-dimensional gelelectrophoresis (2-DE), 76 protein spots at difference level of expression(p＜0.05) have been found out, including 41 spots decreasing intreatment group, and 35 spots increasing in control groups.â‘¡Among theidentified 14 of the total of protein spots, the expression level of proteinswhich up-regulates in treatment group is 8 in total, including: PPIA,Lamin-β1, FRase, TBP2, SAS, GAPDH, P²¹ and Gal-7 and whichdown-regulates in treatment group is 6 proteins in total, including:Keratin (CK-19), Actin, TERATPase, GSTO1-1, PFN1 and ALP. It hintthat ECP can inhibit HCC Bel-7404 cell line to growth by inducing it toapoptosis or directly to death in multi-channels. Chapterâ…£: Verification of the differential expression levelsof the partial proteins by western-blotting analysis and itsfunction studyObjective: To verify the differential expression levels of the partialproteins Tbp2,1aminB1 and Gal-7 which identified with comparedproteomic techniques.Method: To verify the differential expression levels of Tbp2,1aminB1 and Gal-7 again by Western-blotting methods, which expressin HCC Bel-7404 treatment with ECP group and control group everidentified with comparative proteomic techniques.Result: The proteins of Tbp2,1aminB1 and Gal-7 expressup-regulation in treatment group with ECP, and the results were identicalwith the proteome analysis.Conclusion: The differential expression proteins of Tbp2,1aminB1and Gal-7 screened by proteome analysis are indeed differentialexpression in treatment group and control group of HCC Bel-7404.