Toxicological Study Of The Ethanol Extract Of Centipede And The Influence On The Biological Behaviors To Hepatocellular Carcinoma Cell Line BEL-7402

Background and Objective:Hepatocellular carcinoma(HCC)is one of the most common alignant cancer in the World.The prognosis of HCC is very poor,and the average life time is very short.At present,hepatic resection with other therapy(eg, chemotherapy and radio therapy)play an important role in the systemic treatment of HCC in China.However,HCC usually presented with simultaneously underlying liver disease,eg,liver cirrhosis and chronic hepatitis which cause continuous liver damage,and the resection rate of the patients who undergo radical hepatic resection is very low.So,it is very important to find an effective drug which may improve the healing effect for HCC.Centipede has applied to prophylaxis and treatment of diseases for more than two thousand years in China,which has some extent toxicity.The reports about the effect in carcinomas' treatment of ethanol extract of centipede(EEC)are in odds and ends.Up to now,little information of its detailed mechanism of action has been known to us.In the following experiment EEC is used as a new type antineoplastic medicine in mice.Its toxicology are studied in a bid to provide theoretical foundation for seeking a safe and efficacious medicine dosage for HCC.Mitogen-activated protein kinase(MAPK)pathway is a important signal pathway that the signal transfer from surface of the cell to the nuclear. The data from other researchers show that the MAPK pathway may participate in the hepatocarcinogenesis.In order to investigate the toxicity,sensibility and mechanisms of EEC, we take HCC Bel-7402 as a object to research treatment with EEC by a series of methods.The of extracellular regulated protein kinase(ERK),c-Jun N-terminal kinase(JNK)and p38 were detected by RT-PCR method.The phosphated protein p-ERK1/2 and p-JNK1 and p-p38 were detected with Western Blot.What we do aim to give its theory to clinic treatment with EEC for HCC. CHAPTERâ… THE TOXICOLOGICAL STUDY OF EECObjective:The ethanol extract of centipede(EEC)was used as an antineoplastic medicine in Kunming mice and Sprague-Dawlay rats,and it's toxicology was studied in a bid to provide theoretical foundation for seeking a safe and efficacious dosage for HCC.Method:1.Toxicological experimentAcute oral toxicity experiment:40 heathy mice of Kuming strain(20 males and 20 females)were randomly divided into 2 groups(EEC group and contrast group).The animals were lavaged EEC with the Test of maximum tolerated dose(80000mg/kg)in EEC group.All mice were observed for one week.2 Bone marrow cell micronucleus experiment50 healthy mice of Kuming strain(25 males and 25 females)were randomly divided into 5 groups.Cyclophosphamide positive control goup(40mg/kg),EEC group(10000mg/kg,20000mg/kg, 40000mg/kg),negative control group.The animals were given the medicine with the lavage method for one week.Then EEC bone marrow cell micronucleus experiment 24 hours after the seventh application of medicine were conducted.3.30 days feeding test80 SD rats were randomly divided into four groups:EEC group (2500mg/kg,5000mg/kg,10000mg/kg),negative control group.SD rats were killed in 30 days and 14 days after stopping lavaging EEC.And the main organs(liver,kidney,lung,heart,testis,ovary)were taken out and weighted.The organ coefficient was calculated.The haematology and biochemistry were measured.These tissues of rats dyed with HE and pathomorphological observation were carried out.4 Measure MDAThe content of MDA in the homogenate of left liver tissues of the mice were determined in every group in 30 days and 14 days after stopping lavaging EEC.Result:Test of maximum tolerated dose was 80000mg/10g/d,no one Kunming mouse died.Bone marrow cell micronucleus experiment:The results showed that the micronucleus rate of bone marrow cell in the 40000mg/kg group and the Cyclophosphamide positive control goup were higher than that of the negative contrl group(P＜0.01).There were no significant differences in the 10000mg/kg group,20000mg/kg group in comparison with the negative control group(P＞0.05)No significant differences existed among various concentration groups in the testing results(weight increment,weight of the main organs,organ coefficient,haematology)in comparison with the corresponding data of negative control group(P＞0.05).ALT and AST measured in 10000mg/kg concentration group were higher than that of the contrl group in 30 days feeding test(P＜0.01).Pathomorphological observation of liver cells were found in 10000mg/kg concentration group mice in the pathological examination.The pathologic section showed hepatic sinusoid dilated congestion in different degrees,infiltration inflammatory cell sometimes. These situation had been disappeared after the EEC stopped for 14 days. There was no damage founded in other groups.The results showed that in comparison with the corresponding data of the negative control group,significant differences existed in the 10000mg/kg concentration group in the content of MDA in liver(P＜0.01).In comparison with the corresponding data of the negative control group,no significant differences existed among 2500mg/kg concentration group and 5000mg/kg concentration group in the content of MDA in liver(P＞0.05).Conclusion:Test of maximum tolerated dose is 80000mg/kg/d.40000mg/kg ECC is some extent and capable of mutagenesis.10000mg/kg ECC probably injure the liver cells by peroxidization of lipid and MDA.EEC is a compound with low toxicity. Chapterâ…¡Assay sensibility and mechanism of HCC Bel-7402 cell line to EEC in vitroObjective:To investigate the toxicity and the sensibility of EEC,liver cancer cell line Bel-7402 were treated with EEC,which could provide evidences for clinic therapeutic theory.Method:1.Bel-7402 were cultured in vitro.EEC was applied to interfere the growth of Bel-7402 with different drug concentrations(64mg/ml,32mg/ml,16mg/ml,8mg/ml,4mg/ml,2mg/ml):MTT method and different drug concentration-time growth curve were employed to investigate the toxicity and sensibility of EEC.The human liver cancer cell line Bel-7402 and human normal liver cell line L-O2 were treated with different concentration EEC.2.Hoechst 33258 fluorescence staining method was applied to determining the ability of EEC induce Bel-7402 cell apoptosis.3.The cell morphosis were observed by the inverted optical microscope.4.We detected apoptosis ratio in EEC group by flow cytometry(FCM).Result:1.EEC could inhibit the growth of Bel-7402 cell obviously at different concentration with concentration-gradient dependent.The result of MTT method showed that the inhibition ratio of various concentration of EEC at 64mg/ml,32mg/ml,16mg/ml,8mg/ml,4mg/ml and 2mg/ml were (80.59±2.95)%,(71.96±3.36)%,(64.57±2.93)%,(53.35±1.85)%,(33.77±0.88)%and(6.37±0.44)%respectively,and IC₅₀of EEC to Bel-7402 was 7.83mg/ml.Inhibition ratio increased when EEC concentration added with direct correlation tendency.2.The growth curve showed that the OD of Bel-7402 had direct relations with EEC concentration and administrative time.The growth curve for the contrast group showed the OD of Bel-7402 cell proliferation was fast. As for the treatment group of the concentration at 4mg/ml,the curve also showed the OD of Bel-7402 elevated firstly,and then descented gradually after 2^nddays.For the groups of 8mg/ml and 16mg/ml,the OD of Bel-7402 sharply decreased in 1^stday(p＜0.01).The growth curve of L-O2 showed that cell proliferation was fast.There was no differences existed among the contrast and 4mg/ml,8mg/ml EEC concentration groups(p＞0.05).The growth curve of L-O2 in 16mg/ml concentration group showed that cell proliferation was no difference with contrast group before the 6^th day(p＞0.05).However,the cell proliferation was slower than that of contrast group in the 6^thand 7^thday(p＜0.01).3.Observed through inverted optical microscope after 48 hours,we found out that these was no evident depressant effect for cells Bel-7402 at the EEC concentration of 2mg/ml and 4mg/ml groups.At the EEC concentration of 8mg/ml and 16mg/ml,viable counts of Bel-7402 decrease, and cytomorph transform to round and shrink.Over 32mg/ml concentration of the EEC,dead cells of Bel-7402 increased,and many irregular cells could be discovered.Using a Hoechst 33258 fluorescence staining method,we observed the Bel-7402 in 8mg/ml and 16mg/ml concentration group:the increase of cells density,chromatic agglutination,chromatic margination, nuclear pyknosis and nuclear fragmentation,through inverted optical microscope in treatment groups. 4 The result of FCM displayed for treatment groups showed apoptosis ratio:The apoptosis ratio for the contrast was(2.37±0.37)%after Bel-7402 cells treated with EEC 24 hours.For the treatment groups at the concentration of 4mg/ml,8mg/ml and 16mg/ml,it was(4.99±0.76)%, (10.59±1.11)%and(15.80±1.46)%respectively.It was significant difference form treatment groups and control group(p＜0.01).After 48 hours,the apoptosis ratio for the contrast was(3.44±0.45)%.For the treatment groups at the concentration of 4mg/ml,8mg/ml and 16mg/ml,it was(5.87±0.93)%, (12.48±1.75)%,and(20.29±1.90)%respectively.It was significant difference form 8mg/ml and 16mg/ml groups and control group(p＜0.01). The apoptosis ratio in 4mg/ml,8mg/ml concentration groups were different between 24h with 48h(p＜0.01).While,there was no difference at the 4mg/ml concentration group between 24h and 48h(P＞0.05).Conclusion:1.EEC can inhibit the growth of the Bel-7402.The inhibitory effect is time-concentration dependent.2.EEC can induce Bel-7402 cell line to apoptosis at 8mg/ml concentration,and had no transparent inhibiting on L-O2. Chapterâ…¢To explore the mechanisms of apoptosis in human Bel-7402 cell line treated with EECObjectives:To explore the effect of antitumor molecular mechanisms,the HCC Bel-7402 cell line were treated with various concentraitons of EEC and the MAPK signal transduction pathway were detected by RT-PCR and western blot.Methods:Bel-7402 cells were treated with various concentrations of EEC(4mg / ml,8mg/ml,16mg/ml).The level of ERK,JNK,p38,p53,Bcl-2,Bax and caspase-3 were measured by reverse transcfiption-polymerase chain reaction analysis(RT-PCR).The expression of phosphorylated ERK1/2, JNK1,p38 in Bel-7402 cells were measured by western blot,which were treated with concentrations of EEC and MAPK inhibitors respectively.(ERK MAPK inhibitor:PD98059,JNK MAPK inhibitor:SP600125,P38 MAPK inhibitor:SB203580).All the detection items in this study were repeated 3 times.Results:The expression of JNK,p53,Bax and caspase-3 in EEC groups was upgrade significantly than that of contrast group(P＜0.01),and the expression of ERK and Bcl-2 was significantly weaker than that of contrast group(P＜0.05).The expression of p38 was no significant difference among EEC groups and contrast group(P＞0.05).It was shown that the activity of p-ERK1/2 in EEC groups were weaker than that of contrast group(P＜0.01). The activity of p-ERK1/2 in EEC+ PD98059 group were weaker than those of in contrast group(P＜0.01).The activity of p-JNK1 in EEC groups were higher than that of contrast group(P＜0.01).But,the activity of p- JNK1 in EEC+SP600125 group were weaker than those of in 16mg/ml concentration group,it were higher than those in contrast group(P＜0.01).The activity of p-p38 was no significant difference among EEC groups,but the activity of p-p38 in EEC+ SB203580 group were weaker than those of in normal control group(P＜0.01).The linear correlation analysis results showed that the expression of ERK in EEC group were negatively related with p53,Bax and caspase-3 (r=-0.965,P＜0.01;r=-0.974,P＜0.01;r=-0.974,P＜0.01),and were positively related with Bcl-2(r=0.946,P＜0.01).The expression of JNK in EEC group were positively related with p53,Bax and caspase-3(r=0.913,P＜0.01; r=0.931,P＜0.01,r=0.937,P＜0.01),and were negatively related with Bcl-2 (r=-0.883,P＜0.01).Conclusion:1.EEC maybe activate JNK1 pathway and enactivate ERK1/2 pathway to induce apoptosis of Bel-7402.2.The activation of JNK1 pathway and enactivation of ERK1/2 pathway may upgrade the expression of p53,Bax and caspase-3 and downgrade the expression of Bcl-2 in EEC group.It hint that EEC maybe inhibit Bel-7402 cell line to growth by inducing it to apoptosis in multi-channels.