| Objective:The mice model of human gastric cancer cell MGC-803 transplanted in nude was established in this study to observe the effect of EGCG which is the extraction of green tea on the growth of gastric cancer cells transplanted nude mice.In this transplanted nude mice,we studied the expression of SMO/Gli-1 signal pathway and apoptotic relevant indices affected by EGCG.We also cultivated the human gastric cancer cell line MGC-803 in vitro to observe the effect of EGCG on the microstructures and apoptotic morphologic variations of gastric cancer cells.More important,our study explored the impact of EGCG on the expression of apoptotic relevant factors and the relationships between EGCG and SMO/Gli-1 signal pathway.Using both in vivo and in vitro experiments,we researched if the drug of EGCG had the role of inhibiting tumor and explored its functional mechanisms.Using EGCG to treat gastric cancer may become a new treatment method for clinic.Our results may provide the experimental evidences and scientific supports to the clinical use of traditional Chinese medicine to treat malignant tumors and for further relevant studies.Materials and methods:Thesis 1We used human gastric cancer MGC-803 cell line for subcutaneous injection into the axilla of nude mice to establish animal model of tumor transplantation.Eight days after injection of gastric cancer MGC-803 cell,we chose the successfully transplanted mice model and recorded the weight of each mouse.All mice were divided into four groups at random: the control group,intraperitoneal injection of tri-distilled water everyday;low-dose of EGCG group,intraperitoneal injection 10mg/kg of EGCG everyday;high-dose of EGCG group, intraperitoneal injection 20mg/kg of EGCG everyday;5-FU group,injection of 5-FU(50mg/kg)near the tumor every two days.5-FU group was the positive control group,and different concentrations of EGCG groups were the experimental groups.Administrating of different drugs was continued for 2 weeks.The consumption,drinking,spirit and activity aspects of nude mice were observed in each group.In the meanwhile,to observe the tumor growth status,we measured long diameter(major axis A)and short diameter(major axis B)of the superficial masses for calculating the sizes according to the formula(V=1/2×A~2×B).The body weight of each nude mouse should be measured before and after drug intervention,and then made comparisons between them.We sacrificed all the mice 2 weeks after drug intake and removed the subcutaneous tumors.The tumor growth inhibition rates were calculated by the weight of tumors,using the formula =(weight of control group –weight of experimental group)/ weight of control group×100%.HE staining method was used to observe the cell composition and morphologic variations of lump sections in the tumor samples,and compared the changes among groups.Thesis 2We explored the specific mechanisms of the changes of gastric cancer cell affected by EGCG using the tumors specimens from thesis 1,exploring the mechanisms of the effect of EGCG on Shh signal pathway and its impact on apoptotic relevant indicators.First,we observed the changes of tiny structures of the gastric cancer cell in the tumors specimens of different groups under transmission electron microscope,evaluating whether the characteristic changes of apoptosis existed in tumors specimens and comparing the differences between groups.Second,we did immunohistochemistry staining for tumors specimens of each group to detect the expression of apoptosis related indicators,such as bcl-2,bax and caspase-3.We evaluated the expression of each indicators according to the dyeing intensity and density.Third,we applied RT-PCR method to detect the mRNA expression of Gli-1,SMO,bcl-2,bax and caspase-3 in different groups of tumor specimen to compare if there were statistical significant differences.Fourth,we applied Western blotting method to detect the protein expression of Gli-1,SMO,bcl-2,bax and cleaved caspase-3 between each group.Thesis 3We cultured human gastric cancer cell MGC-803 in vitro to observe whether the experimental drug of EGCG had the effect of inhibiting the growth of gastric cancer cells as well as inducing process of apoptosis,more important,to explore its mechanisms.At first,MTT method was used to observe tumor cells survival inhibiting rate according to the OD value in different groups with different concentrations of EGCG.We selected more appropriate concentration of EGCG used in vitro according to the results of the MTT experiment.We divided the cultured gastric cancer cells into four groups:(1)the control group,this group is the cultured gastric cancer cells without any medicine administrated;(2)the EGCG group,the cultured gastric cancer cells were dealt with 20μmol/L EGCG for 24 hours;(3)the Gli-1 transinfected group,the gastric cancer cells of this group was transinfected by the Gli-1 plasmid to make the Gli-1 overexpressed;(4)Gli-1 transinfected + EGCG group,after successfully transinfection with Gli-1,the cultured cells were administrated with 20μmol/L EGCG for 24 hours.We used TUNEL staining to observe apoptosis situation of gastric cancer cells between different groups to evaluate if EGCG can induce apoptosis of gastric cancer cell in vitro.By FCM technique we wanted to study whether cell cycle of gastric cancer cells is altered significantly by EGCG and compare the difference among groups.Annexin V-PI staining method was adopted to compare the apoptotic assay between different groups.The ratio of early stages apoptosis and later stages apoptosis between different groups was also calculated.We used RT-PCR and Western blotting methods to detect mRNA and the protein expression of Gli-1,SMO,bcl-2,bax and caspase-3.The results of RT-PCR and Western blotting could reveal the effect of EGCG on the SMO/Gli-1 signal pathway.We overexpressed Gli-1 by Gli-1 plasmid transinfection to confirm the cancer inhibiting effect of the experimental drug of EGCG and the relationship between EGCG and SMO/Gli-1 signal pathway in gastric cancer cell.Results:Thesis 11.The effects of medicine intervention on the general state of gastric cancer cell transplanted nude miceThere were no side effects such as significant redness,swollen and ulceration on the local injection site of human gastric cancer cell line MGC-803 transplanted nude mice.The lump grew up gradually and up to 0.8cm in diameter 8 days after human gastric cancer cell line transplanted.The transplanted nude mice of this situation were considered as successfully transplanted.Successfully transplanted nude mice were divided into four groups randomly for administrated with different drugs,without significant differences in body weight among groups.There were no anorexia,uneasy,diarrhea in the control group and EGCG groups with normal activities and communication in these groups.However,were almost the same among control group and different concentration of EGCG groups.The mice of 5-FU group were with poor spirit and less meal,easy to be disturbed,and diarrhea occasionally,less communications and activities,less consuming and drinking than other groups.The average weight of mice in 5-FU group(19.30±1.54g)was significant less than other groups(P<0.05)after 2 weeks of medicine administration.There were no dead mice during the whole experimental process.2.The effect of drug intervention on body weight of transplanted nude miceThere were no significant difference in body weight among four groups before the administration of medicine(B),the average value of which was about 18.6g.We weighed mice in each group again 2 weeks after administration of medicine(A).The average value of body weight in control group was 21.13±1.43 g,21.73±1.05 g in low concentration of EGCG group,21.85±1.52 g in high concentration of EGCG group.There were no significant statistic changes among these three groups(P>0.05).But the average mice weight of 5-FU group was 19.3±1.54 g,much less than any other group,with significant statistic differences compared with any other group(P<0.05).3.The effect of drug intervention on lump volume of transplanted nude miceWe sacrificed all the mice and resected the lump from the mice intactly after 2 weeks of intervene,calculated the tumor inhibiting ratio according to the lump volume.The tumor grew up gradually with the time.The growth rates of the lump in groups of EGCG and 5-FU administration were significantly slower than control group.From the third day of medicine intervene,the lump volume of 5-FU group was significantly smaller than control group(P<0.05).The same result was found in the the high concentration of EGCG group.From the fifth day of intervene,the the lump volume of low concentration of EGCG group was significantly smaller than control group(P<0.05).From the seventh day of intervene,the the lump volume of high concentration of EGCG group was much smaller than the low concentration of EGCG group(P<0.05).The lump volumes were 1921±183mm~3 in control group,1174±294mm~3 in low concentration of EGCG group,730±123mm~3 in high concentration of EGCG group,679±202mm~3 in 5-FU group.The lump volume of 5-FU group was a little smaller than the high concentration of EGCG group,but with no significant statistic difference(P>0.05).4.The effect of drug intervention on weight of lump and tumor inhibiting ratioWe weighed the lump weight and calculated the average value of each group.The values of lump weight were 1.13±0.09 mg in control group,0.89±0.13 mg in low concentration of EGCG group,0.58±0.06 mg in high concentration of EGCG group,0.52±0.07 mg in 5-FU group.There were significant statistic difference between the control group and the other three groups(P<0.05).However the weight of high concentration EGCG group and 5-FU group are almost the same,without significant difference(P>0.05),but both of them were much less than the low concentration of EGCG group,with significant statistic difference(P<0.05).According to the weight of lump,we used the following formula to calculate the tumor inhibiting ratio of each group.The ratio of tumor inhibiting=(weight of control group-weight of experimental group)/ weight of control group×100%,by which we can qualify the difference among groups.We set the tumor inhibiting ratio of control group as 0%.The ratio of low concentration of EGCG group was 21.24%,but 48.67% in high concentration of EGCG group,53.98% in 5-FU group.5.Using HE staining method to observe the morphologic change of tumor tissueUnder the microscope,we found that the cell structure of control group was clear,which suggested that gastric cancer cell growth in good condition.However,in the other three groups,the tumor cell became round,pyknosis,and cell spacing changed obviously.Nucleus edge setting was similar to the change of cell apoptosis.This phenomenon became more obvious in the group of high dose of EGCG,but the most significant change was found in 5-FU group.Thesis 21.Observing the micro-structure changes of the cells under transmission electronic microscopeWe found normal status MGC-803 gastric cancer cells in the control group.Low concentration of EGCG group showed a nuclear heterochromatin increased and edge set,enriched cell volume,round shape.In high concentration of EGCG group,cell volume became much more smaller,vacuolated,uneven pyknotic nuclei present and increased surrounding spaces.Sprout and bubbles in the cell membrane of apoptotic body could be found.In 5-FU group,the nucleus were broken,degraded and digested.The formation of cavitation and high electron density material also existed.Apoptotic body increased obviously in 5-FU group.2.Apoptosis of the gastric cancer cell in immunohistochemical staining slicesThe results of immunohistochemical staining method showed that EGCG groups and 5-FU group can obviously inhibit the expression of apoptosis gene bcl-2,and increase the expression of bax and caspase-3.The effect became more apparent in high concentration of EGCG than that of lower concentration,which indicated dose-effect relationship.But there was no significant statistic difference between 5-FU group and high concentration of EGCG group.3.Expression of SMO and Gli-1 and apoptotic relevant indicators of bax,caspase-3 and bcl-2 in RT-PCRThe results of RT-PCR showed that EGCG could significantly reduce the mRNA expression of SMO,Gli-1 and bcl-2,but increase the mRNA expression of bax and caspase-3. This effect became more obvious as the concentration of EGCG increased(P<0.05).These results between high dose of EGCG group and 5-FU group were similar,without any significant statistical difference.4.Expression of SMO and Gli-1 and apoptotic relevant indicators of bax,caspase-3 and bcl-2 in Western blottingAccording to the results of Western blotting,we could clearly observed that EGCG could reduce the protein expression of SMO,Gli-1 and bcl-2 significantly compared with the control group,but increase the protein expression of bax and cleaved caspase-3,which became more apparent as the concentration of EGCG increased(P<0.05).The results between high dose of EGCG group and 5-FU group were similar,without any significant statistical difference.Thesis 31.Selected the appropriate concentration of EGCG by MTT assayTook 0,10,20,40 μmol/L of EGCG to do the MTT experiment,which can be used to observe the surviving situation of tumor cells under different concentrations of EGCG.Results of MTT showed that the survival rate of gastric cancer cells(OD value)declined gradually with the increase of drug concentrations and administration time.For 24 hs,OD values of 20 μmol/L and 40 μmol/L EGCG were 0.50±0.07 and 0.45±0.06 respectively(P>0.05).For 48 hs,OD value of 20 μmol/L and 40 μmol/L EGCG were 0.45 ± 0.06 and 0.35± 0.06(P>0.05).The concentration of 20 umol/L EGCG was chosen for the following experiment in vitro according to the the principle of low dose and equivalent outcome.2.Gli-1 plasmid transinfection to overexpress the expression of Gli-1Set the control group,the negative reagent transinfection group and Gli-1 plasmid transinfection group.We dectected the Gli-1 expression by Western blotting to evaluate the transfection efficiency After transinfection.The results of Western blotting showed that there was no significant statistic difference between control group and negative transinfection group(P>0.05)indicating no obvious influence by transinfection.The expression of Gli-1 protein in transinfection group was significantly increased compared with the control group(>2 times),which proved successfully transinfection.3.TUNEL stainResults of TUNEL staining experiment showed that little apoptotic green cells were found in control group,but less in Gli-1 transinfection group.The apoptotic cells became increased when we administrated EGCG.There was a decreased in the number of apoptotic cells in the group of Gli-1 transinfection + EGCG,which illustrated the fact that the transinfection of plasmid Gli-1 can reduce the function of inducing apoptosis created by EGCG.4.FCM analysis the cell cycleFlow cytometry technique showed that the apoptotic cells in EGCG group was obviously increased and number of cells in DNA duplication phase(S phase)cells was reduced compared with control group.The apoptotic cells became decreased dramatically in transinfection group,with decreasing number in G1 phase but increasing number in S phase.Administration of EGCG after transinfection,the ratio of apoptosis became increased again,but less than EGCG group which suggested the function of inducing gastric cancer cell apoptosis by EGCG can be inhibited by Gli-1 plasmid transinfection.5.The analysis of Annexin V-PI double dyeingResults of Annexin V-PI double dyeing showed the cell ratio of early apoptosis(Annexin V+ PI-)is 10.4% and later apoptosis(Annexin V+ PI+)is 5.6% in control group.The cell ratio is up to 45.0% of early apoptosis and 16.7% of later apoptosis in EGCG group indicating that EGCG can induce apoptosis of hunman gastric cancer cell line MGC-803,as well as the spontaneous necrosis of cancer cells.Transinfection of Gli-1 plasmid decreased the ratio of both early and later apoptosis when compared with the control group,which suggested overexpression of Gli-1 can inhibit the cell apoptosis created by EGCG.When we administrate the EGCG to the Gli-1 transinfected gastric cancer cell,the ratio of apoptosis increased again,with 37.2% of early apoptosis ratio and 9.6% of later apoptosis ratio.The total ratio of apoptosis was up to 46.8%,less than the total ratio of EGCG group,which meant that Gli-1 was tightly related to the function of inducing apoptosis exerted by EGCG.6.RT-PCR to acquire mRNA quantityAccording to the results of RT-PCR,EGCG could obviously increase the mRNA expression of bax and caspase-3,and reduce mRNA expression of of SMO,Gli-1 and bcl-2 compared with the control group(P<0.05).Administration of EGCG after successful transinfection of Gli-1,the increased expressions of bax and caspase-3 were less than EGCG group(P<0.05).Meanwhile,the decreased expressions of SMO,Gli-1 and bcl-2 were also less than EGCG group(P<0.05).Experimental results illustrated that the function of inducing apoptosis induced by EGCG were relevant to Gli-1.7.Western blotting to acquire protein quantityAccording to the results of Western blotting,EGCG group could obviously increase protein expression of bax and cleaved caspase-3,and decrease protein expression of SMO,Gli-1 and bcl-2 compared with control group(P<0.05).Administration of EGCG after successful transinfection of Gli-1,the increased expressions of bax and cleaved caspase-3 were less than EGCG group(P<0.05).Meanwhile,the decreased expressions of SMO,Gli-1 and bcl-2 were also less than EGCG group(P<0.05).In accord with the results of RT-PCR,EGCG induced apoptosis of gastric cancer cells through the changes of Shh signal pathway.Conclusions:1.EGCG could inhibit the growth of lump from gastric cancer cells MGC-803 transplanted nude mice,which showed a dose-effect relationship.The tumor inhibiting effect of high concentration of EGCG(20mg/kg)was similar to 5-FU group,but EGCG had less side effects than 5-FU.2.EGCG could block the cell cycle and inhibit cell proliferation through affecting the SMO/Gli-1 signal pathway,making the proliferation of human gastric cancer cell cycle arrest in G1 phase from getting into S phase(DNA duplicating stage).3.EGCG can induce the apoptosis of human gastric cancer cell line MGC-803 through suppressing the SMO/Gli-1 signal pathway,up-regulating the expression of pro-apoptotic gene of bax and caspase-3 and down-regulating the inhibiting-apoptotic gene of bcl-2. |
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