| Tea is a favorite beverage containing a variety of bioloigical active substances,and the core component is epigallocatechin-3-gallate(EGCG).In this study,we are focusing on antitumor effects and their mechanisms,in order to find valuable variety with application potential of EGCG-rich tea.The following approaches have been applied in these studies:first of all,by using High Performance Liquid Chromatography(HPLC),we screened a series of tea varieties to find the best ones with rich EGCG Secondly,after the preparation of tea extract and tea medicated rat serum,we cultured tumor cell lines and investigated the inhibition effects of tea extract and tea medicated rat serum on the growth of tumor cells.Third,according to the results of experiments in vitro,the best variety was chosen for further studies.We established the xenograft model of tumor cell,and study on in vivo antitumor effects of the tea extract with EGCG-rich tea.Finally,through the detection of cell cycle and apoptosis by flow cytometry,observation of the ultrastructure of tumor cells and tumor tissue by scanning electron microscope,we drew the conclusion of the anti-tumor mechanism of EGCG rich tea.At the same time,in order to better study on the antitumor mechanism,we used virtualcomputer software to mimicmolecular docking of5 kinds of bioactive substances in tea(EGCG as the core components and 4 others,EC analysis,ECG,EGC,L-theanine)to the proteins in the MAPK and NF-B signaling pathways.We confirmed the potentical receptor proteins for the active substance by Western Blotting and RT-PCR experiments.EGCG contents in different varieties were detected by high performance liquid chromatography,and the variety 09-5 was found with highest EGCG at 13.91%?Therefore,the variety of 09-5 was chosen for the tea extra and tea medicated rat serum preparation for the following studies.The in vitro inhibition experiment showed that the most sensitive cell line to tea extract and medicated serum was human hepatoma cell line HepG2 among the tested tumor cell lines.Then,we selected HepG2 line as the experimental material for the further study on the mechanism of the antitumor effect of EGCG in tea.In vitro tests showed that the teas extract and tea medicated serum significantly inhibited the growth of human hepatocellular carcinoma cell line HepG2 in a concentration dependent manner.The highest inhibition rate of the medicated serum on the cell line in vitro was54.82%detected at the best treatment.When the tea extracts at high,middle dosages were applied to the cell cultures,theinhibition rates were detected at 86.95%and 60.42%respectively.The cell cycle and cell apoptosis was detected by the flow cytometry,the treatments of the tea extract,tea group and drug serum showed that some impacts on the human hepatocellular carcinoma cell line HepG2 when they werecompared to the minuscontrol group without tea component.While the percentage of cells in G2/M phase increased,the percentage of cells in G0/G1 phase was significantly reduced,which showed the cell cycle arrests on the cell line.The apoptosis rate was significantly increased when they were compared to minus control(P<0.05).The Western blotting showed that the high and middle dosage of EGCG tea extracts significantly inhibited on the protein levels of ERK1 and P38 alpha.In the EGCG rich tea extract doseage and the medicated serum treatment group,the P65 protein level was significantly lower than that in the control group(P<0.05).The results of RT-PCR showed that the transcription levels of P65 mRNAin high dosage tea extract treatment and tea medicated serum group were significantly decreased(P<0.05),the expression of ERK1 and P38 mRNA were significantly lower than the control group(P<0.05).Hower,the expression of K-ras and Raf-1 transcription level and proteinlevels were not obviously differentIn vivo study showed that inhibition of EGCG richtea on transplantation tumor in nude mice was 84.11%.The commonly used antitumor drug,fluorouracil,was used as the positive control.Results showed that inhibition of fluorouracil chemotherapy was 80.84%,whilethe combination treatment of tea and the chemical drug increased inhibition efficiency to 94.26%,which is significantly different from the effect of the fluorouracil treatment(P<0.01).The detection methods were used by Quantitative PCR for transcription expression,Western blotting for protein,flow cytometry for cell cycle and apoptosis,the results showed the similar to the data of in vitro experiments.Compared to the control group,P65 mRNA expression in tumor tissues was significantly reduced(P<0.05),tea plus fluorouracil group ERK1 and P38 alpha mRNA transcription decreased significantly(P<0.05).The expression of ERK1 and P38 alpha protein in EGCG richtea treatment group was significantly lower than that of the control group(P<0.05).However,the expression of mRNA and protein of K-ras and Raf-1 in tumor tissue were no significant difference among all the treatments,which were the same as the in vitro data.Compared with the control group,treatment group with EGCG rich tea in tumor tissue,cell cycle arrest in G2/M phase,the cell apoptosis rate increased significantly(P<0.05).Drug toxicology tests of the red blood cell index showed that no toxic effect of tea on blood system.Dectection of biochemical indicators in serum showed that the function of liver and kidney,liver and kidney were normal under tea treatment.Whilethe common chemical drug showed obvious toxicity,the tea plus fluorouracil combination treatment improved significantly functions of liver and kidney functions with detected differences of ALT,AST and BUN(P<0.05).Ultrastructural observationunder transmission electron microscope on tumor tissuefound that tea treatement induced more swelling mitochondria and endoplasmic reticulum,poor cell membrane and nuclear membrane integrity,obvious marginalizedchromatin condensation,apoptotic fragmentation.This study suggests that EGCG rich tea has significant anti-tumor effects in vitro and in vivo,and little toxic side effect;combined with chemical drugs,not only can enhance the antitumor effect of chemotherapy drugs,but also can reduce the liver and kidney toxicity of chemicals.Tea extract and serum can down-regulate the expression of multiple target proteins in the MAPK and NF-kB signal pathways with protein activity inhibition,cell cycle arrest,apoptosis,then achieve the anti tumor effects.In conclusion,we found that EGCG richtea cultivar 09-5 has strong anti-tumor effect,and our studies further proved that tea could be used as a kind of special food for human health benefits. |
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