| BackgroundThyroid cancer(TC)accounts for approximately 2.1%of all cancer diagnoses worldwide,with 77% of those diagnoses occurring in women.About two hundred and thirty thousand women and 70,000men worldwide were newly diagnosed with thyroid cancer only in 2012,with annual incidence rate of6.1/100,000 for women and 2.0/100,000 for men;The mortality is equivalent to about 0.6/100,000 females and 0.3/100,000 males.The thyroid cancer is a major public health problem worldwide because of its high morbidity,and it brings great inconvenience to the entire human life.TC can now be treated with surgical excision,radioiodine therapy,and drug targeting,but it is usually not curable,so finding more convenient and effective drug is a huge challenge for thyroid cancer.Epigallocatechin gallate(EGCG)is the main component of green tea catechins,accounting for about 59%of the total catechins.It has moderate inhibitory effects on bacteria,viruses,thrombin,inflammation,and tumors.The anti-cancer properties of EGCG has been the focus of scientific research for a long time.At present,studies have shown that it has significant inhibitory effects on malignant cancers such as stomach,rectum,liver,leukemia and pancreas.However,the anti-cancer effect of EGCG on thyroid cancer has rarely been reported.Therefore,the anti-cancer effect of EGCG on human thyroid cancer cells was studied in order to reduce the incidence of thyroid cancer in the process of drinking tea.ObjectiveTo study the inhibitory effect of EGCG on human thyroid cancer cells and its mechanisms.Methods1.Cell studies in vitro1.1 MTT cell viability test and EdU cell proliferation testMTT Cell Proliferation Assay were used to generate water-insoluble blue-purple formazan by succinate dehydrogenase reaction in living cells and store them in Mitochondria.The Dimethyl sulfoxide can dissolves formazan,and the color of the solution is proportional to the amount of formazan within a certain range,and the absorbance is proportional to the content of formazan at 490 nm by enzyme labeling.Therefore,it is often used as a method to determine the number and activity of living cells.EdU(5-Ethynyl-2'-deoxyuridine)is a thymidine analogue.In the process of DNA replication,EdU can replace thymidine(T)to infiltrate into the newly synthesized DNA strand,through a"Click"reaction,and then through the specific fluorescence reaction between EdU and Apollo dye,the quantity of newly synthesized DNA can be analyzed by detecting the fluorescence.1.2 One Step TUNEL Apoptosis AssayIn the process of cell apoptosis,specific DNA endonucleases are activated,which can break the DNA strands of chromosomes,thus forming many exposed sticky ends.Under the catalysis of TdT,deoxyribonucleotides are labeled with fluorescein(FITC)dUTP,which can be used to detect cell apoptosis.The control or surviving cells were less likely to produce 3'-OH without labeled with dUTP,and could not be stained by TUNEL reagent.The apoptosis rate of cells treated with EGCG was observed and analyzed.1.3 Cell Scratch TestThe cell scratch test is a relatively simple experimental scheme to detect the characteristics of cancer cell wound healing.By artificial scratching on the cultured monolayer cells,the cells at the bottom of the dish were exfoliated and presented as a"groove mark".Then the cells were cultured to simulate the wound healing experiment in vitro,and then the ability of drugs to move and migrate cancer cells was tested.When the cells were cultured in logarithmic phase,the serum-free complete medium with equal final concentration of EGCG of 10mM,25mM,50mM,100mM and 200mM was added.Scratch photographs were taken at 0 h,wound healing status was recorded at 12 and 24 hours respectively,and wound healing rate was analyzed and discussed.1.4 Cell migration and invasion experimentsThe bottom of the Transwell chamber was 8mm in diameter.Cancer cells were laid in the upper layer of the chamber.The process of cancer cells entering the blood vessel and lymphatic vessel through the small hole was detected,and a matrix was used to simulate the invasion process of cancer cells in the bottom of the Invasion chamber.At the bottom of the 24-well plate,600-800ml complete culture medium containing 20%serum and 200ml cell suspension was added to the upper chamber,and EGCG solution was added at the same time.The final concentration of EGCG was 0,10,25,50,100,200mM.The cells were kept at 37 C,5%CO2 incubator for 24 hours.The migration and invasion of cells were counted by crystal violet staining.1.5 Cell Cycle Detection ExperimentDNA synthesis is a necessary process in cell cycle:G1 phase cells have a copy of genome,S phase cells actively participate in DNA synthesis,and the amount of DNA in the G2 phase nucleus is twice that of the G1 phase.Cell DNA content can be measured by dyeing with fluorescent dyes and quantifying with flow cytometry.1.6 Western blotWestern blotting is a method for detecting protein expression.Currently,Bcl-2/Bax,Cleaved Caspase-3 and Cleaved PARP are the main target proteins for the mechanism of apoptosis,and the EGFR-RAS/RAF/MEK/ERK signaling pathway protein expression levels were used to study anticancer mechanisms.2.Animal studies in vivo2.1 Construction of tumor model and AdministrationTT/TPC-1/ARO of human thyroid cells in logarithmic growth phase was digested and collected. The cells were suspended with sterile saline.The cell density was 5×10~6/200ml.The cell suspension was injected subcutaneously into the right forelimb of nude mice with a disposable sterile syringe.After the transplanted tumor was 200?l of a sterile physiological saline solution having an EGCG concentration of10,25,50,100,and 200?M was injected subcutaneously into each tumor per group per day,and each controlwhile the Control group was injected with 200?l of sterile physiological solution.brine.The dosing cycle is four weeks(28 days).2.2 HE staining and immunohistochemistryHE staining and immunohistochemical can be used to observe and detect the morphological structure of healthy tissue sections and pathological sections,which is the most basic way of pathological detection.Immonohistochemistry is based on the theory of specific reaction between antigen and antibody.It uses color reaction to analyze the proteins in tissues and cells.Results1.MTT and EdU results manifested that,with the increase of EGCG concentration,the cell viability of human thyroid cancer cells decreased gradually(p<0.05),and the cell proliferation rate decreased gradually(p<0.05)in a dose-dependent manner.2.One-step TUNEL apoptosis experiment revealed that the apoptotic rate of human thyroid cancer cells increased with the increase of EGCG concentration(p<0.05).3.The results of cell scratch,migration and invasion experiments exhibited that the wound healing rate of cell scratch decreased gradually with the increase of EGCG concentration(p<0.05),while the cell migration and invasion rate decreased gradually(p<0.05)compared with Control group.4.The results of cell cycle assay displayed that compared with Control group,the DNA content of the S phase increased with the increase of EGCG concentration of EGCG(p<0.05)and the G2 phase decreased significantly(p<0.05),which means G1/S phase is blocked in the cell cycle.5.The results of HE staining showed that compared with Control group,EGCG treated group had lighter HE staining,looser cell arrangement and larger intercellular space,and obvious tissue vacancy and apoptosis areas were observed at the concentration of 100-200mM6.Immunohistochemical results illustrated that,compared with the control group,KI67 staining area decreased and KI67 positive cells decreased with the increase of EGCG concentration,which indicated that the growth of human thyroid cancer cells(p<0.05);the density of microvessels decreased and the number of microvessels decreased with the increase of EGCG concentration(p<0.05),the percentage of Cleaved-PARP positive cells increased,and the higher EGCG concentration,the stronger apoptosis of tissue cells(p<0.05).7.Western blot showed that compared with Control group,the relative expression of apoptosis-related Proteins Bax/Bcl-2,Cleaved Caspase-3 and Cleaved PARP increased with the increase of EGCG concentration(p<0.05)The WB results of the mechanism pathway showed that the expression of p-EGFR,Ras,p-RAF,p-MEK and p-Erk1/2 protein decreased gradually and decreased with the increase of EGCG concentration(p<0.05).Conclusion1.EGCG can significantly reduced the proliferation,survival,migration,invasion and arrested cell cycle progression of human thyroid cancer cells.2.EGCG can significantly inhibit the growth of allograft tumors,significantly reduce the proliferation of human thyroid cancer cells in nude mice,and reduce the degree of infiltration of CD-31labeled microvessels in thyroid tumors.3.In terms of mechanism of action,EGCG may inhibit the expression of Ras,p-RAF,p-ErK,and p-MEK by inhibiting the relative expression of p-EGFR,thereby exerting an anti-human thyroid cancer effect.4.The Anti cancer effect of EGCG was dose dependent,that is,the Anti cancer effect of EGCG increased with the increase of the concentration. |
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