The Function And Mechanism Of EGCG On Canine Breast Tumor Cells In Vivo And In Vitro

This study explored the anti-tumor function of EGCG through stimulation test invitro and mice test in vivo respectively, canine mammary cell line CMT1211as amodel.The cell line CMT1211was cultured in Dulbecco's Modified Eagle's medium(DMEM) containing10%of FBS (fetal bovine serum), and incubated at37℃, with5%CO2and constant humidity. When cell had a confluence of about80%,intervention treatment was given. The CMT1211cells were divided into a controlgroup and a treatment group of different concentrations of EGCG(0,20,40,60,80,100,120μg/mL). At6,12,24,48hours after intervention, the changes of cell number,morphology and state were observed by phase contrast microscope. The inhibitions ofcell proliferation were detected by MTT assay. The change of cell cycle phase and cellapoptosis was analyzed by flow cytometry. The transcription and translation of thegenes p53,bax,bcl-2were observed in canine mammary gland tumor cells after24htreated by EGCG and when EGCG concentration was80μg/mL, the transcription andtranslation of those genes were also detected at6h,12h,24h,48h by RT-PCR, obtainedthe results:After being treated with EGCG solution of different concentrations, the number,morphology and state of CMT1211cells all changed correspondingly. Compared withthe control group, the cell proliferation of treatment group became slower, and cellsbecame weaker. The cell number decreased and cell shapes were slender andcollapsed observing under the microscope. Besides, these changes were time and dosedependent. From the results of MTT assay, we can see that of EGCG had theinhibitory effect on the CMT1211cell proliferation, IC50=83.437μg/mL.Detected the cell cycle of CMT1211by flow cytometry, it indicated that the cellcycle of treated group was blocked in the G1phase and then inhibited the cellproliferation to apoptosis. The apoptosis was related to the increase of mRNA ofgenes p53,bax and the decrease of gene bcl-2expression.In vivo tests, inoculated ICR mice using CMT1211cell to establish the transplantation tumor in ICR mice. The mice were divided into one control groupEGCG (50mg/kg) and one test group. Detected the mRNA expression of p53, bax,bcl-2of mammary cancer and studied the function of EGCG anti-cancer, finallyobtained the following results.The EGCG(50mg/kg)can effectively inhibit the growth of the canine mammarytumor. The mRNA expression of wild-type p53, bax increased, and bcl-2reduced.Conclusions：EGCG had the functions of inhibiting the growth and promotingapoptosis in CMT1211cell line. These functions were related to the up-regulating ofbax,p53and the down-regulating of bcl-2.