유방암 세포 주 MCF-7에서의 녹차 추출물이 p53 경로에 미치는 영향

Effects of Green Tea Extract on the p53 Pathway in the MCF-7 Breast Cancer Cell Line

Abstract

녹차(GT) 추출물의 효과를 인간 유방암 유래 세포인 MCF-7 세포를 사용하여 조사 하였다. GT추출물의 세포 독성 효과를 MTT 방법을 사용하여 관찰한 결과, MCF-7 세포는 현저한 세포 독성 효과를 보였고, 이 독성 효과는 GT추출물 농도 의존적으로 증가하였다. p53과 관련 단백질인 p21/cip1과 CDK2의 연관성을 조사하기 위해 GT추출물 처리 후 웨스턴 분석법을 통해 이들 단백질의 발현을 조사하였다. GT추출물 처리 후, MCF-7 세포에서 p53 단백질의 양은 농도에 따라 현저하게 증가 하였다. p21/cip1 단백질의 발현은 낮은 농도의 GT추출물에서 증가되며, 고농도에서도 감소하지 않았다. 그러나 CDK2의 단백질의 양은 높은 농도의 GT추출물에서 CDK2 발현의 급격한 감소가 관찰되었다. 이 결과는 GT추출물의 처리는 MCF-7 세포에서 p53와 p21/cip1를 증가시켜, 그 결과로 활성화 된 p21/cip1는 CDK2의 발현을 억제 함을 나타내고 있다. GT추출물이 MCF-7 세포의 세포주기에 어떤 영향을 미치는지 확인하기 위하여 FACS 분석으로 관찰한 결과, MCF-7 세포에서 세포주기의 G1 단계가 점차 증가하는 결과를 보였다. 이 결과는 GT추출물의 유방암 세포에서의 항암 효과는 세포주기의 G1 단계에서 MCF-7 세포를 정지시키는 p53에 의해 조절된다는 사실을 명확하게 보여 주고 있다.

The effects of a green tea extract (GTE) were examined using the MCF-7 human breast cancer cell line. Cell viability assays using 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays revealed that GTE had a significant cytotoxic effect on MCF-7 cells, depending on the concentration of GTE. Western blotting of p53 and its related proteins, p21/cip1 and CDK2, after GTE treatment revealed that a significant and concentration dependent increase in p53 protein in response to GTE. The levels of p21/cip1 proteins were also increased at low GTE concentrations were significantly increased even at the highest GTE concentrations. However, the level of CDK2 was significantly decreased by treatment with high concentrations of GTE. These results indicate that treatment with GTE increased the p53 level in MCF-7 cells, and this activation of p53 markedly elevated the levels of p21/cip1proteins, which, in turn, inhibited CDK2 expression in the MCF-7 cells. The inhibition of CDK2 expression might then affect cell cycle progression. Subsequent FACS analysis indicated that GTE treatment the gradually increased progression of the MCF-7 to the G1 phase. These results clearly demonstrate that the anti-tumor effect of GTE in MCF-7 cells is regulated by p53 arrest of the MCF-7 cells at the G1 stage of cell cycle.

Fig. 1. Cytotoxicity of MCF-7 cells by different concentrations of green tea extract. MCF-7 cells were treated with GT extract (3, 6, 9, 12, and 20 ug/ml) and 50% of DMSO was used as control (Ct: 0) for 16 hr and cell viability was measured by the MTT assay.

Fig. 2. Effect of green tea extracts on the level of p53, p21/cip1 and CDK2 in MCF-7 cells. MCF-7 cells were treated with GT extract (0, 3, 9 and 20 ug/ml) for 16 hr and the expression of p53, p21/cip1 and CDK2 protein was analyzed by Western blot.

Fig. 3. The distribution of MCF-7 cells in the cell cycle after treating with green tea extract for 16 hr. MCF-7 cells were treated with 0 and 3, 9 and 20 ug/ml of GT extract and cells were evaluated for DNA contents by flow cytometric analysis.

Fig. 4. Schematic representation of the effects of green tea extract on the cell cycle of MCF-7 cells. The treatment of GT extract increases p53 and p21/cip1 in MCF-7 cells. GT inhibits the CDK2 expression and induces growth arrest at the G1 stage of cell cycle in MCF-7 cells.

Table 1. The distribution of MCF-7 cells in the cell cycle after treating with 0 and 3, 9 and 20 ug/ml of GT extract