金雀异黄素对肾癌细胞GRC-1增殖及p27表达的影响

Effect of Genistein on Proliferation of Renal Cell Carcinoma Cell Line GRC-1 and Its Influence to p27 Expression

背景及目的:肾癌发病率在东西方国家间有较大差异,而东方国家人群中豆制品的消耗量较高可能是其肾癌发生率较低的原因之一。本实验拟研究大豆来源的酪氨酸蛋白激酶抑制剂金雀异黄素(genistein)对肾癌细胞系GRC-1细胞的增殖的影响,并初步探讨此种影响与抑癌基因p27的关系。方法:使用倒置显微镜、MTT法、流式细胞仪观察肾癌细胞系GRC-1细胞在受到金雀异黄素作用后其细胞增殖情况的变化;并用Westernblot法测量GRC-1细胞中P27蛋白表达水平的变化。结果:(1)经20μmol/L金雀异黄素处理的细胞变为梭形,伪足减少,核分裂相减少,细胞间连接减少;而经过40μmol/L金雀异黄素处理出现大量细胞碎片,细胞形态极度不规则,有丝分裂相少见,细胞间少有连接。(2)经20μmol/L金雀异黄素处理72h的细胞与对照组相比,其存活率为45.72%,其中有73.8%的细胞处在G1期,26.2%的细胞处在G2期;对照组为G1期31.6%,G2期3.8%。(3)经20μmol/L金雀异黄素处理72h的细胞裂解液中P27蛋白条带的灰度值为65.4±4.7,对照组为52.3±6.3。结论:金雀异黄素可显著抑制肾肿瘤细胞的增殖,使细胞周期阻滞于G1/M、G2/S期,其机理可能是通过提高肿瘤细胞中P27蛋白的表达水平实现的。

BACKGROUND &OBJECTIVE: There has always been a significant morb id ity difference of renal cell carcinoma between the Oriental and Occidental count ries. The high soybean consumption in Oriental countries may be one of the reaso ns. We investigated the effect of a tyrosine protein kinase inhibitor extracted from soybean, namely genistein, on the proliferation of a renal cell carcinoma c ell line, GRC-1; and analyzed the correlation between this effect and a well-k nown anti-oncogene, p27. METHODS: Inverted microscopy, MTT method, and flow cyt ometry (FCM) were used to examine the changes in proliferation of GRC-1 cells a fter treatment with genistein; and the intracellular p27 protein expression was determined by Western blot analysis. RESULTS: (1)After treatment with genistein, changed morphology of the GRC-1 cells was observed. Cell junctions decreased. In the presence of 20 μmol/L genistein, GRC-1 cells showed shuttle-shaped, an d fewer pseudopodia, mitoses and cell junctions were observed. In the 40 μmol/L genistein group, many cells broke into debris, and became extremely irregular i n shape. Meanwhile, mitoses and cell junctions were rarely seen. (2)Treated with 20 μmol/L genistein, 73.8%GRC-1 cells were in G1 phase, 26.2%in G2 phase 72 hours after treatment; while in control group, 31.6%in G1 phase and 3.8%in G2 phase, respectively. (3)After exposure to 20 μmol/L genistein for 72 hours, We stern blot suggested that the band of p27 was 65.4±4.7 in gray scale value, whi le the control group was 52.3±6.3. CONCLUSION: Genistein can inhibit the prolif eration of renal cell carcinoma cells, and cause cell cycle arrest at G1/M, G2/S phase. The possible underlying mechanism may involve its upregulation of p27 ex pression in the renal cancer cells.