金雀异黄素的体内与体外抗结肠癌作用及其机制

目的 探讨金雀异黄素(Gen)对结肠癌细胞增殖、凋亡和相关蛋白表达的影响,及其抗结肠癌的作用机制.方法 采用四甲基偶氮唑蓝(MTT)法检测结肠癌细胞增殖情况,光镜和电镜下观察细胞形态学和超微结构变化,流式细胞术检测Gen对结肠癌细胞周期和凋亡的影响,流式细胞术和免疫组化法检测结肠癌细胞凋亡相关蛋白的表达情况.结果 MTT法检测结果显示,Gen对SW480细胞增殖具有抑制作用,呈明显的剂量-效应和时间-效应关系,抑制率以80μg/ml Gen作用72 h为最高(60.2%).光镜下,药物作用后细胞皱缩、破裂,胞浆中出现空泡.透射电镜下,细胞体积缩小,核染色质高度浓缩,形成沿核膜收缩的新月状小体.流式细胞术检测结果显示,0、20、40、80μg/ml Gen作用SW480细胞48 h后,增殖细胞核抗原(PCNA)蛋白的荧光指数(F1)值分别为1.49±0.02、1.28±0.04、1.14 4±0.03和0.93±0.08,血管内皮生长因子(VEGF)蛋白的F1值分别为1.75 4±0.02、1.34 4±0.06、1.32 4±0.04和1.23 4±0.04,p21蛋白的F1值分别1.26±0.05、1.36 4±0.06、1.61 4±0.03和1.73±0.03,20、40、80 μg/ml Gen组与μg/ml Gen组比较,差异有统计学意义(P<0.05或P<0.01).免疫组化检测结果显示,Gen处理组中PCNA和VEGF蛋白的表达降低,p21蛋白表达升高,与对照组差异有统计学意义(P<0.05).结论 Gen抑制结肠癌细胞的生长与诱导细胞凋亡、阻滞细胞周期于G_2/M期有关.Gen抗结肠癌的作用机制可能与下调PCNA和VEGF蛋白的表达、上调p21蛋白的表达有关.

Effects of genistein on colon cancer cells in vitro and in vivo and its mechanism of action

Objective To study the effects of genistein on the proliferation, apoptosis induction and expression of related gene proteins of human colon cancer cells in vitro and in vivo, and its mechanisms of action. Methods MTT colorimetric assay was used to detect the effects of genistein on the proliferation of human colon adenocarcinoma SW480 cells. Light and transmission electron microscopy were used to study the histological and ultrastructural changes. Flow cytometry was used to determine the effects of genistein on cell cycle and apoptosis. Flow cytometry and immunohistochemistry were used to determine the effects of genistein on apoptosis induction and expression of related gene proteins of colon cancer cells. Results The MTT colorimetric assay showed that genistein inhibited the proliferation of SW480 cells in a dose-dependent and time-dependent manner, and the highest inhibition rate was 60. 2% after 80 μg/ml genistein treatment for 72 h. The light microscopy revealed that many genistein-treated cancer cells were shrunken, disrupted,or showing cytoplasmic vacuolization. The electron microscopic examination showed cell shrinkage, nuclear fragmentation and pronounced chromatin condensation, sometimes formed crescent chromatin condensation attached to the nuclear membrane. The results of flow cytometry showed that: after SW480 cells were treated with 0, 20, 40, 80 μg/ml genistein for 48 h, the FI values of PCNA were 1.49 ±0.02, 1.28 ±0.04, 1.14 ±0.03, and 0.93±0.08; the FI values of VEGF were 1.75 ±0.02, 1.34±0.06, 1.32 ±0.04, andl.23±0.04; the fluorescence index (FI) values of p21 were 1.26 ±0.05, 1.36±0.06, 1.61 ±0.03, and 1.73 ±0.03, respectively. There were statistically significant differences between the control group and each treatment group (P<0.05 or P < 0.01). The scores of immunohistochemical staining of PCNA and VEGF proteins were decreased, while p21 increased. There were statistically significant differences between the control group and each treatment group (P < 0. 05 or P < 0. 01). Conclusion Genistein can inhibit the growth of colon cancer cells via apoptosis induction and cell cycle arrest at G_2/M phase. The anti-tumor mechanisms of genistein may be related with the down-regulation of expression of VEGF and PCNA, and upregulation of the expression of p21.