核因子κB在乳腺癌他莫昔芬耐药中的作用研究

The role of nuclear factor-kappa B in tamoxifen-resistance of breast cancer

目的 :探讨核因子κB(nuclear factor-kappa B,NF-κB)信号通路在乳腺癌对他莫昔芬耐药中的作用。方法 :采用MTT法检测0～25μmol/L他莫昔芬对激素受体阳性乳腺癌细胞MCF7及其耐药细胞MCF7R/LCC9增殖的抑制作用。采用实时荧光定量PCR和蛋白质印迹法分别检测以上2种乳腺癌细胞中NF-κB信号通路中NF-κB m RNA及其抑制蛋白α(inhibitory subunitαof NF-κB,IKBα)的表达水平。分别将携带突变质粒p CDH-CMV-IKBα(S32A,S262A)和p CDH-CMV-IKKβ(S177E,S181E)以及空载体p CDH-CMV-Vector的重组慢病毒感染乳腺癌MCF7-GC3AI细胞,成功构建NF-κB信号通路失活、激活和正常状态的乳腺癌细胞系,然后采用MTT法检测他莫昔芬以及联合NF-κB信号通路抑制剂ACT001处理对各组细胞增殖的抑制作用,荧光显微镜下观察他莫昔芬作用下各组细胞的凋亡情况,细胞克隆形成实验检测各组细胞克隆形成能力。结果 :与MCF7细胞相比,乳腺癌耐药细胞MCF7R/LCC9对他莫昔芬明显耐药(P<0.01),而且耐药细胞MCF7R/LCC9中NF-κB信号通路负调节因子IKBα的表达水平明显降低(P<0.01),而NF-κB m RNA水平明显升高(P<0.01)。他莫昔芬处理48 h后,与MCF7-GC3AI-Vector组相比,MCF7-GC3AI-IKKβ转染组细胞的增殖抑制能力明显降低(P<0.01),细胞凋亡率也明显降低(P<0.05),细胞克隆形成能力明显增强(P<0.05);而MCF7-GC3AI-IKBα转染组细胞的增殖抑制能力明显升高(P<0.05),细胞凋亡率也明显升高(P<0.01),细胞克隆形成能力明显降低(P<0.05)。联合ACT001作用可以增加他莫昔芬对MCF7R/LCC9细胞的细胞毒性(P<0.01)。结论 :NF-κB信号通路在激素受体阳性乳腺癌细胞产生他莫昔芬耐药性的过程中发挥重要作用。

Objective： To explore the role of nuclear factor-kappa B（NF-κB） signaling pathway in tamoxifen resistance of breast cancer cells. Methods： MTT assay was used to detect the inhibitory effect of 0-25 μmol/L tamoxifen on the proliferation of hormone recept or-positive breast cancer MCF7 and MCF7 R/LCC9 cells. Real-time fluorescence quantitative PCR and Western blotting were used to detect the levels of NF-κB m RNA and its inhibitory subunit α（IKBα） protein in these two breast cancer cells, respectively. MCF7-GC3 AI cells were infected with the lentivirus carrying the mutant plasmid p CDH-CMV-IKBα（S32 A, S262 A）, p CDH-CMV-IKKβ（S177 E, S181 E） and the empty vector p CDH-CMV-Vector. Then the inhibitory effect of tamoxifen in combination with NF-κB signaling pathway inhibitor ACT001 on proliferation of NF-κB signaling pathway-inactivated,-activated and normal MCF7-GC3 AI cells was detected by MTT assay. The apoptosis of MCF7-GC3 AI cells in each group was observed under a fluorescence microscope, and the clone formation ability of these cells was detected by colony formation assay. Results： MCF7 R/LCC9 cells were resistant to tamoxifen as compared with MCF7 cells（P〈0.01）. The expression level of negative regulator IKBα of NF-κB signaling pathway in MCF7 R/LCC9 cells was decreased（P〈0.01）, and the m RNA level of NF-κB was increased（P〈0.01） as compared with MCF7 cells. After treatment with tamoxifen for 48 h, the proliferation inhibitory ability and apoptosis rate of MCF7-GC3 AI-IKKβ cells were decreased（P〈0.01, P〈0.05）, and the cell colony formation ability was increased（P〈0.05） as compared with MCF7-GC3 AI-Vector cells. But the proliferation inhibitory ability and apoptosis rate of MCF7-GC3 AI-IKBα cells were increased（P〈0.05, P〈0.01）, and the cell colony formation ability was decreased（P〈0.05） after tamoxifen treatment for 48 h. ACT001, an inhibitor of NF-κB signaling pathway could increase the cytotoxicity of tamoxifen in MCF7 R/LCC9 cells（P〈0.01）.Conclusion： NF-κB signaling pathway maybe play an important role in the development of tamoxifen resistance in hormone receptor-positive breast cancer cells.