干扰素-γ对乳腺癌细胞Her2/neu表达及^(131)I-Herceptin抑制肿瘤细胞增殖的影响

Effects of Interferon-γ on Her-2/neu Expression and Antitumor Activity of (131)~I-Herceptin in Breast Cancer Cell Lines

背景与目的:利用Herceptin对Her2/neu的靶向特性,将放射性核素131I联结到Herceptin进行放免靶向治疗,是治疗转移性乳腺癌的方法之一。而肿瘤摄取标记抗体的量与肿瘤细胞靶位分子的表达量密切相关。本研究应用IFN-γ上调乳腺癌细胞系Her2/neu的表达,以提高131I-Herceptin在乳腺癌细胞系的结合,及131I-Herceptin对乳腺癌细胞增殖的抑制作用。方法:取对数生长期乳腺癌细胞系MCF-7、SKBR-3和BT-474,实验组以终浓度为500U/ml的IFN-γ诱导培养48h,对照组加入不含IFN-γ的等量培养液。诱导前后采用流式细胞仪(FACS)检测3种细胞Her2/neu的表达率及平均荧光强度(MFI)。采用Iodogen法对Herceptin进行131I标记,以高压液相层析法(HPLC)测定131I-Herceptin的放射化学纯度(RCP),以非竞争性饱和结合法测定3种细胞诱导前后131I-Herceptin的结合率(B/T)。采用克隆形成实验评价诱导后131I-Herceptin对3种细胞杀伤效应的变化。实验组与对照组间Her2/neu表达率、MFI、B/T及克隆形成率的差异采用t检验。结果:MCF-7细胞Her2/neu基础表达率为(8.5±1.9)%,诱导后升高至(15.2±2.7)%(t=3.515,P<0.05),MFI从38±7升高至121±17(t=7.823,P<0.01);SKBR-3细胞和BT-474细胞的基础表达率分别为(98.9±1.1)%和(98.1±0.9)%,诱导后分别为(99.7±0.9)%和(99.5±1.2)%,无明显改变(P>0.05),但MFI分别从952±125、1020±98增高至1608±201(t=4.802,P<0.01)和1968±192(t=7.614,P<0.002)。131I-Herceptin在MCF-7、SKBR-3和BT-474的基础结合率分别为(5.2±1.4)%、(35.8±4.5)%和(37.2±3.6)%,诱导后分别升高为(12.3±3.4)%、(48.9±7.1)%和(59.5±8.7)%,对131I-Herceptin的结合倍增比分别为2.4、1.4和1.6倍。实验组3种细胞的克隆形成率分别为(30±4)%、(23±5)%及(19±6%),均显著低于对照组[分别为(49±3)%、(37±6)%、(34±5)%](t=6.574、3.105、3.323,P<0.05)。结论:IFN-γ可以上调乳腺癌细胞系Her-2/neu的表达和肿瘤细胞对131I-Herceptin的结合量,因此也提高了131I-Herceptin对乳腺癌细胞的增殖抑制作用。

BACKGROUND ＆ OBJECTIVE： Herceptin plays an important role in treating metastatic breast cancer by targeting Her2/neu, therefore, combining Herceptin with iodine-131 （1311） might enhance its antitumor activity. This study was to up-regulate Her2/neu expression by interferon-y （IFN-y）, and explore its effect on binding and antitumor activity of 13~l-Herceptin in breast cancer cell lines MCF-7, SKBR-3 and BT-474. METHODS： MCF-7, SKBR-3 and BT-474 cells were cultured with or without IFN-y（500 U/ml） for 48 h. The positive rate and mean fluorescence intensity （MFI） of Her2/neu on the 3 cell lines were tested by flow cytometry. Herceptin was labeled with 13~1 by Iodogen method, and its radiochemical purity （RCP） was tested by size- exclusion high-pressure liquid chromatography （HPLC）. The binding rate of ~3~l-Herceptin on cells was measured by non-competitive saturation analysis, and its killing effect was estimated by colony-forming assay. The positive rate and MFI of Her2/neu, binding rate of ^131I-Herceptin, and colony-forming rate were compared between IFN-γ-induced group and control group by t test. RESULTS： For MCF-7 cells, the positive rate and MFI of Her2/neu were significantly higher in IFN-γ-induced cells than in control cells [（15.2±4.7）% vs. （8.5±1.9）%, t=3.515, P〈0.05; 121±17 vs. 38±7, t=7.823, P〈0.002]; for SKBR-3 and BT-474 cells, no obvious difference of Her2/neu positive rate was observed between IFN-γ-induced cells and control cells [（99.7±0.9）% vs. （98.9±1.1）%, P〉0.05; （99.5±1.2）% vs. （98.1±0.9）%, P〉0.05], but the MFI of Her2/neu was significantly higher in IFN-γ-induced cells than in control cells （1 608±201 vs. 952±125, t=4.802, P〈0.01; 1 968±192 vs. 1 020±98, t=7.614, P〈0.002）. The binding rates of Her2/neu were increased from （5.2±1.4）% to （12.3±3.4）% by 2.4 folds in MCF-7 cells, from （35.8±4.5）% to （48.9±7.1）% by 1.4 folds in SKBR-3 cells, and from （37.2±3.6）% to （59.5±8.7）% by 1.6 folds in BT-474 cells after inducement with IFN-γ. The colony-forming rates were significantly lower in IFN-γ-induced MCF-7, SKBR- 3 and BT-474 cells than in control cells [（30±4）% vs. （49±3）%, t=6.574, P〈0.05; （23±5）% vs. （37±6）% t=3.105, P〈0.05; （19±6）% vs. （34±5）%, t=3.323, P〈0.05]. CONCLUSION： IFN-γ can up-regulate Her-2/neu expression and increase the binding of ^131I-Herceptin, hence, improve the inhibitory effect of ^131Il-Herceptin on proliferation of breast cancer cells.