不同干扰素-γ浓度上调乳腺癌细胞系Her2/neu表达的实验研究

Effect on Her2/neu expression of breast cancer cells by different concentration of interferon-γ

背景与目的:Herceptin是目前治疗Her2/neu高表达晚期转移性乳腺癌的常用分子靶向药物。如将具有强烈细胞毒作用的放射性核素131I联结到Herceptin进行放免靶向治疗,可提高Herceptin的药效。放射免疫治疗的疗效主要取决于定位在瘤体的抗体量。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。本研究探讨不同浓度IFN-γ对乳腺癌细胞系Her2/neu的诱导效应,以选择最适IFN-γ浓度对乳腺癌细胞系Her2/neu的表达以及131I-Herceptin结合率的影响。方法:取对数生长期乳腺癌细胞系MCF-7、SK-BR-3和BT-474,实验组以终浓度为10、100、500、1000U/m l的IFN-γ诱导培养48h,对照组加入等量不含IFN-γ的培养液。每组设置3个复管。各组三种细胞Her2/neu的表达率及平均荧光强度(MFI)采用流式细胞仪(FACS)检测。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三种细胞诱导前后对131I-Herceptin的结合量(Ncpm)及结合率(BR)。诱导前后三种细胞Her2/neu表达率、MFI和BR的差异采用t检验。结果:MCF-7、SK-BR-3和BT-474细胞Her2/neu基础表达率分别为8.5%、97.8%和98.2%,IFN-γ浓度分别为10U/m l、100U/m l时,三种细胞Her2/neu表达率以及MFI均未见显著性增高(t值未列出,P>0.05)。IFN-γ为500U/m l以上时,MCF-7细胞Her2/neu表达率显著提高(t=3.892,P<0.02),其它两种细胞表达率无明显变化(t值未列出,P>0.05),但三种细胞MFI均显著提高(P<0.05)。而500U/m l和1000U/m l组间Her2/neu表达率、MFI比较未见显著性差异(P>0.05)。131I-Herceptin在MCF-7、SK-BR-3及BT-474细胞的基础结合率分别为(5.3±1.5)%、(34.8±4.9)%和(36.2±4.7)%。经IFN-γ诱导后三种细胞对131I-Her-ceptin的结合率均增高,当IFN-γ浓度在500U/m l以上以及SK-BR-3经IFN-γ浓度为1000U/m l诱导时,结合率均显著提高(t值未列出,P<0.05)。Ncpm均不同程度提高,其中MCF-7增幅最明显,倍增比为2.8倍,SKBR-3和BT-474分别为1.5、1.6倍。结论:IFN-γ可以上调乳腺癌细胞Her-2/neu的表达,在一定范围内IFN-γ浓度与Her2/neu表达率及对131I-Herceptin的结合量存在相关性。

Background and Purpose： Herceptin could be a potential candidate for targeted therapy in Her2/neu overexpression metastatic breast cancer, but its efficacy has proven to be limited when used as a single agent. So binding Herceptin with a potent toxic drug such as iodine-131 may enhance its anticancer activity. In radioimmunotherapy, the quantity of molecule target sites is strongly associated with the binding of radiolabelled antibody. Here we reported the effect on Her2/neu expression of breast cancer cells treated by different concentrations of interferon-γ. Methods： The human breast cancer cell lines MCF-7, SK-BR-3 and BT-474 were in logarithmic growth phase and cultured in the presence of 10, 100, 500 or 1000U/ml interferon-γ for 48 hours. The corresponding controls encountered no interferon-γ. Cell surface expression rate and mean fluorescence intensity（MFI） of Her2/neu on three cell lines were measured by FACS. Herceptin was labelled with iodine-131 by Iodogen method and its radiochemical purity（RCP） was tested by size-exclusion high pressure liquid chromatography（ HPLC）. The binding rate（BR） and net radiocounts per minute（Ncpm） of ^131I-Herceptin on cells were measured by non-competetive saturation analysis. The indices such as expression rate, MFI and BR were compared between groups with different concentration of interferon-γ by t-test. Results： The basal expression rate of MCF-7, SKBR-3 and BT-474 were 8.5%, 97.8% and 98.2%, respectively. When cells were treated with either 10U/ml or 100U/ml of IFN-γ,the indices such as expression rate and MFI showed no significant enhancement as compared to the corresponding groups without interferon-γ treatment in the three cells（ t values not showed, P 〉 0. 05 ）. However, when the concentration of IFN-γ was used at above 500U/ml, the expression rate increased remarkably in MCF-7 （ t = 3. 892, P 〈 0. 02 ）, and the other two cell lines showed no significant changes of expression rate （t values not showed, P 〉 0. 05 ）. However, MFI increased significantly in the three cell lines than the controls. As to the two indices mentioned above, no significant tlifference was shown between the cell lines treated with 500U/ml and 1000U/ml of IFN-γ/（ P 〉0.05 ）. And the basal binding rates of ^131I-Herceptin on MCF-7, SK-BR-3 and BT-474 were （ 5.3±1.5 ） %, （ 34.8±4.9 ） % and （ 36.2±4.7 ） %, respectively. By the induction of IFN-γ the binding rate and Ncpm of ^1311-Herceptin in the three cell lines increased by different de- grees. When IFN-γ was 500U/ml or 1000U/ml,the binding rate increased significantly（P 〈0.05 ）. The highest multiplica- tion ratio of ^131I-Herceptin binding to the cell was remarkable, which reached by 2.8 time in MCF-7,and 1.5 time in SK- BR-3,1.6 time in BT-474. Conelusions：Interferon-γ can up-regulate Her2/neu expression and increase the binding of ^131I- Herceptin to breast cancer cells. There is relevance between the induction effect and concentration of IFN-γ.