反义核酸药物癌泰得与单克隆抗体曲妥珠单抗对乳腺癌细胞株SKBR3的体外杀伤作用

Effects of single versus combined use of transtuzumab and cantide on breast cancer cells SKBR3 over-expressing HER2

目的探讨反义核酸药物（癌泰得）与单克隆抗体靶向药物（曲妥珠单抗，商品名赫赛汀），两者单独或联合应用对HER2过表达乳腺癌细胞株的体外杀伤作用。方法采用间接免疫荧光法观察HER2和hTERT在SKBR3细胞中的分布及药物对两种蛋白亚细胞定位的影响；MTT法测定不同浓度癌泰得与赫赛汀单独或联合应用对SKBR3的抑制作用；流式细胞仪检测细胞凋亡比率。结果（1）SKBR3细胞中同时表达HER2和hTERT蛋白，HER2分布于细胞膜表面，hTERT分布于细胞核，加入赫赛汀后，HER2出现了胞质迁移，hTERT定位无明显变化；（2）MTT法观察到赫赛汀与癌泰得单用和联用均对SKBR3细胞有抑制作用，联用时抑制率增加；当0．4μmol／L癌泰得与0．85μg/ml赫赛汀联用时为相加作用；0．4μmol／L癌泰得分别与1．70、3．40、6．88、13．75μg／ml赫赛汀联用时为协同作用；（3）流式细胞仪检测：单独应用赫赛汀，细胞凋亡率为11．26％，单独应用癌泰得，凋亡率为25．75％，两药联用凋亡率为41．41％，以晚期凋亡为主。结论（1）HER2与hTERT在SKBR3中的亚细胞定位不同；（2）单独应用赫赛汀或癌泰得，对SKBR3细胞有抑制作用，抑制率与药物浓度呈线性相关；（3）两药联用具有相加或协同作用，联用可提高SKBR3细胞的凋亡率。

Objective To evaluate the in vitro cytotoxic effects of cantide or herceptin on human breast cancer SKBR3 ceils over-expressing HER2. Methods The distribution of HER2 and hTERT protein in SKBR3 cells and the effects of cantide and/or herceptin on the subcellular localization of HER2 and hTERT were observed by indirect immunofluorescent assay. The inhibition rate of herceptin and/or cantide at different concentrations on SKBR3 ceils was detected by MTF assay. And the apoptotic rate of cells was evaluated by flow cytometer. Results （ 1 ） The expressions of both HER2 and hTERT proteins in SKBR3 ceils were found. HER2 protein was predominant in cell membranes while hTERT protein in nuclei. After the addition of herceptin, the cytoplasmic migration of I-IER2 was found while there was no distinct location change of cantide. （2） In MTr assay, the single use of cantide or herceptin and the combined use of both produced inhibitory effects on SKBR3 cells while the inhibition rate was higher for combined use. The inhibitory effects became additive in the combined use of 0.4 p^mol/L cantide and 0. 85 ~g/ml herceptin. And there were synergistic effects in the combined use of 0. 4 p^mol/L cantide and 1.70, 3.40, 6. 88 or 13.75 /~g/ml herceptin. （3） The apoptotic rate was 25.75% for cantide alone, 11.26% for herceptin alone and 41.41% for their combined use （apoptotic cells predominant in advanced stage）. Conclusion Due to different localizations, cantide and herceptin have different action sites in their combined use. When in single use, the inhibition rate is linearly correlated with the concentration of herceptin or cantide. And their combined use produces additive or synergistic antitumor effects on SKBR3 breast cancer cells.