曲妥珠单抗对HER-2胞外域水平不同的肿瘤细胞系的影响

目的探讨曲妥珠单抗(trastuzumab)对细胞膜p185人表皮生长因子受体2(HER-2)强阳性表达,以及HER-2胞外域(ECD)水平不同的肿瘤细胞系SKBR3和SKOV3细胞的生长、克隆形成及细胞内HER-2蛋白水平的影响。方法SKBR3和SKOV3细胞经曲妥珠单抗处理后,统计细胞数目及克隆形成率。双抗夹心ELISA法检测细胞培养上清中HER-2 ECD水平,Western blot检测细胞HER-2蛋白水平。结果高HER-2 ECD水平的SKBR3细胞生长及克隆形成率明显被曲妥珠单抗抑制,在相对分子质量为90 000和40 000左右分别有1条未知磷酸化蛋白明显降低或基本消失,而细胞生长及克隆形成率未受影响的SKOV3细胞中此蛋白无明显变化。SKBR3和SKOV3细胞中p185 HER-2蛋白、磷酸化-p185蛋白、磷酸化-p95蛋白水平并未见明显降低。曲妥珠单抗不仅与SKOV3细胞培养上清中的HER-2 ECD反应,也与p68/ECDⅢa蛋白反应。经曲妥珠单抗处理后,SKBR3细胞HER-2 ECD水平明显降低,但将曲妥珠单抗处理前、后的细胞数目调整到基本一致时,HER-2 ECD水平未发生明显变化。结论曲妥珠单抗抑制肿瘤细胞生长可能与阻止相对分子质量为90 000和40 000左右的未知磷酸化蛋白表达有关;p68/ECDⅢa蛋白也可能存在曲妥珠单抗识别位点;HER-2 ECD水平降低可能与SKBR3细胞数目减少有关。

Effect of trastuzumab on tumor cell lines shedding high or low level of HER-2 ECD

OBJECTIVE: To examine the effect of trastuzumab on cell proliferation, colony formation and changes of HER-2 proteins in human breast cancer cell line SKBR3 and human ovarian cancer cell line SKOV3 cells which overexpress p185 HER-2 but shed high or low HER-2 extracellular domain (ECD) levels. METHODS: SKBR3 cells and SKOV3 cells were treated with or without trastuzumab. Cell number and the rate of colony formation were calculated. Western blot analysis was used to detect p185 HER-2, HER-2 ECD and phospho-HER-2. Two-site ELISA assay was used for the detection of HER-2 ECD. RESULTS: Trastuzumab inhibited cell proliferation, colony formation, and decreased or eliminated the levels of two uncharacterized phospho-proteins (molar weight about 90 000 and 40 000) in SKBR3 cells shedding high level of HER-2 ECD expression. These responses were not observed in SKOV3 cells shedding low level of HER-2 ECD expression. But total p185, phospho-p185 and phospho-p95 proteins did not appear to change in SKBR3 and SKOV3 cells after treatment with trastuzumab. Trastuzumab reacts not only with proteolytic cleavage HER-2 ECD containing HER-2 ECD I , II , III and IV subdomains of p185 HER-2 extracellular domain, but also with the secreted autoinhibitor p68/ECD III a specifying 340 residues, identical to subdomains I and II from the extracellular domain of p185 HER-2, followed by a unique C-terminal sequence of 79 aa encoded by intron 8, which suggested that there may be a trastuzumab binding site on p68/ECD III a protein. Comparing with HER-2 ECD levels of the same number of SKBR3 cells, there was no significant decrease of HER-2 ECD shedding level after treatment with or without trastuzumab for 4 days in serum-free medium. CONCLUSION: Antitumor effects of trastuzumab may be related to the two uncharacterized phospho-p90 and/or phospho-p40 proteins. There is probably a trastuzumab epitope on p68/ECD III a. The decrease of HER-2 ECD levels may be positively correlated with the number of SKBR3 cells.