Department of Urology, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, USA.
J Urol. 2013 Mar;189(3):1104-9. doi: 10.1016/j.juro.2012.08.218. Epub 2012 Oct 8.
Prior study has shown that bacillus Calmette-Guérin binds to and cross-links α5β1 integrins present on the surface of urothelial carcinoma cells. Antibody mediated cross-linking of α5β1 integrins can reproduce signal transduction, gene transactivation and phenotypic changes, similar to those observed in response to bacillus Calmette-Guérin. We evaluated the effect of a synthetic polyvalent ligand for α5β1 on these elements of the tumor response to bacillus Calmette-Guérin.
The consensus α5β1 integrin binding tripeptide RGD was linked to a MAP8 backbone to result in an octavalent construct targeting α5β1 integrin. RGD-MAP8 was used to determine its effect on signaling pathway activation (nuclear factor-κB, NRF2 and CEBP), gene expression (p21, interleukin-6 and 8, CXCL1, CXCL2 and CCL20) and cytotoxicity (trypan blue exclusion and HMGB1 release) in human urothelial carcinoma cells. Results were compared to those of treatment with bacillus Calmette-Guérin or the missense peptide GRD-MAP8.
The RDG-MAP8 construct significantly increased nuclear factor-κB signaling and p21 expression relative to controls. Compared to bacillus Calmette-Guérin treatment, only p21 expression was comparable for cells treated with RGD-MAP8, averaging 70% of bacillus Calmette-Guérin induced expression. RGD-MAP8 failed to have a significant effect on CEBP or NRF2 activation, gene expression or cell viability.
Intracellular signaling, gene transactivation and phenotypic changes in response to RGD-MAP8 were qualitatively and quantitatively different than those observed in response to bacillus Calmette-Guérin. Results suggest that while α5β1 integrin cross-linking contributes to the bacillus Calmette-Guérin response, it alone is insufficient to duplicate the full spectrum of bacillus Calmette-Guérin induced changes in urothelial carcinoma cell biology.
先前的研究表明，卡介苗与尿路上皮癌细胞表面的α5β1 整联蛋白结合并交联。抗体介导的α5β1 整联蛋白交联可以重现信号转导、基因转激活和表型变化，类似于对卡介苗的反应。我们评估了一种用于α5β1 的合成多价配体对卡介苗诱导的肿瘤反应的这些因素的影响。
将共识α5β1 整联蛋白结合三肽 RGD 连接到 MAP8 骨架上，得到靶向α5β1 整联蛋白的八价构建体。RGD-MAP8 用于确定其对信号通路激活（核因子-κB、NRF2 和 CEBP）、基因表达（p21、白细胞介素-6 和 8、CXCL1、CXCL2 和 CCL20）和细胞毒性（台盼蓝排除和 HMGB1 释放）的影响在人尿路上皮癌细胞中。结果与卡介苗或错义肽 GRD-MAP8 的治疗结果进行了比较。
与对照组相比，RDG-MAP8 构建体显著增加了核因子-κB 信号和 p21 表达。与卡介苗治疗相比，只有 RGD-MAP8 处理的细胞中 p21 表达可与卡介苗诱导的表达相媲美，平均为卡介苗诱导表达的 70%。RGD-MAP8 对 CEBP 或 NRF2 激活、基因表达或细胞活力没有显著影响。
与对卡介苗的反应相比，RGD-MAP8 引起的细胞内信号转导、基因转激活和表型变化在性质和数量上均有所不同。结果表明，尽管α5β1 整联蛋白交联有助于卡介苗的反应，但它本身不足以复制卡介苗诱导的尿路上皮癌细胞生物学变化的全部范围。
