Department of Pathology, University of Verona, Strada Le Grazie 8, Verona, Italy.
Mol Cancer. 2010 Mar 17;9:61. doi: 10.1186/1476-4598-9-61.
Our understanding of the mechanism regulating pancreatic cancer metastatic phenotype is limited. We analyzed the role of RHOA and PRKCZ in the motility attitude of two subclones of the pancreatic adenocarcinoma cell line SUIT-2 (S2), with different in vivo metastatic potential in nude mice: S2-m with a low metastatic potential and highly metastatic S2-CP9 using RHOA and PRKCZ cell-permeable inhibitory peptides.
Adhesion assays, cell permeable peptides, RHOA activity assay, western blotting
When used in combination cell-permeable inhibitory peptides partially inhibited cell adhesion by about 50% in clone S2-CP9. In clone S2-m, the effect was limited to 15% inhibition. In a wound healing assay, S2-CP9 was sensitive only to treatment with the combination of both RHOA and PRKCZ inhibitory peptides. Conversely, S2-m was unable to migrate toward both ends of the wound in basal conditions. Migration of cells through a membrane with 8 mum pores was completely abolished in both clones by individual treatment with RHOA and PRKCZ inhibitory peptides.
Herein, we demonstrate a critical role for RHOA and PRKCZ in the regulation of different aspects of cell motility of pancreatic adenocarcinoma and demonstrate the need to inhibit both pathways to obtain a functionally relevant effect in most assays. These results indicate that RHOA and PRKCZ, and their downstream effectors, can represent important pharmacological targets that could potentially control the highly metastatic attitude of PDAC.
我们对调节胰腺癌转移表型的机制的了解有限。我们分析了 RHOA 和 PRKCZ 在胰腺腺癌细胞系 SUIT-2（S2）的两个亚克隆的运动状态中的作用，这两个亚克隆在裸鼠体内具有不同的转移潜能：具有低转移潜能的 S2-m 和高转移性 S2-CP9，使用 RHOA 和 PRKCZ 细胞通透性抑制肽。
粘附试验、细胞通透性肽、RHOA 活性测定、western blot
当联合使用细胞通透性抑制肽时，克隆 S2-CP9 的细胞粘附被部分抑制约 50%。在克隆 S2-m 中，效果仅限于 15%的抑制。在划痕愈合试验中，S2-CP9 仅对 RHOA 和 PRKCZ 抑制肽的联合处理敏感。相反，在基础条件下，S2-m 无法向伤口的两端迁移。在两个克隆中，通过单独用 RHOA 和 PRKCZ 抑制肽处理，细胞通过 8μm 孔膜的迁移完全被阻断。
本文证明了 RHOA 和 PRKCZ 在调节胰腺腺癌细胞运动的不同方面的关键作用，并证明了需要抑制两条途径，才能在大多数试验中获得功能相关的效果。这些结果表明，RHOA 和 PRKCZ 及其下游效应物可以作为重要的药理靶点，有可能控制 PDAC 的高度转移状态。
