Department of Biochemistry, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand.
Cancer Sci. 2010 Mar;101(3):658-65. doi: 10.1111/j.1349-7006.2009.01427.x. Epub 2009 Nov 7.
Myristoylated alanine-rich C kinase substrate (MARCKS), a substrate of protein kinase C (PKC) has been suggested to be implicated in cell adhesion, secretion, and motility through the regulation of the actin cytoskeletal structure. The quantitative real-time-polymerase chain reaction analysis revealed that MARCKS is significantly overexpressed in Opisthorchis viverrini-associated cholangiocarcinoma (CCA) (P = 0.001) in a hamster model, which correlated with the results of mRNA in situ hybridization. An immunohistochemical analysis of 60 CCA patients revealed a significant increase of MARCKS expression. Moreover, the log-rank analysis indicated that CCA patients with a high MARCKS expression have significantly shorter survival times than those with a low MARCKS expression (P = 0.02). This study investigated whether MARCKS overexpression is associated with CCA metastasis. Using a confocal microscopic analysis of CCA cell lines that had been stimulated with the PKC activator, 12-0-tetradecanoyl phorbol-13-acetate (TPA), MARCKS was found to be translocated from the plasma membrane to the perinuclear area. In addition, phosphorylated MARCKS (pMARCKS) became highly concentrated in the perinuclear area. Moreover, an adhesion assay demonstrated that the exogenous overexpression of MARCKS remarkably promoted cell attachment. Interestingly, after TPA stimulation, the CCA cell line-depleted MARCKS showed a decrease in migration and invasion activity. It can be concluded that in non-stimulation, MARCKS promotes cell attachment to the extracellular matrix. After TPA stimulation, PKC phosphorylates MARCKS leading to cell migration or invasion. Taken together, the results of this study reveal a prominent role for MARCKS as one of the key players in the migration of CCA cells and suggest that cycling between MARCKS and pMARCKS can regulate the metastasis of biliary cancer cells.
肌醇多磷酸化蛋白 MARCKS 是蛋白激酶 C（PKC）的底物，已有研究表明其通过调节肌动蛋白细胞骨架结构参与细胞黏附、分泌和运动。实时定量聚合酶链反应分析显示，在肝吸虫相关胆管癌（CCA）的仓鼠模型中 MARCKS 显著过表达（P=0.001），与原位杂交的 mRNA 结果一致。对 60 例 CCA 患者的免疫组织化学分析显示 MARCKS 表达显著增加。此外，对数秩分析表明 MARCKS 高表达的 CCA 患者的生存时间明显短于 MARCKS 低表达的患者（P=0.02）。本研究探讨了 MARCKS 过表达是否与 CCA 转移有关。通过用 PKC 激活剂 12-0-十四烷酰佛波醇-13-乙酸盐（TPA）刺激 CCA 细胞系的共聚焦显微镜分析，发现 MARCKS 从质膜转位到核周区。此外，磷酸化 MARCKS（pMARCKS）在核周区高度浓缩。此外，黏附实验表明 MARCKS 的外源性过表达显著促进细胞黏附。有趣的是，在 TPA 刺激后，MARCKS 耗尽的 CCA 细胞系的迁移和侵袭活性下降。由此可以得出结论，在非刺激状态下，MARCKS 促进细胞附着到细胞外基质上。在 TPA 刺激后，PKC 使 MARCKS 磷酸化，导致细胞迁移或侵袭。综上所述，本研究结果揭示了 MARCKS 作为 CCA 细胞迁移的关键因子之一的重要作用，并表明 MARCKS 和 pMARCKS 之间的循环可以调节胆管癌细胞的转移。
