Department of Clinical Chemistry & Toxicology, School of Pharmaceutical Sciences, University of São Paulo, São Paulo, SP, Brazil.
J Cell Biochem. 2010 May;110(1):52-61. doi: 10.1002/jcb.22472.
RECK is an anti-tumoral gene whose activity has been associated with its inhibitory effects regulating MMP-2, MMP-9, and MT1-MMP. RECK level decreases as gliobastoma progresses, varying from less invasive grade II gliomas to very invasive human glioblastoma multiforme (GBM). Since RECK expression and glioma invasiveness show an inverse correlation, the aim of the present study is to investigate whether RECK expression would inhibit glioma invasive behavior. We conducted this study to explore forced RECK expression in the highly invasive T98G human GBM cell line. Expression levels as well as protein levels of RECK, MMP-2, MMP-9, and MT1-MMP were assessed by qPCR and immunoblotting in T98G/RECK+ cells. The invasion and migration capacity of RECK+ cells was inhibited in transwell and wound assays. Dramatic cytoskeleton modifications were observed in the T98G/RECK+ cells, when compared to control cells, such as the abundance of stress fibers (contractile actin-myosin II bundles) and alteration of lamellipodia. T98G/RECK+ cells also displayed phosphorylated focal adhesion kinase (P-FAK) in mature focal adhesions associated with stress fibers; whereas P-FAK in control cells was mostly associated with immature focal complexes. Interestingly, the RECK protein was predominantly localized at the leading edge of migrating cells, associated with membrane ruffles. Unexpectedly, introduced expression of RECK effectively inhibited the invasive process through rearrangement of actin filaments, promoting a decrease in migratory ability. This work has associated RECK tumor-suppressing activity with the inhibition of motility and invasion in this GBM model, which are two glioma characteristics responsible for the inefficiency of current available treatments.
RECK 是一种抗肿瘤基因，其活性与其抑制 MMP-2、MMP-9 和 MT1-MMP 的抑制作用有关。随着神经胶质瘤的进展，RECK 水平降低，从侵袭性较低的二级神经胶质瘤到非常侵袭性的多形性胶质母细胞瘤（GBM）。由于 RECK 表达与神经胶质瘤侵袭性呈负相关，本研究旨在探讨 RECK 表达是否会抑制神经胶质瘤的侵袭行为。我们进行了这项研究，以探索在高度侵袭性的 T98G 人 GBM 细胞系中强制表达 RECK。通过 qPCR 和免疫印迹评估 T98G/RECK+细胞中 RECK、MMP-2、MMP-9 和 MT1-MMP 的表达水平和蛋白水平。在 Transwell 和划痕实验中，RECK+细胞的侵袭和迁移能力受到抑制。与对照细胞相比，T98G/RECK+细胞观察到明显的细胞骨架修饰，例如应力纤维（收缩性肌动球蛋白 II 束）的丰度增加和片状伪足的改变。T98G/RECK+细胞还显示与应力纤维相关的成熟焦点黏附激酶（P-FAK）；而对照细胞中的 P-FAK 主要与不成熟的焦点复合物相关。有趣的是，RECK 蛋白主要定位于迁移细胞的前缘，与膜皱襞相关。出乎意料的是，引入 RECK 的表达通过重排肌动蛋白丝有效地抑制了侵袭过程，降低了迁移能力。这项工作将 RECK 的肿瘤抑制活性与该 GBM 模型中运动和侵袭的抑制联系起来，这两个是导致当前可用治疗方法无效的两个神经胶质瘤特征。
