Division of Pharmacology and Toxicology, Faculty of Pharmacy, University of Helsinki, FI-00014 Helsinki, Finland.
Institute of Biotechnology, University of Helsinki, FI-00014 Helsinki, Finland.
Eur J Pharm Sci. 2014 May 13;55:46-57. doi: 10.1016/j.ejps.2014.01.002. Epub 2014 Jan 29.
Diacylglycerol (DAG) is a central mediator of signaling pathways that regulate cell proliferation, survival and apoptosis. Therefore, C1 domain, the DAG binding site within protein kinase C (PKC) and other DAG effector proteins, is considered a potential cancer drug target. Derivatives of 5-(hydroxymethyl)isophthalic acid are a novel group of C1 domain ligands with antiproliferative and differentiation-inducing effects. Our previous work showed that these isophthalate derivatives exhibit antiproliferative and elongation-inducing effects in HeLa human cervical cancer cells. In this study we further characterized the effects of bis(3-trifluoromethylbenzyl) 5-(hydroxymethyl)isophthalate (HMI-1a3) on HeLa cell proliferation and morphology. HMI-1a3-induced cell elongation was accompanied with loss of focal adhesions and actin stress fibers, and exposure to HMI-1a3 induced a prominent relocation of cofilin-1 into the nucleus regardless of cell phenotype. The antiproliferative and morphological responses to HMI-1a3 were not modified by pharmacological inhibition or activation of PKC, or by RNAi knock-down of specific PKC isoforms, suggesting that the effects of HMI-1a3 were not mediated by PKC. Genome-wide gene expression microarray and gene set enrichment analysis suggested that, among others, HMI-1a3 induces changes in small GTPase-mediated signaling pathways. Our experiments revealed that the isophthalates bind also to the C1 domains of β2-chimaerin, protein kinase D (PKD) and myotonic dystrophy kinase-related Cdc42-binding kinase (MRCK), which are potential mediators of small GTPase signaling and cytoskeletal reorganization. Pharmacological inhibition of MRCK, but not that of PKD attenuated HMI-1a3-induced cell elongation, suggesting that MRCK participates in mediating the effects of HMI-1a3 on HeLa cell morphology.
二酰基甘油 (DAG) 是调节细胞增殖、存活和凋亡的信号通路的核心介质。因此，蛋白激酶 C (PKC) 的 C1 结构域和其他 DAG 效应蛋白的 DAG 结合位点被认为是潜在的癌症药物靶点。5-(羟甲基)异酞酸的衍生物是一类新型的 C1 结构域配体，具有抗增殖和诱导分化的作用。我们之前的工作表明，这些异酞酸衍生物在 HeLa 人宫颈癌细胞中表现出抗增殖和伸长诱导作用。在这项研究中，我们进一步研究了双(3-三氟甲基苄基)5-(羟甲基)异酞酸 (HMI-1a3) 对 HeLa 细胞增殖和形态的影响。HMI-1a3 诱导的细胞伸长伴随着焦点黏附的丧失和肌动蛋白应力纤维的消失，并且无论细胞表型如何，HMI-1a3 诱导的 cofilin-1 明显向核内迁移。HMI-1a3 引起的抗增殖和形态变化不受 PKC 的药理学抑制或激活，或特定 PKC 同工型的 RNAi 敲低的修饰，表明 HMI-1a3 的作用不是由 PKC 介导的。全基因组基因表达微阵列和基因集富集分析表明，HMI-1a3 诱导了小 GTPase 介导的信号通路的变化。我们的实验表明，异酞酸还与β2-奇美拉蛋白、蛋白激酶 D (PKD) 和肌强直性营养不良相关 CDC42 结合激酶 (MRCK) 的 C1 结构域结合，这些蛋白是小 GTPase 信号和细胞骨架重排的潜在介质。MRCK 的药理学抑制，而不是 PKD 的抑制，减弱了 HMI-1a3 诱导的细胞伸长，这表明 MRCK 参与介导 HMI-1a3 对 HeLa 细胞形态的影响。
