Kallergi Galatea, Tsapara Anna, Kampa Marilena, Papakonstanti Evangelia A, Krasagakis Kostas, Castanas Elias, Stournaras Christos
Department of Biochemistry, School of Medicine, University of Crete, GR-71110 Heraklion, Greece.
Exp Cell Res. 2003 Aug 1;288(1):94-109. doi: 10.1016/s0014-4827(03)00210-6.
The mechanisms through which opioids regulate the activity of malignant breast epithelial cells are currently unknown. In the present study we report the differential actin cytoskeleton reorganization induced by opioids in malignant (MCF7) and nonmalignant (MCF12A) breast epithelial cells expressing functional opioid receptors. Exposure of MCF7 cells to the opioid agonist alpha(s1) casomorphin induced important actin assembly and reorganization, including the formation of filopodia and lamellipodia. In contrast, incubation of MCF12A cells with alpha(s1) casomorphin revealed a partial but transient disassembly of actin microfilaments. Immunoprecipitation and immunoblot analyses showed rapid phosphorylation of focal adhesion kinase (FAK) and vinculin in opioid-treated MCF7 cells. Moreover, FAK associates with phosphatidylinositol-3 (PI-3 kinase), the latter being subsequently phosphorylated and activated. In addition, a substantial activation of the small GTPase Rac1 was observed. Pretreatment of MCF7 cells with the specific PI-3 kinase inhibitor wortmannin abolished both the activation of Rac1 and actin reorganization, while the opioid-induced phosphorylation of FAK and vinculin remained unaffected. Interestingly, in opioid-treated MCF12A cells this signaling cascade remained inactive, while we identified rapid phosphorylation of actin regulating the protein villin. Finally, opioids differentially inhibited cell motility in each cell line. Our data suggest a distinct, opioid-induced, signaling pathway activated in malignant breast epithelial cells, leading to important actin reorganization. These findings may indicate a potential antineoplastic role of opiates, based on the activation of differential signaling mechanisms.
目前尚不清楚阿片类药物调节恶性乳腺上皮细胞活性的机制。在本研究中，我们报告了阿片类药物在表达功能性阿片受体的恶性（MCF7）和非恶性（MCF12A）乳腺上皮细胞中诱导的肌动蛋白细胞骨架的差异重组。将MCF7细胞暴露于阿片类激动剂α(s1)酪蛋白吗啡可诱导重要的肌动蛋白组装和重组，包括丝状伪足和片状伪足的形成。相比之下，用α(s1)酪蛋白吗啡孵育MCF12A细胞则显示肌动蛋白微丝有部分但短暂的解聚。免疫沉淀和免疫印迹分析表明，在经阿片类药物处理的MCF7细胞中，粘着斑激酶（FAK）和纽蛋白迅速磷酸化。此外，FAK与磷脂酰肌醇-3（PI-3激酶）结合，后者随后被磷酸化并激活。另外，还观察到小GTP酶Rac1的大量激活。用特异性PI-3激酶抑制剂渥曼青霉素预处理MCF7细胞可消除Rac1的激活和肌动蛋白重组，而阿片类药物诱导的FAK和纽蛋白的磷酸化不受影响。有趣的是，在经阿片类药物处理的MCF12A细胞中，这一信号级联反应仍然不活跃，而我们发现了调节肌动蛋白的蛋白绒毛蛋白的快速磷酸化。最后，阿片类药物对每种细胞系的细胞运动性有不同程度的抑制作用。我们的数据表明，在恶性乳腺上皮细胞中激活了一条独特的、由阿片类药物诱导的信号通路，导致重要的肌动蛋白重组。基于不同信号机制的激活，这些发现可能表明阿片类药物具有潜在的抗肿瘤作用。
