Rice R L, Tang D G, Haddad M, Honn K V, Taylor J D
Department of Biological Sciences, Wayne State University, Detroit, MI 48202, USA.
Int J Cancer. 1998 Jul 17;77(2):271-8. doi: 10.1002/(sici)1097-0215(19980717)77:2<271::aid-ijc17>3.0.co;2-e.
12(S)-hydroxyeicosatetraenoic acid [12(S)-HETE], a lipoxygenase metabolite of arachidonic acid, has been shown to be involved in a wide variety of cellular activities (i.e., adhesion, spreading, motility, invasion) which promote metastasis to occur in tumor cells. In this study, several techniques (Western blotting, flow cytometry and DNase I assay) were performed to examine the alterations in the distribution of G- and F-actin expressed in B16a melanoma cells. Each of these methods independently revealed that 12(S)-HETE treatment (0.1 mM, 15 min) resulted in an increase in the F-actin content in the cytoskeletal preparations. Since the integrity of cytoskeletal networks (i.e., actin filaments) can be dynamically regulated through protein phosphorylation, we investigated the potential role of several protein kinases in the 12(S)-HETE-induced actin polymerization. By flow cytometric analysis, 12(S)-HETE was found to increase the actin filament contents. This effect could be inhibited by protein kinase C (PKC) inhibitors (calphostin C and staurosporine) as well as by protein tyrosine kinase (PTK) inhibitor (genistein) but not by protein kinase A inhibitor (H8), suggesting that the 12(S)-HETE effect involves PKC and PTK. This conclusion is consistent with the observations that phorbol 12-myristate-13-acetate (PMA) mimics the biological effect of 12(S)-HETE in promoting the F-actin formation in B16a cells. As a final analysis, direct protein phosphorylation studies indicate that 12(S)-HETE treatment led to enhanced phosphorylation of myosin light chain, which may contribute to the increased stress fiber formation following 12(S)-HETE stimulation.
12(S)-羟基二十碳四烯酸[12(S)-HETE]是花生四烯酸的脂氧合酶代谢产物，已被证明参与多种促进肿瘤细胞发生转移的细胞活动（如黏附、铺展、运动、侵袭）。在本研究中，采用了几种技术（蛋白质免疫印迹法、流式细胞术和脱氧核糖核酸酶I测定法）来检测B16a黑色素瘤细胞中G-肌动蛋白和F-肌动蛋白分布的变化。这些方法中的每一种都独立显示，12(S)-HETE处理（0.1 mM，15分钟）导致细胞骨架制剂中F-肌动蛋白含量增加。由于细胞骨架网络（即肌动蛋白丝）的完整性可通过蛋白质磷酸化进行动态调节，我们研究了几种蛋白激酶在12(S)-HETE诱导的肌动蛋白聚合中的潜在作用。通过流式细胞术分析，发现12(S)-HETE可增加肌动蛋白丝含量。这种作用可被蛋白激酶C（PKC）抑制剂（钙泊三醇C和星形孢菌素）以及蛋白酪氨酸激酶（PTK）抑制剂（染料木黄酮）抑制，但不能被蛋白激酶A抑制剂（H8）抑制，这表明12(S)-HETE的作用涉及PKC和PTK。这一结论与佛波醇12-肉豆蔻酸酯-13-乙酸酯（PMA）模拟12(S)-HETE促进B16a细胞中F-肌动蛋白形成的生物学效应的观察结果一致。作为最终分析，直接蛋白质磷酸化研究表明，12(S)-HETE处理导致肌球蛋白轻链磷酸化增强，这可能有助于12(S)-HETE刺激后应力纤维形成增加。
