Grabacka Maja, Plonka Przemyslaw M, Urbanska Krystyna, Reiss Krzysztof
Center for Neurovirology, Department of Neuroscience, School of Medicine, Temple University, Philadelphia, Pennsylvania 19122, USA.
Clin Cancer Res. 2006 May 15;12(10):3028-36. doi: 10.1158/1078-0432.CCR-05-2556.
Peroxisome proliferator-activated receptors (PPAR) regulate lipid and glucose metabolism but their anticancer properties have been recently studied as well. We previously reported the antimetastatic activity of the PPARalpha ligand, fenofibrate, against melanoma tumors in vivo. Here we investigated possible molecular mechanisms of fenofibrate anti metastatic action.
Monolayer cultures of mouse (B16F10) and human (SkMell88) melanoma cell lines, soft agar assay, and cell migration assay were used in this study. In addition, we analyzed PPARalpha expression and its transcriptional activity in response to fenotibrate by using Western blots and liciferase-based reporter system.
Fenofibrate inhibited migration of B16F10 and SkMel188 cells in Transwell chambers and colony formation in soft agar. These effects were reversed by PPAR inhibitor, GW9662. Western blot analysis revealed time-dependent down-regulation of Akt and extracellular signal-regulated kinase l/2 phosphorylation in fenofibrate-treated cells. A B16F10 cell line stably expressing constitutively active Akt mutant was resistant to fenofibrate. In contrast, Akt gene silencing with siRNA mimicked the fenofibrate action and reduced the migratory ability of B16F1O cells. In addition, fenofibrate strongly sensitized BI6FIO cells to the proapoptotic drug staurosporine, further supporting the possibility that fenofibrate-induced down-regulation of Akt function contributes to fenofibrate-mediated inhibition of metastatic potential in this experimental model.
Our results show that the PPAR-dependent antimetastatic activity of fenofibrate involves down-regulation of Akt phosphorylation and suggest that supplementation with this drug may improve the effectiveness of melanoma chemotherapy.
过氧化物酶体增殖物激活受体（PPAR）调节脂质和葡萄糖代谢，但最近也对其抗癌特性进行了研究。我们之前报道过PPARα配体非诺贝特在体内对黑色素瘤肿瘤的抗转移活性。在此，我们研究了非诺贝特抗转移作用可能的分子机制。
本研究使用了小鼠（B16F10）和人（SkMel188）黑色素瘤细胞系的单层培养、软琼脂试验和细胞迁移试验。此外，我们通过蛋白质免疫印迹法和基于荧光素酶的报告系统分析了非诺贝特作用下PPARα的表达及其转录活性。
非诺贝特抑制了B16F10和SkMel188细胞在Transwell小室中的迁移以及在软琼脂中的集落形成。这些作用被PPAR抑制剂GW9662逆转。蛋白质免疫印迹分析显示，在非诺贝特处理的细胞中，Akt和细胞外信号调节激酶1/2磷酸化呈时间依赖性下调。稳定表达组成型活性Akt突变体的B16F10细胞系对非诺贝特具有抗性。相反，用小干扰RNA使Akt基因沉默模拟了非诺贝特的作用，并降低了B16F10细胞的迁移能力。此外，非诺贝特使B16F10细胞对促凋亡药物星形孢菌素强烈敏感，进一步支持了非诺贝特诱导的Akt功能下调有助于该实验模型中非诺贝特介导的转移潜能抑制这一可能性。
我们的结果表明，非诺贝特依赖PPAR的抗转移活性涉及Akt磷酸化的下调，并提示补充该药物可能提高黑色素瘤化疗的疗效。
