Xu Haifang, Washington Sharlene, Verderame Michael F, Manni Andrea
Department of Medicine, Division of Endocrinology, The Pennsylvania State University College of Medicine, Hershey, PA 17033, USA.
Breast Cancer Res Treat. 2008 Jan;107(1):63-70. doi: 10.1007/s10549-007-9536-5. Epub 2007 Feb 27.
We have shown that alpha-difluoromethylornithine (DFMO), an inhibitor of ornithine decarboxylase, the first and rate-limiting enzyme in polyamine synthesis, has significant antiproliferative and antiinvasive effects in breast cancer cells. We have also reported that these antitumor effects are associated with activation of multiple signaling pathways, including STAT-3, STAT-1, Jun-N-Terminal kinase (JNK), and Mitogen activated protein kinase (MAPK), the latter being found to mediate its antiinvasive action in MDA-MB-435 cells. The present experiments were designed to test the effect of DFMO on the protein kinase A (PKA) pathway and determine its biological significance. We found that DFMO administration (1 mM) to MDA-MB-435 breast cancer cells significantly increased cAMP response element (CRE)-binding protein (CREB) phosphorylation as well as the transactivation of pCRE-luc, a CREB-dependent promoter activated by PKA. To determine the significance of this biochemical effect of DFMO, we used the PKA inhibitor H89 which, as expected, suppressed in a dose-dependent manner (1 and 10 microM) basal and DFMO-induced CREB phosphorylation in our system. Administration of H89 alone was able to suppress proliferation of MDA-MB-435 cells when used at a concentration (10 microM) shown to completely block basal CREB phosphorylation. At concentrations of 0.5 and 1 muM, H89 treatment, while having no antiproliferative effect of its own, potentiated in a dose-dependent fashion the growth inhibitory action of a suboptimal concentration of DFMO (0.01 mM). Ten micromoles of H89 reduced invasiveness of MDA-MB-435 cells in matrigel by approximately 40% (an effect similar to that of 1 mM DFMO). The combination treatment further reduced invasiveness by approximately 80% (P < 0.01 versus the individual treatments). H89 treatment (10 microM) partially reduced DFMO-induced phosphorylation of STAT-3 but not that of STAT-1, Extracellular regulated kinase (ERK), and JNK. In conclusion, our results indicate that PKA signaling exerts proproliferative and proinvasive effects in our experimental system. Therefore, its activation by DFMO represents a compensatory mechanism which should be blocked in order to maximize the antitumor action of the drug. Our data are also consistent with the notion that STAT-3 activation by DFMO is at least in part mediated through the PKA pathway.
我们已经表明，α-二氟甲基鸟氨酸（DFMO）是鸟氨酸脱羧酶的抑制剂，鸟氨酸脱羧酶是多胺合成中的首个限速酶，它在乳腺癌细胞中具有显著的抗增殖和抗侵袭作用。我们还报道了这些抗肿瘤作用与多种信号通路的激活有关，包括STAT-3、STAT-1、Jun-N端激酶（JNK）和丝裂原活化蛋白激酶（MAPK），后者被发现介导其在MDA-MB-435细胞中的抗侵袭作用。本实验旨在测试DFMO对蛋白激酶A（PKA）通路的影响并确定其生物学意义。我们发现，向MDA-MB-435乳腺癌细胞施用DFMO（1 mM）可显著增加环磷酸腺苷反应元件（CRE）结合蛋白（CREB）的磷酸化以及pCRE-luc的反式激活，pCRE-luc是一种由PKA激活的依赖CREB的启动子。为了确定DFMO这种生化作用的意义，我们使用了PKA抑制剂H89，正如预期的那样，它在我们的系统中以剂量依赖的方式（1和10 microM）抑制基础和DFMO诱导的CREB磷酸化。单独施用H89（10 microM）时，其浓度能够完全阻断基础CREB磷酸化，此时能够抑制MDA-MB-435细胞的增殖。在0.5和1 microM的浓度下，H89处理虽然自身没有抗增殖作用，但却以剂量依赖的方式增强了次优浓度DFMO（0.01 mM）的生长抑制作用。10微摩尔的H89使MDA-MB-435细胞在基质胶中的侵袭能力降低了约40%（与1 mM DFMO的效果相似）。联合处理进一步将侵袭能力降低了约80%（与单独处理相比，P < 0.01）。H89处理（10 microM）部分降低了DFMO诱导的STAT-3磷酸化，但没有降低STAT-1、细胞外调节激酶（ERK）和JNK的磷酸化。总之，我们的结果表明，PKA信号在我们的实验系统中发挥促增殖和促侵袭作用。因此，DFMO对其的激活代表了一种补偿机制，为了最大化该药物的抗肿瘤作用，应该阻断这种机制。我们的数据也与DFMO激活STAT-3至少部分是通过PKA通路介导的这一观点一致。
