Activation of protein kinase A (PKA) signaling mitigates the antiproliferative and antiinvasive effects of alpha-difluoromethylornithine in breast cancer cells.
Activation of protein kinase A (PKA) signaling mitigates the antiproliferative and antiinvasive effects of alpha-difluoromethylornithine in breast cancer cells.
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蛋白激酶 A (PKA) 信号传导的激活可减轻 α-二氟甲基鸟氨酸在乳腺癌细胞中的抗增殖和抗侵袭作用。
DOI：
10.1007/s10549-007-9536-5
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发表时间：
2008
影响因子：
3.8
通讯作者：
Manni,Andrea
中科院分区：
文献类型：
--
作者：
Xu,Haifang;Washington,Sharlene;Verderame,MichaelF;Manni,Andrea
我们已经证明，α-二氟甲基鸟氨酸（DFMO），一种鸟氨酸脱羧酶的抑制剂，多胺合成的第一个和限速酶，在乳腺癌细胞中具有显着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-末端激酶（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。本实验旨在检测DFMO对蛋白激酶A（PKA）通路的影响，并确定其生物学意义。我们发现给予MDA-MB-435乳腺癌细胞DFMO（1 mM）显著增加cAMP反应元件（CRE）结合蛋白（CREB）磷酸化以及pCRE-luc（PKA激活的CREB依赖性启动子）的反式激活。为了确定DFMO这种生化作用的意义，我们使用PKA抑制剂H89，正如预期的那样，它以剂量依赖性方式（1和10 μM）抑制我们系统中DFMO诱导的CREB磷酸化。当以显示完全阻断基础CREB磷酸化的浓度（10 μM）使用时，单独施用H89能够抑制MDA-MB-435细胞的增殖。在0.5和1 μM的浓度下，H89处理虽然本身没有抗增殖作用，但以剂量依赖性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性降低了约40%（与ImM DFMO的效果相似）。联合治疗进一步降低侵袭性达80%（与单独治疗相比P< 0.01）。H89处理（10 μM）部分减少DFMO诱导的STAT-3磷酸化，但不减少STAT-1、细胞外调节激酶（ERK）和JNK的磷酸化。总之，我们的研究结果表明，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，其通过DFMO的激活代表了一种补偿机制，应阻断该机制以最大限度地发挥药物的抗肿瘤作用。我们的数据也与DFMO激活STAT-3至少部分通过PKA途径介导的观点一致。
We have shown that α-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 μM) 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 μM) shown to completely block basal CREB phosphorylation. At concentrations of 0.5 and 1 μM, 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 ∼40% (an effect similar to that of 1 mM DFMO). The combination treatment further reduced invasiveness by ∼80% (P< 0.01 versus the individual treatments). H89 treatment (10 μM) 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.