Navarro Rosaura, Martínez Rosa, Busnadiego Idoia, Ruiz-Larrea M Begoña, Ruiz-Sanz José Ignacio
Department of Physiology, Faculty of Medicine and Dentistry, University of the Basque Country, 48080-Bilbao, Spain.
Ann N Y Acad Sci. 2006 Dec;1090:408-18. doi: 10.1196/annals.1378.044.
Doxorubicin (DOX) is a potent anticancer drug, whose clinical use is limited on account of its toxicity. DOX cytotoxic effects have been associated with reactive oxygen species (ROS) generated during drug metabolism. ROS induce signaling cascades leading to changes in the phosphorylation status of target proteins, which are keys for cell survival or apoptosis. The mitogen-activated protein kinase (MAPK) cascades are routes activated in response to oxidative stress. In this work, the effects of DOX on cytotoxicity, indicators of oxidative stress (malondialdehyde -MDA- and GSH), and the phosphorylation status of extracellular signal-regulated kinases (ERKs), c-Jun N-terminal kinases (JNKs), and p38 kinases were analyzed in primary cultures of rat hepatocytes. DOX (1-50 microM) did not modify lactate dehydrogenase (LDH) release into the medium, the levels of MDA (determined by high-performance liquid chromatography [HPLC]) or the intracellular GSH during the incubation time up to 6 h. GSH levels from mitochondria extracted by Percoll gradient from cultured hepatocytes were not modified by DOX, thus excluding its depletion or any impaired mitochondrial uptake. Characterization of proteins by Western blot analysis revealed that DOX increased phosphorylation of p38 kinases and JNK1 and JNK2 in a dose- and time-dependent manner. DOX also increased ERK2 phosphorylation at latter time points. In conclusion, DOX triggers activation of ERK, JNK, and p38 kinases in primary cultures of rat hepatocytes independently of oxidant damage.
阿霉素（DOX）是一种强效抗癌药物，但其临床应用因毒性而受到限制。DOX的细胞毒性作用与药物代谢过程中产生的活性氧（ROS）有关。ROS诱导信号级联反应，导致靶蛋白磷酸化状态发生变化，而靶蛋白磷酸化状态的变化是细胞存活或凋亡的关键。丝裂原活化蛋白激酶（MAPK）级联反应是响应氧化应激而激活的信号通路。在本研究中，我们分析了DOX对大鼠原代肝细胞毒性、氧化应激指标（丙二醛-MDA-和谷胱甘肽-GSH）以及细胞外信号调节激酶（ERK）、c-Jun氨基末端激酶（JNK）和p38激酶磷酸化状态的影响。在长达6小时的孵育时间内，DOX（1-50 microM）未改变乳酸脱氢酶（LDH）释放到培养基中的水平、MDA水平（通过高效液相色谱法-HPLC-测定）或细胞内GSH水平。通过Percoll梯度从培养的肝细胞中提取的线粒体GSH水平未被DOX改变，因此排除了其耗竭或线粒体摄取受损的情况。通过蛋白质印迹分析对蛋白质进行表征，结果显示DOX以剂量和时间依赖性方式增加p38激酶以及JNK1和JNK2的磷酸化。DOX在后期时间点也增加了ERK2的磷酸化。总之，DOX在大鼠原代肝细胞培养物中触发ERK、JNK和p-38激酶的激活，且与氧化损伤无关。
