Luna Marian, Wong Sam, Ferrario Angela, Gomer Charles J
Saban Research Institute, Childrens Hospital Los Angeles, Los Angeles, CA, USA.
Photochem Photobiol. 2008 Mar-Apr;84(2):509-14. doi: 10.1111/j.1751-1097.2007.00299.x. Epub 2008 Feb 11.
Photodynamic therapy (PDT), using the porphyrin photosensitizer Photofrin (PH), is approved for the clinical treatment of solid tumors. In addition to the direct cytotoxic responses of PH-PDT-mediated oxidative stress, this procedure also induces expression of angiogenic and prosurvival molecules including cyclooxygenase-2 (COX-2). In vivo treatment efficacy is improved when PH-PDT is combined with inhibitors of COX-2. In the current study we evaluated the signaling pathways involved with PH-PDT-mediated COX-2 expression in a mouse fibrosarcoma cell line. COX-2 promoter reporter constructs with mutated transcription elements documented that the nuclear factor kappa B (NFkappaB) element, cyclic-AMP response element 2 (CRE-2), CCAAT/enhancer binding protein (C/EBP) element and activator binding protein-1 (AP-1) element were responsive to PH-PDT. Transcription factor binding assays demonstrated that nuclear protein binding to NFkappaB, CRE-2, c-fos and c-jun elements were elevated following PH-PDT. Kinase phosphorylation upstream of COX-2 expression was also examined following PH-PDT. Stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) and c-Jun were phosphorylated following PH-PDT but the SAPK/JNK inhibitor SP600125 failed to attenuate COX-2 expression. In contrast, p38 mitogen-activated protein kinase (MAPK), which activates CRE-2 binding, was phosphorylated following PH-PDT and inhibitors of p38 MAPK, SB203580 and SB202190, decreased PH-PDT-induced COX-2 expression at both the mRNA and protein levels. Extracellular signal-regulated kinase (ERK1/2) phosphorylation, which also increases CRE-2 binding activity, was initially high in untreated cells, decreased immediately following PH-PDT and then rapidly increased. MEK1/2 is immediately upstream of ERK1/2 and the MEK1 inhibitor PD98059 failed to attenuate COX-2 expression while the MEK1/2 inhibitor U0126 induced a slight decrease in COX-2 expression. The NFkappaB inhibitor SN50 failed to reduce COX-2 expression. These results demonstrate that multiple protein kinase cascades can be activated by oxidative stress and that the p38 MAPK signaling pathway and CRE-2 binding are involved in COX-2 expression following PH-PDT.
光动力疗法（PDT），使用卟啉光敏剂光卟啉（PH），已被批准用于实体瘤的临床治疗。除了PH-PDT介导的氧化应激的直接细胞毒性反应外，该程序还诱导包括环氧合酶-2（COX-2）在内的血管生成和促生存分子的表达。当PH-PDT与COX-2抑制剂联合使用时，体内治疗效果会得到改善。在本研究中，我们评估了小鼠纤维肉瘤细胞系中与PH-PDT介导的COX-2表达相关的信号通路。具有突变转录元件的COX-2启动子报告构建体表明，核因子κB（NFκB）元件、环磷酸腺苷反应元件2（CRE-2）、CCAAT/增强子结合蛋白（C/EBP）元件和激活剂结合蛋白-1（AP-1）元件对PH-PDT有反应。转录因子结合试验表明，PH-PDT后，与NFκB、CRE-2、c-fos和c-jun元件结合的核蛋白增加。在PH-PDT后还检测了COX-2表达上游的激酶磷酸化。PH-PDT后，应激激活蛋白激酶/c-Jun氨基末端激酶（SAPK/JNK）和c-Jun被磷酸化，但SAPK/JNK抑制剂SP600125未能减弱COX-2的表达。相反，激活CRE-2结合的p38丝裂原活化蛋白激酶（MAPK）在PH-PDT后被磷酸化，p38 MAPK抑制剂SB203580和SB202190在mRNA和蛋白质水平上均降低了PH-PDT诱导的COX-2表达。细胞外信号调节激酶（ERK1/2）磷酸化也增加CRE-2结合活性，在未处理细胞中最初较高，在PH-PDT后立即降低，然后迅速增加。MEK1/2在ERK1/2的上游，MEK1抑制剂PD98059未能减弱COX-2的表达，而MEK1/2抑制剂U0126使COX-2表达略有降低。NFκB抑制剂SN50未能降低COX-2的表达。这些结果表明，氧化应激可激活多种蛋白激酶级联反应，并且p38 MAPK信号通路和CRE-2结合参与了PH-PDT后的COX-2表达。
