Sutter Andreas P, Maaser Kerstin, Gerst Bastian, Krahn Antje, Zeitz Martin, Scherübl Hans
Charité-Universitätsmedizin Berlin, Campus Benjamin Franklin, Medical Clinic I, Berlin, Germany.
Biochem Pharmacol. 2004 May 1;67(9):1701-10. doi: 10.1016/j.bcp.2004.01.009.
Specific ligands of the peripheral benzodiazepine receptor (PBR) activate pro-apoptotic and anti-proliferative signaling pathways. Previously, we found that PBR ligands activated the p38 mitogen-activated protein kinase (MAPK) pathway in esophageal cancer cells, and that the activation of p38MAPK contributed to tumor cell apoptosis and cell cycle arrest. Here, we report that PBR ligands also activate the pro-survival MAPK/ERK kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway in esophageal cancer cells, which might compromise the efficacy of PBR ligands. Hence, a combination treatment of PBR ligands and MEK inhibitors, which are emerging as promising anticancer agents, was pursued to determine whether this treatment could lead to enhanced apoptosis and cell cycle arrest. Using Western blotting we demonstrated a time- and dose-dependent phosphorylation of ERK1/2 in response to PBR ligands. Apoptosis was investigated by assessment of mitochondrial alterations and caspase-3 activity. Cell cycle arrest was measured by flow cytometric analysis of stained isolated nuclei. The inhibition of MEK/ERK with a pharmacologic inhibitor, 2'-amino-3'-methoxyflavone (PD 98059), resulted in a synergistic enhancement of PBR-ligand-induced growth inhibition, apoptosis and cell cycle arrest. Specifity of the pharmacologic inhibitor was confirmed by the use of 1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene (U 0126), a second MEK/ERK inhibitor, and 1,4-diamino-2,3-dicyano-1,4-bis(methylthio)butadiene (U 0124), a structural analogue of it which does not display any affinity to MEK. Enhanced pro-apoptotic and anti-proliferative effects were observed both in KYSE-140 esophageal squamous cancer and OE-33 adenocarcinoma cells, suggesting that this effect was not cell-type specific. In addition, the PBR-mediated overexpression of the stress response gene (growth arrest and DNA-damage-inducible gene gadd153) was synergistically enhanced by MEK inhibition. This is the first report of enhanced PBR-ligand-mediated apoptosis and cell cycle arrest by simultaneous MEK inhibition, suggesting a new anticancer strategy.
外周苯二氮䓬受体（PBR）的特异性配体可激活促凋亡和抗增殖信号通路。此前，我们发现PBR配体可激活食管癌细胞中的p38丝裂原活化蛋白激酶（MAPK）通路，且p38MAPK的激活有助于肿瘤细胞凋亡和细胞周期停滞。在此，我们报告PBR配体还可激活食管癌细胞中的促生存MAPK/细胞外信号调节激酶（ERK）激酶（MEK）/细胞外信号调节激酶（ERK）通路，这可能会削弱PBR配体的疗效。因此，我们采用PBR配体与正成为有前景的抗癌药物的MEK抑制剂联合治疗，以确定这种治疗是否能增强细胞凋亡和细胞周期停滞。通过蛋白质印迹法，我们证明了ERK1/2对PBR配体有时间和剂量依赖性的磷酸化反应。通过评估线粒体改变和半胱天冬酶-3活性来研究细胞凋亡。通过对染色的分离细胞核进行流式细胞术分析来测量细胞周期停滞。用一种药理抑制剂2'-氨基-3'-甲氧基黄酮（PD 98059）抑制MEK/ERK，导致PBR配体诱导的生长抑制、细胞凋亡和细胞周期停滞协同增强。通过使用第二种MEK/ERK抑制剂1,4-二氨基-2,3-二氰基-1,4-双（2-氨基苯硫基）丁二烯（U 0126）和其结构类似物、对MEK无任何亲和力的1,4-二氨基-2,3-二氰基-1,4-双（甲硫基）丁二烯（U 0124），证实了该药理抑制剂的特异性。在KYSE-140食管鳞癌和OE-33腺癌细胞中均观察到促凋亡和抗增殖作用增强，表明这种作用并非细胞类型特异性的。此外，MEK抑制可协同增强PBR介导的应激反应基因（生长停滞和DNA损伤诱导基因gadd153）的过表达。这是关于通过同时抑制MEK增强PBR配体介导的细胞凋亡和细胞周期停滞的首次报道，提示了一种新的抗癌策略。
