Nakada S, Kawano T, Saito-akita S, Iwase S, Horiguchi-Yamada J, Ohno T, Yamada H
Department of Molecular Genetics, Institute of DNA Medicine, Jikei University School of Medicine, 3-25-8 Nishi-Shinbashi, Minato-ku, Tokyo 105-8461, Japan.
Anticancer Res. 2001 Jan-Feb;21(1A):167-71.
TNF-alpha is one of the key inflammatory cytokines and it modulates various events through several pathways. U937 myelomonocytic leukemia cells are sensitive to TNF-alpha and about 20% of these cells undergo apoptosis within 6 hours after treatment. Co-treatment of these cells with actinomycin D or cycloheximide enhances TNF-alpha induced apoptosis, suggesting that some TNF-alpha-derived signals can augment apoptosis. We investigated whether mitosis-activating protein kinases (MAPKs) had an influence on TNF-alpha induced apoptosis.
U937 cells were treated by TNF-alpha with or without MEK or p38MAPK inhibitors. Apoptosis was assessed morphologically by fluorescence microscopy and caspase-3 was studied by immunoblotting. Expression of apoptosis-inhibitory proteins was studied by RT-PCR whilst the activation of JNKs was investigated by detecting their phosphorylation.
TNF-alpha treatment induced apoptosis in about 23% of the cells, while pretreatment with a MEK inhibitor (PD98059) caused 69% of the cells to undergo apoptosis. The inhibition of p38MAPK by SB203580 scarcely enhanced apoptosis, although another p38MAPK inhibitor (PD169316) induced apoptosis in 37% of the cells. Simultaneous pretreatment of cells with PD98059 and PD169316 resulted in the highest level of TNF-alpha induced apoptosis and 90% of the cells underwent apoptosis after 6 hours. In cells pretreated with PD98059 plus PD169316, caspase-3 was completely cleaved at 6 hours and early induction of c-IAP2/HIAP 1 mRNA was not observed. JNKs showed rapid and extensive phosphorylation in these cells.
TNF-alpha induced apoptosis was potentiated by the inhibition of either MEK alone, or MEK plus p38MAPK, suggesting that the MAPK pathway may be a promising target for cancer therapy.
肿瘤坏死因子-α（TNF-α）是关键的炎性细胞因子之一，它通过多种途径调节各种事件。U937髓单核细胞白血病细胞对TNF-α敏感，约20%的这些细胞在处理后6小时内发生凋亡。用放线菌素D或环己酰亚胺联合处理这些细胞可增强TNF-α诱导的凋亡，这表明一些TNF-α衍生的信号可增强凋亡。我们研究了丝裂原活化蛋白激酶（MAPKs）是否对TNF-α诱导的凋亡有影响。
用TNF-α处理U937细胞，同时加入或不加入MEK或p38MAPK抑制剂。通过荧光显微镜对凋亡进行形态学评估，通过免疫印迹研究半胱天冬酶-3。通过逆转录-聚合酶链反应（RT-PCR）研究凋亡抑制蛋白的表达，同时通过检测其磷酸化来研究应激活化蛋白激酶（JNKs）的激活。
TNF-α处理诱导约23%的细胞凋亡，而用MEK抑制剂（PD98059）预处理导致69%的细胞发生凋亡。SB203580对p38MAPK的抑制几乎没有增强凋亡，尽管另一种p38MAPK抑制剂（PD169316）诱导37%的细胞凋亡。用PD98059和PD169316同时预处理细胞导致TNF-α诱导的凋亡水平最高，6小时后90%的细胞发生凋亡。在用PD98059加PD169316预处理的细胞中，半胱天冬酶-3在6小时时完全裂解，未观察到c-IAP2/HIAP 1 mRNA的早期诱导。JNKs在这些细胞中显示出快速而广泛的磷酸化。
单独抑制MEK或MEK加p38MAPK可增强TNF-α诱导的凋亡，这表明MAPK途径可能是癌症治疗的一个有前景的靶点。
