Jin Xinghua, Wu Xiu-Xian, Abdel-Muneem Nouh Mohammed Ahmed, Kakehi Yoshiyuki
Department of Urology, Faculty of Medicine, Kagawa University, Kagawa, Japan.
J Urol. 2007 May;177(5):1894-9. doi: 10.1016/j.juro.2007.01.018.
TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) triggers apoptosis in various tumor cells by engaging death receptors 4 and 5. We investigated the effect of chemotherapeutic agents on death receptor 4 mediated apoptosis in human renal cell carcinoma cells using HGS-ETR1, which is a human monoclonal agonistic antibody specific for death receptor 4.
Cytotoxicity was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Synergy was assessed by isobolographic analysis.
Treatment of the ACHN human renal cell carcinoma cell line with HGS-ETR1 combined with 5-fluorouracil, vinblastine or gemcitabine did not overcome resistance to these agents. However, treatment with HGS-ETR1 combined with doxorubicin had a synergistic cytotoxic effect. Synergy was also achieved in another human renal cell carcinoma cell line, Caki-1, and in 5 freshly derived renal cell carcinoma cell cultures. A synergistic effect was also observed with HGS-ETR1 combined with the doxorubicin derivatives epirubicin, pirarubicin or amrubicin. The synergy achieved in cytotoxicity with HGS-ETR1 and doxorubicin was also achieved in apoptosis. Sequential treatment with doxorubicin followed by HGS-ETR1 induced significantly more cytotoxicity than reverse treatment or simultaneous treatment (p<0.05). Doxorubicin remarkably increased the cell surface expression of death receptor 4 in renal cell carcinoma cells. The combination of doxorubicin and HGS-ETR1 significantly activated the caspase cascade, including caspase-8, 9, 6 and 3, which are the downstream molecules of death receptors.
These findings indicate that doxorubicin sensitizes renal cell carcinoma cells to death receptor 4 mediated apoptosis through the induction of death receptor 4 and the activation of caspases, suggesting that combination therapy of doxorubicin and HGS-ETR1 might be effective as renal cell carcinoma therapy.
肿瘤坏死因子相关凋亡诱导配体（TRAIL）通过与死亡受体4和5结合，触发多种肿瘤细胞的凋亡。我们使用HGS-ETR1（一种针对死亡受体4的人源单克隆激动抗体）研究了化疗药物对人肾癌细胞中死亡受体4介导的凋亡的影响。
通过3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四氮唑溴盐法测定细胞毒性。通过等效线图分析评估协同作用。
用HGS-ETR1联合5-氟尿嘧啶、长春碱或吉西他滨处理ACHN人肾癌细胞系，并未克服对这些药物的耐药性。然而，用HGS-ETR1联合阿霉素具有协同细胞毒性作用。在另一人肾癌细胞系Caki-1以及5种新鲜分离的肾癌细胞培养物中也实现了协同作用。HGS-ETR1联合阿霉素衍生物表柔比星、吡柔比星或氨柔比星也观察到协同作用。HGS-ETR1和阿霉素在细胞毒性方面实现的协同作用在凋亡方面也得以实现。阿霉素后接HGS-ETR1的序贯处理比反向处理或同时处理诱导的细胞毒性显著更高（p<0.05）。阿霉素显著增加肾癌细胞中死亡受体4的细胞表面表达。阿霉素和HGS-ETR1的联合显著激活了包括半胱天冬酶-8、9、6和3在内的半胱天冬酶级联反应，这些是死亡受体的下游分子。
这些发现表明，阿霉素通过诱导死亡受体4和激活半胱天冬酶，使肾癌细胞对死亡受体4介导的凋亡敏感，提示阿霉素和HGS-ETR1联合治疗可能作为肾细胞癌治疗有效。
