Hikami Shoichiro, Shiozaki Atsushi, Kitagawa-Juge Maki, Ichikawa Daisuke, Kosuga Toshiyuki, Konishi Hirotaka, Komatsu Shuhei, Fujiwara Hitoshi, Okamoto Kazuma, Otsuji Eigo
Division of Digestive Surgery, Department of Surgery, Kyoto Prefectural University of Medicine, 465 Kajii-cho, Kamigyo-ku, Kyoto, 602-8566, Japan.
Dig Dis Sci. 2017 Mar;62(3):652-659. doi: 10.1007/s10620-016-4430-9. Epub 2017 Jan 3.
The inhibitor of apoptosis protein (IAP) family are reported to play important roles in cancer cells evading apoptosis. However, the significance of their expression in human esophageal squamous cell carcinoma (ESCC) cells remains uncertain.
The present study aimed to investigate the role of the IAP family members in tumor necrosis factor-α (TNF-α)-induced apoptosis of human ESCC cells.
Five human ESCC cell lines were pretreated with TNF-α, cycloheximide (CHX, protein synthesis inhibitor), epoxomicin (proteasome inhibitor). Apoptosis assay and protein study with Western blot testing were conducted. Knockdown experiments with IAP siRNA were conducted, and the effect on cell apoptosis was analyzed.
Significant apoptosis was induced in five ESCC cell lines by TNF-α plus CHX stimulation, but not when treated with TNF-α or CHX alone. The protein expression levels of cIAP1 and XIAP were decreased by treatment with TNF-α in the presence of CHX, and the degree of cIAP1 and XIAP expression decrease was correlated with sensitivity to TNF-α plus CHX-induced apoptosis. Epoxomicin suppressed TNF-α plus CHX-induced degradation of survivin, cIAP1, and XIAP, in addition to apoptosis. A caspase inhibitor (z-VAD-fmk) suppressed TNF-α plus CHX-induced apoptosis, but did not suppress degradation of survivin, cIAP1, and XIAP. Furthermore, cIAP1 or XIAP siRNA transfected cells underwent apoptosis in response to treatment with TNF-α alone. Double knockdown of both genes resulted in further increased apoptosis.
cIAP1 and XIAP play an essential role in the resistance of ESCC cells against apoptosis.
据报道，凋亡抑制蛋白（IAP）家族在癌细胞逃避凋亡过程中发挥重要作用。然而，其在人食管鳞状细胞癌（ESCC）细胞中的表达意义仍不明确。
本研究旨在探讨IAP家族成员在肿瘤坏死因子-α（TNF-α）诱导的人ESCC细胞凋亡中的作用。
用TNF-α、环己酰亚胺（CHX，蛋白质合成抑制剂）、环氧霉素（蛋白酶体抑制剂）预处理5种人ESCC细胞系。进行凋亡检测及蛋白质免疫印迹分析。采用IAP siRNA进行敲低实验，并分析对细胞凋亡的影响。
TNF-α加CHX刺激可诱导5种ESCC细胞系显著凋亡，但单独用TNF-α或CHX处理时则无此现象。在CHX存在的情况下，TNF-α处理可降低cIAP1和XIAP的蛋白表达水平，且cIAP1和XIAP表达降低的程度与对TNF-α加CHX诱导凋亡的敏感性相关。环氧霉素除抑制凋亡外，还可抑制TNF-α加CHX诱导的生存素、cIAP1和XIAP的降解。一种半胱天冬酶抑制剂（z-VAD-fmk）可抑制TNF-α加CHX诱导的凋亡，但不抑制生存素、cIAP1和XIAP的降解。此外，转染cIAP1或XIAP siRNA的细胞在单独用TNF-α处理时会发生凋亡。两种基因的双重敲低导致凋亡进一步增加。
cIAP1和XIAP在ESCC细胞抗凋亡过程中起重要作用。
