Evdokiou Andreas, Bouralexis Stelios, Atkins Gerald J, Chai Fugui, Hay Shelley, Clayer Mark, Findlay David M
Department of Orthopaedics, Level 4 Bice Building, Royal Adelaide Hospital, North Terrace, Adelaide 5000, South Australia, Australia.
Int J Cancer. 2002 Jun 1;99(4):491-504. doi: 10.1002/ijc.10376.
Apo2L/TRAIL is a member of the tumor necrosis factor (TNF) family of cytokines that induces death of cancer cells but not normal cells. Its potent apoptotic activity is mediated through its cell surface death domain-containing receptors, DR4 and DR5. Apo2L/TRAIL interacts also with 3 "decoy" receptors that do not induce apoptosis, DcR1, DcR2, which lack functional death domains, and osteoprotegerin (OPG). The aim of our study was to investigate the cytotoxic activity of Apo2L/TRAIL on established osteogenic sarcoma cell lines (BTK-143, HOS, MG-63, SJSA-1, G-292 and SAOS2) and in primary cultures of normal human bone (NHB) cells. When used alone, Apo2L/TRAIL at 100 ng/ml for 24 hr induced greater than 80% cell death in only 1 (BTK-143) of the 6 osteogenic sarcoma cell lines. In contrast, Apo2L/TRAIL-resistant cells were susceptible to Apo2L/TRAIL-mediated apoptosis in the presence of the anticancer drugs, Doxorubicin (DOX), Cisplatin (CDDP) and Etoposide (ETP) but not Methotrexate (MTX) or Cyclophosphamide (CPM). Importantly, neither Apo2L/TRAIL alone nor in combination with any of these drugs affected primary normal human bone cells under equivalent conditions. Apo2L/TRAIL-induced apoptosis, and its augmentation by chemotherapy in the resistant cell lines was mediated through caspase-8 and caspase-3 activation. Furthermore, Apo2L/TRAIL-induced apoptosis and its augmentation by chemotherapy was effectively inhibited by caspase-8 zIETD-fmk and caspase-3 zDEVD-fmk protease inhibitors and by the pan-caspase inhibitor zVAD-fmk. The pattern of basal Apo2L/TRAIL receptor mRNA expression, or expression of the intracellular caspase inhibitor FLICE-inhibitory protein, FLIP, could not be readily correlated with resistance or sensitivity to Apo2L/TRAIL-induced apoptosis. However, the augmentation of Apo2L/TRAIL effects by chemotherapy was associated with drug-induced up-regulation of death receptors DR4 and DR5 mRNA and protein. No obvious correlation was seen between the expression of OPG mRNA or protein and susceptibility of cells to Apo2L/TRAIL-induced apoptosis. Stable over-expression of a dominant negative form of the Fas-associated death domain protein (FADD) in the Apo2L/TRAIL-sensitive BTK-143 cells completely inhibited Apo2L/TRAIL-induced cell death. Our results indicate that chemotherapy and Apo2L/TRAIL act synergistically to kill cancer cells but not normal bone-derived osteoblast-like cells, which has implications for future therapy of osteosarcoma.
Apo2L/TRAIL是细胞因子肿瘤坏死因子（TNF）家族的成员，可诱导癌细胞死亡，但不会诱导正常细胞死亡。其强大的凋亡活性是通过其含细胞表面死亡结构域的受体DR4和DR5介导的。Apo2L/TRAIL还与3种不诱导凋亡的“诱饵”受体相互作用，即缺乏功能性死亡结构域的DcR1、DcR2，以及骨保护素（OPG）。我们研究的目的是调查Apo2L/TRAIL对已建立的骨肉瘤细胞系（BTK-143、HOS、MG-63、SJSA-1、G-292和SAOS2）以及正常人骨（NHB）细胞原代培养物的细胞毒性活性。单独使用时，100 ng/ml的Apo2L/TRAIL作用24小时，在6种骨肉瘤细胞系中仅1种（BTK-143）诱导了超过80%的细胞死亡。相比之下，对Apo2L/TRAIL耐药的细胞在存在抗癌药物阿霉素（DOX）、顺铂（CDDP）和依托泊苷（ETP）时易受Apo2L/TRAIL介导的凋亡影响，但对甲氨蝶呤（MTX）或环磷酰胺（CPM）不敏感。重要的是，在同等条件下，单独的Apo2L/TRAIL或与这些药物中的任何一种联合使用均未影响正常人骨原代细胞。Apo2L/TRAIL诱导的凋亡及其在耐药细胞系中通过化疗的增强是通过半胱天冬酶-8和半胱天冬酶-3的激活介导的。此外，半胱天冬酶-8 zIETD-fmk和半胱天冬酶-3 zDEVD-fmk蛋白酶抑制剂以及泛半胱天冬酶抑制剂zVAD-fmk有效抑制了Apo2L/TRAIL诱导的凋亡及其通过化疗的增强。基础Apo2L/TRAIL受体mRNA表达模式，或细胞内半胱天冬酶抑制剂FLICE抑制蛋白FLIP的表达，与对Apo2L/TRAIL诱导凋亡的耐药性或敏感性之间没有明显的相关性。然而，化疗对Apo2L/TRAIL作用的增强与药物诱导的死亡受体DR4和DR5 mRNA及蛋白的上调有关。OPG mRNA或蛋白的表达与细胞对Apo2L/TRAIL诱导凋亡的敏感性之间没有明显的相关性。在对Apo2L/TRAIL敏感的BTK-143细胞中稳定过表达显性负性形式的Fas相关死亡结构域蛋白（FADD）完全抑制了Apo2L/TRAIL诱导的细胞死亡。我们的结果表明，化疗和Apo2L/TRAIL协同作用杀死癌细胞，但不杀死正常的骨源性成骨样细胞，这对骨肉瘤的未来治疗具有重要意义。
