Mitsiades Nicholas, Mitsiades Constantine S, Poulaki Vassiliki, Anderson Kenneth C, Treon Steven P
Department of Adult Oncology, Dana Farber Cancer Institute, Harvard Medical School, 44 Binney Street, Mayer Bldg., Boston, MA 02115, USA.
Blood. 2002 Mar 15;99(6):2162-71. doi: 10.1182/blood.v99.6.2162.
Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL, Apo2 ligand) effectively kills multiple myeloma (MM) cells in vitro irrespective of refractoriness to dexamethasone and chemotherapy. Because clinical trials with this anticancer agent are expected shortly, we investigated the signaling pathway of TRAIL-induced apoptosis in MM. We detected rapid cleavage of caspases-8, -9, -3, and -6, as well as the caspase substrates poly(ADP-ribose) polymerase (PARP) and DNA fragmentation factor-45 (DFF45), but not caspase-10, upon TRAIL treatment in sensitive MM cells, pointing to caspase-8 as the apical caspase of TRAIL signaling in MM cells. These phenomena were not observed or were significantly delayed in TRAIL-resistant MM cells, suggesting that resistance may arise from inhibition at the level of caspase-8 activation. Higher levels of expression for various apoptosis inhibitors, including FLICE-inhibitory protein (FLIP), and lower procaspase-8 levels were present in TRAIL-resistant cells and sensitivity was restored by the protein synthesis inhibitor cycloheximide (CHX) and the protein kinase C (PKC) inhibitor bisindolylmaleimide (BIM), which both lowered FLIP and cellular inhibitor of apoptosis protein-2 (cIAP-2) protein levels. Forced expression of procaspase-8 or FLIP antisense oligonucleotides also sensitized TRAIL-resistant cells to TRAIL. Moreover, the cell permeable nuclear factor (NF)-kappaB inhibitor SN50, which sensitizes TRAIL-resistant cells to TRAIL, also inhibited cIAP2 protein expression. Finally, CHX, BIM, and SN50 facilitated the cleavage and activation of procaspase-8 in TRAIL-resistant cells, confirming that inhibition of TRAIL-induced apoptosis occurs at this level and that these agents sensitize MM cells by relieving this block. Our data set a framework for the clinical use of approaches that sensitize MM cells to TRAIL by agents that inhibit FLIP and cIAP-2 expression or augment caspase-8 activity.
肿瘤坏死因子相关凋亡诱导配体（TRAIL，Apo2配体）在体外可有效杀死多发性骨髓瘤（MM）细胞，无论其对 dexamethasone 和化疗是否耐药。由于预计不久后将开展该抗癌药物的临床试验，我们研究了 TRAIL 诱导 MM 细胞凋亡的信号通路。我们检测到在敏感的 MM 细胞中，经 TRAIL 处理后，caspases-8、-9、-3 和 -6 以及 caspase 底物聚（ADP - 核糖）聚合酶（PARP）和 DNA 片段化因子 - 45（DFF45）迅速裂解，但 caspase - 10 未裂解，这表明 caspase - 8 是 MM 细胞中 TRAIL 信号通路的起始 caspase。在 TRAIL 耐药的 MM 细胞中未观察到这些现象或显著延迟，这表明耐药可能源于 caspase - 8 激活水平的抑制。在 TRAIL 耐药细胞中存在包括 FLICE 抑制蛋白（FLIP）在内的各种凋亡抑制剂的较高表达水平以及较低的 procaspase - 8 水平，并且通过蛋白质合成抑制剂放线菌酮（CHX）和蛋白激酶 C（PKC）抑制剂双吲哚马来酰胺（BIM）恢复了敏感性，这两种抑制剂都降低了 FLIP 和细胞凋亡抑制蛋白 - 2（cIAP - 2）的蛋白水平。procaspase - 8 或 FLIP 反义寡核苷酸的强制表达也使 TRAIL 耐药细胞对 TRAIL 敏感。此外，使 TRAIL 耐药细胞对 TRAIL 敏感的细胞可渗透核因子（NF）-κB 抑制剂 SN50 也抑制了 cIAP2 蛋白表达。最后，CHX、BIM 和 SN50 促进了 TRAIL 耐药细胞中 procaspase - 8 的裂解和激活，证实了 TRAIL 诱导凋亡的抑制发生在此水平，并且这些药物通过解除这种阻断使 MM 细胞敏感。我们的数据为通过抑制 FLIP 和 cIAP - 2 表达或增强 caspase - 8 活性的药物使 MM 细胞对 TRAIL 敏感的方法的临床应用建立了框架。
