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.