Effects of TRAIL , doxorubicin and 4-hydroxy-ifosfamide in a panel of soft-tissue sarcoma cell lines with different sensitivity to tumor necrosis factor-family cytokines

Submitted TRAIL and cytotoxic agents in soft tissue sarcoma cells 116 Abstract Doxorubicin (DOX) and ifosfamide (IFO) are the most active single agents in soft tissue sarcomas (STS). Still, the response rate in metastatic disease is only 20-30%. Tumor necrosis factor-α (TNF-α) is used for STS only in the setting of isolated limb perfusions. Like TNF-α, TNF-related apoptosis-inducing ligand (TRAIL) also induces apoptosis, and preliminary studies indicate that TRAIL lacks systemic side effects. Resistance to TRAIL has been demonstrated, but can be circumvented by combinations of TRAIL with conventional cytotoxic agents, in vitro. The effects of TRAIL alone and in combination with DOX or 4-hydroxy-IFO (i.e. the active metabolite of IFO, 4-OH-IFO) were evaluated in a panel of TNF-α sensitive and resistant human soft tissue sarcoma cells. The rhabdomyosarcoma cell line KYM-1, its five-fold TNF-α sensitive subline KD4 and its >150-fold TNF-α resistant subline 37B8R were used. Membrane expression of the TRAIL-receptors DR4, DR5 (pro-apoptotic) and DcR1, DcR2 (anti-apoptotic) was assessed by flow cytometry. Drug-induced cytotoxicity was determined by a microculture tetrazolium assay. Apoptosis assays using acridine orange were conducted for the combination that was most potent of inducing cytotoxicity. DOX and 4-OH-IFO decreased survival in all cell lines; a twofold resistance was observed for both drugs in 37B8R. All three cell lines expressed DR4 and DR5, but no or very low levels of DcR1 or DcR2. TRAIL single agent decreased survival in KYM-1 and was even more cytotoxic in KD4 and induced massive apoptosis, while 37B8R was >500-fold resistant to TRAIL compared to KYM-1 and little apoptosis could be observed. The combination of TRAIL plus DOX showed synergistic cytotoxic effects in KYM-1 and 37B8R. The combination of TRAIL plus 4-OH-IFO showed to be additive in all three cell lines. DOX plus TRAIL induced more cytotoxicity as well as apoptosis in all three cell lines compared to TRAIL alone. In 37B8R, DOX overcame resistance to TRAIL. In KYM-1 and its sublines KD4 and 37B8R, sensitivity and resistance to TNF-α and TRAIL parallels. TRAIL resistance was independent from expression of TRAIL receptors. DOX with TRAIL could overcome TRAIL-resistance in 37B8R cells, suggesting a therapeutic potential for this combination for TNF-α and TRAIL refractory STS cells.