Induction of apoptosis by chemotherapeutic drugs: the role of FADD in activation of caspase-8 and synergy with death receptor ligands in ovarian carcinoma cells

Abstract

Although ovarian tumours initially respond to chemotherapy, they gradually acquire drug resistance. The aims of this study were to identify how chemotherapeutic drugs with diverse cellular targets activate apoptotic pathways and to investigate the mechanism by which exposure to a combination of drugs plus death receptor ligands can increase tumour cell kill. The results show that drugs with distinct cellular targets differentially up-regulate TRAIL and TNF as well CD95L, but do not require interaction of these ligands with their receptor partners to induce cell death. Factors that were critical in druginduced apoptosis were activation of caspases, with caspase-8 being activated by diverse drugs in a FADD-independent manner. Certain drugs also demonstrated some dependence on FADD in the induction of cell death. Caspase-9 was activated more selectively by chemotherapeutic agents. Combining ligation of death receptors with exposure to drugs increased tumour cell kill in both drug resistant cell lines and primary ovarian carcinoma cells, even though these cells were not sensitive to death receptor ligation alone. CD95L was more consistent at combining with drugs than TRAIL or TNF. Investigation of the mechanism by which a combination of drugs plus CD95 ligation can increase cell death showed that caspase-8 was activated in cells exposed to a combination of cisplatin and anti-CD95, but not in cells exposed to either agent alone.

Mechanism of cell death induced by the novel enzyme-prodrug combination, nitroreductase/CB1954, and identification of synergism with 5-fluorouracil

“…After 48 h, FACS analysis was performed to determine the degree of cell death induced and the relative amounts of apoptosis and necrosis. Figure 2B demonstrates that the mode of cell death induced by activated CB1954 in this setting was predominantly apoptosis, in common with a range of other chemotherapeutic agents (data not shown and Milner et al, 2002). In addition, RAd-NR infected SKOV3 cells were treated with CB1954 and similar analysis was performed.…”

“…We have previously shown that at a concentration of 50 mM, DEVD-FMK (caspase-3 inhibitor), IETD-FMK (caspase-8 inhibitor) and LEHD-FMK (caspase-9 inhibitor) work in a specific manner (Milner et al, 2002). Therefore, this concentration was used for all the inhibitors in these experiments.…”

“…However, not all cell types require death receptorligand interaction for drugs to be effective (Gamen et al, 1997;Eischen et al, 1997;Villunger et al, 1997;Tolomeo et al, 1998;RuizRuiz and Lopez-Rivas, 1999). Recent studies have indicated that caspase-8 can be activated independently of death receptor ligation (Milner et al, 2002). Interestingly, different drugs may engage distinct components of the apoptotic machinery (Milner et al, 2002).…”

“…Recent studies have indicated that caspase-8 can be activated independently of death receptor ligation (Milner et al, 2002). Interestingly, different drugs may engage distinct components of the apoptotic machinery (Milner et al, 2002). This is important since it may allow the use of different drugs to form potentially synergistic combinations.…”

Analysis of changes in the proteome of HL-60 promyeloid leukemia cells induced by the proteasome inhibitor PSI

“…Thus, administration of proteasome inhibitors results in the stimulation of a proapoptotic autocrine loop by signaling via death receptor family members. However, as Milner et al had shown previously [53], genotoxic stress induced by chemotherapeutic drugs can differentially upregulate TRAIL, TNF and CD95L and activate caspase-8 in a FADDindependent manner without engagement of their receptor partners.…”