The CD95/CD95 ligand system is not the major effector in anticancer drug-mediated apoptosis

Abstract

Many anticancer drugs are able to induce apoptosis in tumor cells but the mechanisms underlying this phenomenon are poorly understood. Some authors reported that the p53 tumor suppressor gene may be responsible for drug-induced apoptosis; however, chemotherapy-induced apoptosis can also be observed in p53 negative cells. Recently, doxorubicin (DXR) was reported to induce CD95L expression to mediate apoptosis through the CD95/CD95L system. Thus, an impairment of such a system may be involved in drug resistance. We evaluated the in vitro antitumor activity of several cytotoxic drugs on two human p53-negative T-cell lymphoma cell lines, the HUT78-B1 CD95L-resistant cell line and the HUT78 parental CD95L-sensitive cell line. We demostrated by Western blotting assay that DXR and etoposide (VP-16) were able to induce CD95L expression after 4 h of treatment. In contrast, they were unable to induce the expression of p53. DXR, at concentrations ranging from 0.001 ± 1 mg/ml, and VP16, at concentrations ranging from 0.05 ± 1 mg/ml, were equally cytotoxic and induced apoptosis in both cell lines as assessed by fluorescence microscopy and flow cytometry analyses. Although we observed a slightly reduced percentage of apoptotic cells in HUT78B1 when compared with the parental HUT78 cells after few hours of drug exposure, this difference was no longer evident at 48 or 72 h. Similarly, the exposure of HUT78 cells to a CD95-blocking antibody partially reduced early apoptosis (24 h) without affecting the long-term effects of the drugs including cytotoxicity. Furthermore, as observed with DXR and VP-16, both the CD95L-sensitive and the CD95L-resistant cell lines resulted equally sensitive to the cytotoxic effects of a number of different cytotoxic drugs (vincristine, camptothecin, 5-fluorouracil and methotrexate). The treatment with the Caspase-3 tetrapeptide aldehyde inhibitor, Ac-DEVD-CHO, did not affect the DXR-induced apoptosis whereas it only modestly inhibited apoptosis and cytotoxicity of VP-16, while Z-VAD.FMK, a Caspase inhibitor that prevents the processing of Caspase-3 to its active form, was able to block DXR-induced apoptosis at 24 h but not at 48 h. Thus, our results do not confirm a crucial role for the CD95/CD95L system in druginduced apoptosis and suggest the involvement of alternative p53-independent pathways at least in this experimental model system.

The topoisomerase inhibitors camptothecin and etoposide induce a CD95-independent apoptosis of activated peripheral lymphocytes

“…In agreement with previous reports in different experimental models 19,46 our results indicate that ETO-or CPT-induced apoptosis of activated PBL may be in part independent of caspases activation. It was already reported that some death inducers, such anti-CD2 monoclonal antibodies, staurosporine as well as the pro-apoptotic protein Bax 47,48 which induce caspase activation are however not blocked by the broad spectrum inhibitor zVAD.…”

“…10,15 More recently chemotherapeutic agents such as doxorubicin, ETO and teniposide were shown to induce a rapid up-regulation of CD95-L via the activation of the SAPK/JNK pathway. 12,16 However these results remain controversial 17 and the observation that some Fas-resistant cell lines undergo apoptosis after treatment with ETO and doxorubicin 11,18,19 suggested that in addition to the CD95/CD95-L pathway other mechanisms may be involved in drug-induced apoptosis.…”

Cytotoxic drugs and the CD95 pathway

“…[54][55][56][57] However, induction of CD95-L following drug treatment was also found in these studies. 55,56 The discrepancy between different results may reflect differences in cell lines used, kinetics, cell culture conditions, drug concentrations and concentrations or effectiveness of blocking antibodies. Drug-induced apoptosis may also involve cell type-specific activation of parallel pathways such as death receptor pathways and mitochondrial pathways.…”

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

“…Initial studies revealed that cytotoxic drugs can activate death receptor/caspase-8 signalling pathways and this mechanism may be an essential factor in the early phase of drug-induced cell death depending on the cell type used (Friesen et al, 1996;Fulda et al, 1997Fulda et al, , 1998aFulda et al, , b, 2000Micheau et al, 1997). 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).…”