Chemotherapy augments TRAIL-induced apoptosis in breast cell lines

Expression and function of the TRAIL apoptotic pathway was investigated in normal and malignant breast epithelial cells. Glutathione-S-transferase (GS T)-TRAIL extracellular domain fusion proteins Here produced to analyze TRAI L-induced apoptosis. Only GST-TRAIL constructs containing regions homologou s to the Fas self-association and ligand binding domains could induce apopt osis, GST-TRAIL induced significant (>90%) apoptosis in just one of eight n ormal and one of eight malignant breast cell lines. All other lines were re latively resistant to TRAIL-induced apoptosis, Activating TRAIL receptors D R I and DR5 were expressed in all normal and malignant breast cell lines. T he inhibitory receptor TRID was highly expressed in one of tour normal and two of seven malignant breast cell lines, DR4, DR5, or TRID expression did not correlate with sensitivity to TRAIL-induced apoptosis. Incubation of ce ll lines with doxorubicin or 5-fluorouracil significantly augmented TRAIL-i nduced apoptosis in most breast cell lines, By fractional inhibition analys is, the toxicity of the combination of TRAIL and doxorubicin or 5-fluoroura cil was synergistic compared with either agent alone. In contrast, melphala n and paclitaxel augmented TRAIL-induced apoptosis in few cell lines, and m ethotrexate did not augment it in any cell line. Augmentation of TRAIL-indu ced apoptosis by doxorubicin or 5-fluorouracil was mediated through caspase activation, This was evidenced by the fact that chemotherapy agents that s ynergized with TRAIL (e.g., doxorubicin) themselves caused cleavage of casp ase-3 and poly(ADP-ribose) polymerase (PARP), and their toxicity was blocke d by the caspase inhibitor Z-Val-Ala-Asp(OMe)-CH2 (ZVAD-fmk), The combinati on of TRAIL and doxorubicin caused significantly greater caspase-3 and PARP cleavage, and the combined toxicity also was inhibited by ZVAD-fmk. In con trast, chemotherapy agents that did not augment TRAIL-induced apoptosis (e. g., methotrexate) caused minimal caspase-3 and PARP cleavage by themselves, and their toxicity was not inhibited by ZVAD-fmk, These drugs also did not increase caspase-3 or PARP cleavage when combined with TRAIL. Tn summary, few breast cell lines are sensitive to TRAIL-induced apoptosis, and no diff erence in sensitivity is found between normal and malignant cell lines. Tre atment with chemotherapy provides an approach to sensitize breast cancer ce lls to TRAIL-induced apoptosis.