TRAIL is a TNF family member that engages apoptosis via recruitment and rapid activation of caspase-8. Oxygen-free radicals, more generally known as reactive oxygen species (ROS) are well recognized for playing an important role in the regulation of tumor cell apoptosis. ROS within the cells act as secondary messengers in intracellular signalling cascades therefore function as anti-tumorigenic species. But very little is known about the effect of ROS on TRAIL-induced apoptosis. In this study we investigated the effect of CCCP, a classic uncoupler of oxidative phosphorylation, on TRAIL-induced apoptosis in TRAIL-resistant MCF-7 cells. It was found that pretreatment with CCCP for 6 h and then treatment with TRAIL for additional 3 h markedly enhanced apoptosis by 2-fold as compared with TRAIL alone. The uncoupling effect enhanced TRAIL-induced apoptosis by ROS generation. Moreover, CCCP treatment also reduced mitochondrial transmembrane potential (MTP, ΔΨm) and induced Bax translocation to the mitochondria of its own account. This sensitization was inhibited with N-acetyl-L-cysteine (NAC) treatment by abrogating the ROS which was generated by the combined treatment of CCCP and TRAIL. Of interest, NAC also inhibited reduction of the ΔΨm and Bax translocation after CCCP pretreatment which suggest that the generartion of ROS may precede the loss in MTP. Thus, we demonstrated that CCCP-induced ROS generation enhanced TRAIL induced apoptosis by regulation of Bax translocation and mitochondrial transmembrane potential. The enhancing effect by CCCP-induced ROS generation was restored after NAC treatment. Therefore, our results suggest that uncoupling the cells by CCCP can overcome the resistance to TRAIL protein and can be a very efficient treatment for the tumor cells especially resistant to TRAIL-induced apoptosis.

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