Down-regulation of Cbl-b by bufalin results in up-regulation of DR4/DR5 and sensitization of TRAIL-induced apoptosis in breast cancer cells

TNF-related apoptosis-inducing ligand (TRAIL) is a potential cancer therapeutic agent that preferentially induces apoptosis in cancer cells. However, breast cancer cells are generally resistant to TRAIL. Bufalin is a major active ingredient of the traditional Chinese medicine ChanSu. The present study aimed to assess the synergistic effect of bufalin and TRAIL and elucidate the underlying mechanisms in breast cancer cells. Cell proliferation and apoptosis were measured by MTT assay and flow cytometry, respectively. The expression of proteins was assayed by flow cytometry and/or Western blotting. Transfection studies were used to determine the involvement of DR4, DR5 and Cbl-b in the synergistic effect of bufalin and TRAIL. MCF-7 and MDA-MB-231 cells were resistant to TRAIL. Both cell lines were dramatically sensitized to TRAIL-induced apoptosis by bufalin. Further experiments indicated that bufalin up-regulated DR4 and DR5, activated ERK, JNK and p38 MAPK and down-regulated Cbl-b. Blocking the up-regulation of DR4 and DR5 by siRNA rendered cells less sensitive to apoptosis induced by the combination of bufalin and TRAIL. Inhibition of the activation of ERK, JNK and p38 MAPK by specific inhibitors attenuated DR4 and DR5 up-regulation. Moreover, down-regulation of Cbl-b by shRNA led to stronger activation of ERK, JNK and p38 MAPK, more up-regulation of DR4 and DR5, and a stronger synergistic effect of bufalin and TRAIL. Bufalin enhanced TRAIL-induced apoptosis by up-regulating the expression of DR4 and DR5. Bufalin-induced down-regulation of Cbl-b contributed to the up-regulation of DR4 and DR5, which might be partially mediated by the activation of ERK, JNK and p38 MAPK.