Piperlongumine induces cell death through ROS-mediated CHOP activation and potentiates TRAIL-induced cell death in breast cancer cells

Piperlongumine (PL) has been shown to selectively induce apoptotic cell death in cancer cells via reactive oxygen species (ROS) accumulation. In this study, we characterized a molecular mechanism for PL-induced cell death. Cell viability and cell death were assessed by MTT assay and Annexin V-FITC/PI staining, respectively. ROS generation was measured using the H(2)DCFDA. Small interfering RNA (siRNA) was used for suppressing gene expression. The mRNA and protein expression were analyzed by RT-PCR and Western blot analysis, respectively. We found that PL promotes C/EBP homologous protein (CHOP) induction, which leads to the up-regulation of its targets Bim and DR5. Pretreatment with the ROS scavenger N-acetyl-cysteine abolishes the PL-induced up-regulation of CHOP and its target genes, suggesting an essential role for ROS in PL-induced CHOP activation. The down-regulation of CHOP or Bim with siRNA efficiently attenuates PL-induced cell death, suggesting a critical role for CHOP in this cell death. Furthermore, PL potentiates TRAIL-induced cytotoxicity in breast cancer cells by upregulating DR5, as DR5 knockdown abolished the sensitizing effect of PL on TRAIL responses. Overall, our data suggest a new mechanism for the PL-induced cell death in which ROS mediates CHOP activation, and combination treatment with PL and TRAIL could be a potential strategy for breast cancer therapy.