Piperlongumine is a naturally-occurring small molecule with various biological 
activities. Recent studies demonstrate that piperlongumine selectively kills 
various types of transformed cells with minimal toxicity to non-transformed 
cells by inducing a high level of reactive oxygen species (ROS). ROS generates 
various types of DNA lesions, including base modifications and single strand 
breaks. In order to examine the contribution of ROS-induced DNA damage to the 
cytotoxicity by piperlongumine, various DNA repair-deficient chicken DT40 
cell-lines with a single DNA repair gene deletion were tested for cellular 
sensitivity to piperlongumine. The results showed that cell lines defective in 
homologous recombination (HR) display hyper-sensitivity to piperlongumine, while 
other cell lines with a deficiency in non-homologous end joining (NHEJ), base 
excision repair (BER), nucleotide excision repair (NER), Fanconi anemia (FA) 
pathway, or translesion DNA synthesis (TLS) polymerases, show no sensitivity to 
piperlongumine. The results strongly implicate that double strand breaks (DSBs) 
generated by piperlongumine are major cytotoxic DNA lesions. Furthermore, a 
deletion of 53BP1 or Ku70 in the BRCA1-deficient cell line restored cellular 
resistance to piperlongumine. This strongly supports the idea that 
piperlongumine induces DSB- mediated cell death. Interestingly, piperlongumine 
makes the wild type DT40 cell line hypersensitive to a PARP-inhibitor, Olaparib. 
The results implicate that piperlongumine inhibits HR. Further analysis with 
cell-based HR assay and the kinetic study of Rad51 foci formation confirmed that 
piperlongumine suppresses HR activity. Altogether, we revealed novel mechanisms 
of piperlongumine-induced cytotoxicity.