Note for: Impact of DNA repair pathways on the cytotoxicity of piperlongumine in chicken DT40 cell-lines

Note for: Impact of DNA repair pathways on the cytotoxicity of piperlongumine in chicken DT40 cell-lines

(doi: 10.18632/genesandcancer.26)

One of the paper which was cited by this one, has just been retracted recently (2018, Sep --Selective killing of cancer cells by a small molecule targeting the stress response to ROS. Nature. 

2011;475:231-4). It was the original work which showed that Piperlongumine can generate ROS which eventually was the major mechanism killing the cancer cell lines.

Piperlongumine induces ROS.

Aim;

examine the contribution of ROS-induced DNA damage to the cytotoxicity by piperlongumine.

Method;

various DNA repair-deficient chicken DT40 cell-lines with a single DNA repair gene deletion were tested for cellular sensitivity to piperlongumine.

ROS is necessary for several physiological responses, including differentiation, immunity, metabolic adaptation and autophagy.

A fine balance between the production of ROS and the detoxification of ROS needs to be maintained for proper cell growth.

piperlongumine was identified through a cell-based, high-throughput screening to selectively kill various types of transformed cells with minimal cytotoxicity to non-transformed cells.

piperlongumine increases the level of ROS and apoptotic cell death selectively in cancer cells.

Glutathione S-transferases (GSTs), is up-regulated and piperlongumine directly interacts with GSTs and inhibits their activities.

Our results show that piperlongumine selectively kills cell lines with a defect in homologous recombination (HR). Piperlongumine displays little or no toxicity to cell lines with a defect in other DNA repair pathways, including the base excision repair (BER) that is a major pathway to repair ROS-induced DNA lesions. A deletion of 53BP1 or Ku70 in BRCA1-deficient cell lines restores resistance to piperlongumine, strongly implicating that piperlongumine exerts its cytotoxicity by generating double-strand breaks.

DSBs can be generated directly by ROS and also by DNA interstrand cross-links and protein-DNA cross-links during replication.

Cells deficient in Ku80, LigIV and 53BP1 displayed resistance to piperlongumine. Thus, NHEJ is not the major contributor for the repair of DSBs generated by piperlongumine.

the impact of piperlongumine on HR directly, a cell-based HR assay was performed. SCneo reporter gene with a restriction enzyme I-SceI cutting site was inserted at the Ovalbumin locus.

A functional neomycin-resistant gene is restored only when the disrupted SCEneo is repaired by HR using the 3’-neo gene as a donor.

DNA repair-deficient cell lines showed that HR repair-deficient cell lines display a higher cellular sensitivity to piperlongumine compared with any other DNA repair-deficient cell lines.

The presence of DSBs was confirmed by Rad51 accumulation in chromatin and chromosome breakage by the treatment with piperlongumine.

Beneficial points using DNA-repair deficient KO cell lines -- Our genetic experiments with various DNA repair-deficient cell lines exclude the possibilities that the cytotoxicity of piperlongumine is due to the formation of DNA interstrand cross-links and protein-DNA cross-links as well as bulky DNA lesions.

The source of DSBs and/ or SSBs in piperlongumine-treated cells is currently under investigation.

BRCA1 is known to possess various functions outside of a role in HR. It was reported that BRCA1 plays a yet unidentified role in the repair of DNA interstrand cross-links in mammalian cells.

however, a mechanistic basis of the suppression of HR by piperlongumine remains elusive.

we revealed two novel activities of piperlongumine. One is the induction of DSBs and the other is the suppression of HR.

These findings make piperlongumine the more attractive candidate for chemotherapy of breast and ovarian cancers with defective HR.

These findings make piperlongumine the more attractive candidate for chemotherapy of breast and ovarian cancers with defective HR.

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