Pseudomonas aeruginosa NfsB and nitro-CBI-DEI--a promising enzyme/prodrug combination for gene directed enzyme prodrug therapy

Mol Cancer. 2013 Jun 10;12:58. doi: 10.1186/1476-4598-12-58.

Abstract

Background: The nitro-chloromethylbenzindoline prodrug nitro-CBI-DEI appears a promising candidate for the anti-cancer strategy gene-directed enzyme prodrug therapy, based on its ability to be converted to a highly cytotoxic cell-permeable derivative by the nitroreductase NfsB from Escherichia coli. However, relative to some other nitroaromatic prodrugs, nitro-CBI-DEI is a poor substrate for E. coli NfsB. To address this limitation we evaluated other nitroreductase candidates from E. coli and Pseudomonas aeruginosa.

Findings: Initial screens of candidate genes in the E. coli reporter strain SOS-R2 identified two additional nitroreductases, E. coli NfsA and P. aeruginosa NfsB, as being more effective activators of nitro-CBI-DEI than E. coli NfsB. In monolayer cytotoxicity assays, human colon carcinoma (HCT-116) cells transfected with P. aeruginosa NfsB were >4.5-fold more sensitive to nitro-CBI-DEI than cells expressing either E. coli enzyme, and 23.5-fold more sensitive than untransfected HCT-116. In three dimensional mixed cell cultures, not only were the P. aeruginosa NfsB expressing cells 540-fold more sensitive to nitro-CBI-DEI than pure cultures of untransfected HCT-116, the activated drug that they generated also displayed an unprecedented local bystander effect.

Conclusion: We posit that the discrepancy in the fold-sensitivity to nitro-CBI-DEI between the two and three dimensional cytotoxicity assays stems from loss of activated drug into the media in the monolayer cultures. This emphasises the importance of evaluating high-bystander GDEPT prodrugs in three dimensional models. The high cytotoxicity and bystander effect exhibited by the NfsB_Pa/nitro-CBI-DEI combination suggest that further preclinical development of this GDEPT pairing is warranted.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bystander Effect
  • Cell Line, Tumor
  • Cell Proliferation / drug effects
  • Enzyme Therapy
  • Gene Expression
  • Genetic Therapy
  • HCT116 Cells
  • Humans
  • Inhibitory Concentration 50
  • Nitroreductases / genetics
  • Nitroreductases / metabolism*
  • Prodrugs / metabolism*
  • Prodrugs / pharmacology
  • Prodrugs / toxicity
  • Pseudomonas aeruginosa / enzymology*
  • Pseudomonas aeruginosa / genetics
  • Tumor Stem Cell Assay

Substances

  • Prodrugs
  • Nitroreductases