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PTH-272 Modulation of NRF2 alters the responsiveness of colorectal cancer cells to irinotecan
  1. JP Evans1,
  2. BK Winiarski2,
  3. PA Sutton1,
  4. DH Palmer3,
  5. N Kitteringham2
  1. 1Clinical Cancer Medicine
  2. 2Pharmacology, University of Liverpool
  3. 3Oncology, Royal Liverpool and Broadgreen University Hospital, Liverpool, UK

Abstract

Introduction Chemotherapy is essential to improve outcomes in colorectal cancer (CRC). Irinotecan is a pro-drug commonly used in CRC which requires conversion to the active metabolite Sn38. The cytoprotective protein NRF2 has been associated with chemo-resistance and a worse prognosis in a number of cancers.1It also regulates the activity of enzymes (including CES1 and UGT1A1) that coordinate irinotecan metabolism and efflux. 2NRF2 remains bound to the regulatory protein KEAP1 in its quiescent state.

The aim of this study is to establish the value of modulating NRF2 as a novel strategy for enhancing the efficacy of irinotecan-based chemotherapy either through increased conversion of irinotecan to Sn38 or by sensitisation of CRC cells to its effect.

Method NRF2 was modulated in the human CRC cell line HCT116 using small inhibitory RNA (siRNA) targeting NRF2and KEAP1 or the chemical inhibitor brusatol and inducer CDDO-Me. Cells were then exposed to irinotecan over a range of concentrations for 48 h. Cell viability was measured by MTS assay and expressed as a percentage of control for the calculation of IC50values. GraphPad Prism 6®was used for all statistical analysis and the generation of dose-response curves.

Results Successful NRF2 modulation was confirmed by western blotting for Nrf2 and downstream targets NQO1 and HO-1. The IC50 value of irinotecan in untreated control cells was 90µM. Induction of NRF2 by KEAP1siRNA (IC50 220µM) or CDDO-Me (IC50 325µM) significantly reduced irinotecan cytotoxicity (p < 0.0001). In contrast, inhibition of NRF2 by NRF2siRNA (IC50 11µM) or brusatol (IC50 55µM) had the opposite effect (p < 0.0001).

Conclusion Inhibition of NRF2 sensitises CRC cells to irinotecan cytotoxicity and may allow improved response to treatment, or permit lower doses with fewer side effects. We are currently validating these findings in vivo and examining the contribution of processes that mediate the disposition of irinotecan. The availability of NRF2 inhibiting and inducing compounds may allow for successful modulation in the clinical setting.

Disclosure of interest J. Evans Grant/ Research Support from: Cancer Research UK, B. Winiarski: None Declared, P. Sutton: None Declared, D. Palmer: None Declared, N. Kitteringham: None Declared.

References

  1. Kawasaki Y, Ishigami S, Arigami T, Uenosono Y, Yanagita S, Uchikado Y, et al. Clinicopathological significance of nuclear factor (erythroid-2)-related factor 2 (Nrf2) expression in gastric cancer. BMC Cancer 2015;15(1):5

  2. Wang M, Zhu JY, Chen S, Qing Y, Wu D, Lin YM, et al. Effects of co-treatment with sulforaphane and autophagy modulators on uridine 5’-diphospho-glucuronosyltransferase 1A isoforms and cytochrome P450 3A4 expression in Caco-2 human colon cancer cells. Oncol Lett. 2014;8(6):2407–16

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