MDM2 antagonist nutlin-3a reverses mitoxantrone resistance by inhibiting breast cancer resistance protein mediated drug transport

Fan Zhang, Stacy L. Throm, Laura L. Murley, Laura A. Miller, D. Steven Zatechka, R. Kiplin Guy, Rachel Kennedy, Clinton F. Stewart

Research output: Contribution to journalArticlepeer-review

19 Scopus citations

Abstract

Breast cancer resistance protein (BCRP; ABCG2), a clinical marker for identifying the side population (SP) cancer stem cell subgroup, affects intestinal absorption, brain penetration, hepatobiliary excretion, and multidrug resistance of many anti-cancer drugs. Nutlin-3a is currently under pre-clinical investigation in a variety of solid tumor and leukemia models as a p53 reactivation agent, and has been recently demonstrated to also have p53 independent actions in cancer cells. In the present study, we first report that nutlin-3a can inhibit the efflux function of BCRP. We observed that although the nutlin-3a IC50 did not differ between BCRP over-expressing and vector control cells, nutlin-3a treatment significantly potentiated the cells to treatment with the BCRP substrate mitoxantrone. Combination index calculations suggested synergism between nutlin-3a and mitoxantrone in cell lines over-expressing BCRP. Upon further investigation, it was confirmed that nutlin-3a increased the intracellular accumulation of BCRP substrates such as mitoxantrone and Hoechst 33342 in cells expressing functional BCRP without altering the expression level or localization of BCRP. Interestingly, nutlin-3b, considered virtually "inactive" in disrupting the MDM2/p53 interaction, reversed Hoechst 33342 efflux with the same potency as nutlin-3a. Intracellular accumulation and bi-directional transport studies using MDCKII cells suggested that nutlin-3a is not a substrate of BCRP. Additionally, an ATPase assay using Sf9 insect cell membranes over-expressing wild-type BCRP indicated that nutlin-3a inhibits BCRP ATPase activity in a dose-dependent fashion. In conclusion, our studies demonstrate that nutlin-3a inhibits BCRP efflux function, which consequently reverses BCRP-related drug resistance.

Original languageEnglish
Pages (from-to)24-34
Number of pages11
JournalBiochemical Pharmacology
Volume82
Issue number1
DOIs
StatePublished - Jul 1 2011

Bibliographical note

Funding Information:
This work was supported by US Public Health Service Childhood Solid Tumor Program Grant No. CA23099 , Cancer Center Support (CORE) Grant No. CA21765 , and by American, Lebanese, and Syrian Associated Charities (ALSAC) . We thank Dr. Feng Bai for LC–MS/MS assistance. We thank Jennifer Peters for her help in the confocal and fluorescence imaging.

Keywords

  • ABC transporter
  • Breast cancer resistance protein (BCRP)
  • Multi-drug resistance
  • Nutlin-3a

ASJC Scopus subject areas

  • Biochemistry
  • Pharmacology

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