title: MDM2 antagonist nutlin-3a reverses mitoxantrone resistance by inhibiting breast cancer resistance protein mediated drug transport.
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 IC(50) 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.
authors: Department of Pharmaceutical Sciences, St. Jude Children's Research Hospital, Memphis, TN 38105, USA. fan.zhang@stjude.org
authors: Fan
authors: F
authors: Zhang
authors: Stacy L
authors: SL
authors: Throm
authors: Laura L
authors: LL
authors: Murley
authors: Laura A
authors: LA
authors: Miller
authors: D
authors: D
authors: Steven Zatechka
authors: R
authors: R
authors: Kiplin Guy
authors: Rachel
authors: R
authors: Kennedy
authors: Clinton F
authors: CF
authors: Stewart
mesh_terms: D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters / Q000378:metabolism*; D000970:Antineoplastic Agents / Q000494:pharmacology*; D001562:Benzimidazoles / Q000378:metabolism / Q000494:pharmacology; D001692:Biological Transport / Q000187:drug effects; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D019008:Drug Resistance, Neoplasm / Q000187:drug effects*; D004354:Drug Screening Assays, Antitumor; D004357:Drug Synergism; D004359:Drug Therapy, Combination; D005456:Fluorescent Dyes / Q000378:metabolism / Q000494:pharmacology; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology*; D008942:Mitoxantrone / Q000494:pharmacology*; D009363:Neoplasm Proteins / Q000378:metabolism*; D012516:Osteosarcoma / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D010879:Piperazines / Q000494:pharmacology*
chemical_list: C116614:ABCG2 protein, human; D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters; D000970:Antineoplastic Agents; D001562:Benzimidazoles; D005456:Fluorescent Dyes; D007093:Imidazoles; D009363:Neoplasm Proteins; D010879:Piperazines; C482205:nutlin 3; D008942:Mitoxantrone; C017807:bisbenzimide ethoxide trihydrochloride