title: Lysosomal Sequestration Determines Intracellular Imatinib Levels.
abstract: The intracellular uptake and retention (IUR) of imatinib is reported to be controlled by the influx transporter SLC22A1 (organic cation transporter 1). We recently hypothesized that alternative uptake and/or retention mechanisms exist that determine intracellular imatinib levels. Here, we systematically investigate the nature of these mechanisms. Imatinib uptake in cells was quantitatively determined by liquid chromatography-tandem mass spectrometry. Fluorescent microscopy was used to establish subcellular localization of imatinib. Immunoblotting, cell cycle analyses, and apoptosis assays were performed to evaluate functional consequences of imatinib sequestration. Uptake experiments revealed high intracellular imatinib concentrations in HEK293, the leukemic cell lines K562 and SD-1, and a gastrointestinal stromal tumor cell line GIST-T1. We demonstrated that imatinib IUR is time-, dose-, temperature-, and energy-dependent and provide evidence that SLC22A1 and other potential imatinib transporters do not substantially contribute to the IUR of imatinib. Prazosin, amantadine, NH4Cl, and the vacuolar ATPase inhibitor bafilomycin A1 significantly decreased the IUR of imatinib and likely interfere with lysosomal retention and accumulation of imatinib. Costaining experiments with LysoTracker Red confirmed lysosomal sequestration of imatinib. Inhibition of the lysosomal sequestration had no effect on the inhibition of c-Kit signaling and imatinib-mediated cell cycle arrest but significantly increased apoptosis in imatinib-sensitive GIST-T1 cells. We conclude that intracellular imatinib levels are primarily determined by lysosomal sequestration and do not depend on SLC22A1 expression.
authors.affiliation: Department of Medical Oncology, Erasmus University Medical Center, Erasmus MC Cancer Institute, Rotterdam, The Netherlands (H.B., A.T.D., S.Se., A.W.M.B., P.B., S.Sl., R.H.J.M., E.A.C.W.); Department of Human Genetics, Catholic University Leuven and University Hospitals, Leuven, Belgium (M.D.-R.); Division of Human Health and Medical Science, Graduate School of Kuroshio Science, Kochi University, Nankoku, Kochi, Japan (T.T.); and Department of Pharmaceutical Sciences, St. Jude Children's Research Hospital, Memphis, Tennessee (A.S.).
authors.forename: Herman
authors.initials: H
authors.lastname: Burger
authors.affiliation: Department of Medical Oncology, Erasmus University Medical Center, Erasmus MC Cancer Institute, Rotterdam, The Netherlands (H.B., A.T.D., S.Se., A.W.M.B., P.B., S.Sl., R.H.J.M., E.A.C.W.); Department of Human Genetics, Catholic University Leuven and University Hospitals, Leuven, Belgium (M.D.-R.); Division of Human Health and Medical Science, Graduate School of Kuroshio Science, Kochi University, Nankoku, Kochi, Japan (T.T.); and Department of Pharmaceutical Sciences, St. Jude Children's Research Hospital, Memphis, Tennessee (A.S.).
authors.forename: Alexander T
authors.initials: AT
authors.lastname: den Dekker
authors.affiliation: Department of Medical Oncology, Erasmus University Medical Center, Erasmus MC Cancer Institute, Rotterdam, The Netherlands (H.B., A.T.D., S.Se., A.W.M.B., P.B., S.Sl., R.H.J.M., E.A.C.W.); Department of Human Genetics, Catholic University Leuven and University Hospitals, Leuven, Belgium (M.D.-R.); Division of Human Health and Medical Science, Graduate School of Kuroshio Science, Kochi University, Nankoku, Kochi, Japan (T.T.); and Department of Pharmaceutical Sciences, St. Jude Children's Research Hospital, Memphis, Tennessee (A.S.).
authors.forename: Sandra
authors.initials: S
authors.lastname: Segeletz
authors.affiliation: Department of Medical Oncology, Erasmus University Medical Center, Erasmus MC Cancer Institute, Rotterdam, The Netherlands (H.B., A.T.D., S.Se., A.W.M.B., P.B., S.Sl., R.H.J.M., E.A.C.W.); Department of Human Genetics, Catholic University Leuven and University Hospitals, Leuven, Belgium (M.D.-R.); Division of Human Health and Medical Science, Graduate School of Kuroshio Science, Kochi University, Nankoku, Kochi, Japan (T.T.); and Department of Pharmaceutical Sciences, St. Jude Children's Research Hospital, Memphis, Tennessee (A.S.).
authors.forename: Antonius W M
authors.initials: AW
authors.lastname: Boersma
authors.affiliation: Department of Medical Oncology, Erasmus University Medical Center, Erasmus MC Cancer Institute, Rotterdam, The Netherlands (H.B., A.T.D., S.Se., A.W.M.B., P.B., S.Sl., R.H.J.M., E.A.C.W.); Department of Human Genetics, Catholic University Leuven and University Hospitals, Leuven, Belgium (M.D.-R.); Division of Human Health and Medical Science, Graduate School of Kuroshio Science, Kochi University, Nankoku, Kochi, Japan (T.T.); and Department of Pharmaceutical Sciences, St. Jude Children's Research Hospital, Memphis, Tennessee (A.S.).
authors.forename: Peter
authors.initials: P
authors.lastname: de Bruijn
authors.affiliation: Department of Medical Oncology, Erasmus University Medical Center, Erasmus MC Cancer Institute, Rotterdam, The Netherlands (H.B., A.T.D., S.Se., A.W.M.B., P.B., S.Sl., R.H.J.M., E.A.C.W.); Department of Human Genetics, Catholic University Leuven and University Hospitals, Leuven, Belgium (M.D.-R.); Division of Human Health and Medical Science, Graduate School of Kuroshio Science, Kochi University, Nankoku, Kochi, Japan (T.T.); and Department of Pharmaceutical Sciences, St. Jude Children's Research Hospital, Memphis, Tennessee (A.S.).
authors.forename: Maria
authors.initials: M
authors.lastname: Debiec-Rychter
authors.affiliation: Department of Medical Oncology, Erasmus University Medical Center, Erasmus MC Cancer Institute, Rotterdam, The Netherlands (H.B., A.T.D., S.Se., A.W.M.B., P.B., S.Sl., R.H.J.M., E.A.C.W.); Department of Human Genetics, Catholic University Leuven and University Hospitals, Leuven, Belgium (M.D.-R.); Division of Human Health and Medical Science, Graduate School of Kuroshio Science, Kochi University, Nankoku, Kochi, Japan (T.T.); and Department of Pharmaceutical Sciences, St. Jude Children's Research Hospital, Memphis, Tennessee (A.S.).
authors.forename: Takahiro
authors.initials: T
authors.lastname: Taguchi
authors.affiliation: Department of Medical Oncology, Erasmus University Medical Center, Erasmus MC Cancer Institute, Rotterdam, The Netherlands (H.B., A.T.D., S.Se., A.W.M.B., P.B., S.Sl., R.H.J.M., E.A.C.W.); Department of Human Genetics, Catholic University Leuven and University Hospitals, Leuven, Belgium (M.D.-R.); Division of Human Health and Medical Science, Graduate School of Kuroshio Science, Kochi University, Nankoku, Kochi, Japan (T.T.); and Department of Pharmaceutical Sciences, St. Jude Children's Research Hospital, Memphis, Tennessee (A.S.).
authors.forename: Stefan
authors.initials: S
authors.lastname: Sleijfer
authors.affiliation: Department of Medical Oncology, Erasmus University Medical Center, Erasmus MC Cancer Institute, Rotterdam, The Netherlands (H.B., A.T.D., S.Se., A.W.M.B., P.B., S.Sl., R.H.J.M., E.A.C.W.); Department of Human Genetics, Catholic University Leuven and University Hospitals, Leuven, Belgium (M.D.-R.); Division of Human Health and Medical Science, Graduate School of Kuroshio Science, Kochi University, Nankoku, Kochi, Japan (T.T.); and Department of Pharmaceutical Sciences, St. Jude Children's Research Hospital, Memphis, Tennessee (A.S.).
authors.forename: Alex
authors.initials: A
authors.lastname: Sparreboom
authors.affiliation: Department of Medical Oncology, Erasmus University Medical Center, Erasmus MC Cancer Institute, Rotterdam, The Netherlands (H.B., A.T.D., S.Se., A.W.M.B., P.B., S.Sl., R.H.J.M., E.A.C.W.); Department of Human Genetics, Catholic University Leuven and University Hospitals, Leuven, Belgium (M.D.-R.); Division of Human Health and Medical Science, Graduate School of Kuroshio Science, Kochi University, Nankoku, Kochi, Japan (T.T.); and Department of Pharmaceutical Sciences, St. Jude Children's Research Hospital, Memphis, Tennessee (A.S.).
authors.forename: Ron H J
authors.initials: RH
authors.lastname: Mathijssen
authors.affiliation: Department of Medical Oncology, Erasmus University Medical Center, Erasmus MC Cancer Institute, Rotterdam, The Netherlands (H.B., A.T.D., S.Se., A.W.M.B., P.B., S.Sl., R.H.J.M., E.A.C.W.); Department of Human Genetics, Catholic University Leuven and University Hospitals, Leuven, Belgium (M.D.-R.); Division of Human Health and Medical Science, Graduate School of Kuroshio Science, Kochi University, Nankoku, Kochi, Japan (T.T.); and Department of Pharmaceutical Sciences, St. Jude Children's Research Hospital, Memphis, Tennessee (A.S.) e.wiemer@erasmusmc.nl.
authors.forename: Erik A C
authors.initials: EA
authors.lastname: Wiemer
mesh_terms: D000547:Amantadine / Q000494:pharmacology; D000643:Ammonium Chloride / Q000494:pharmacology; D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis; D001549:Benzamides / Q000494:pharmacology*; D002453:Cell Cycle; D045744:Cell Line, Tumor; D057809:HEK293 Cells; D006801:Humans; D000068877:Imatinib Mesylate; D008247:Lysosomes / Q000187:drug effects / Q000378:metabolism*; D018942:Macrolides / Q000494:pharmacology; D027702:Organic Cation Transporter 1 / Q000378:metabolism; D010879:Piperazines / Q000494:pharmacology*; D011224:Prazosin / Q000494:pharmacology; D011743:Pyrimidines / Q000494:pharmacology*
chemical_list: D000970:Antineoplastic Agents; D001549:Benzamides; D018942:Macrolides; D027702:Organic Cation Transporter 1; D010879:Piperazines; D011743:Pyrimidines; D000643:Ammonium Chloride; C040929:bafilomycin A1; D000068877:Imatinib Mesylate; D000547:Amantadine; D011224:Prazosin