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Nuclear Trafficking of ABCB1‐Daunorubicin Vesicles Initiated by Sphingomyelinase Reverts Multidrug Resistance in Chinese Hamster Fibroblasts
Author(s) -
Lee WingKee,
Kolesnick Richard N
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.807.1
Subject(s) - daunorubicin , sphingomyelin , microbiology and biotechnology , vesicle , multiple drug resistance , intracellular , sphingolipid , biology , nuclear membrane , chemistry , biochemistry , membrane , nucleus , immunology , leukemia , antibiotics
Effective cancer chemotherapy treatment is hampered by multidrug resistance (MDR), defined by upregulation of ABCB1, which effluxes drugs. Intracellular ABCB1 also sequesters anthracycline drugs in acidic vesicles preventing nuclear targeting. Reports indicate sphingolipids affect vesicular trafficking. We hypothesized sphingomyelinase (SMase) activity might mobilize ABCB1/drug vesicles to revert MDR. Using drug‐resistant ADX fibroblasts expressing ABCB1‐EGFP, time‐lapse confocal microscopy revealed daunorubicin (DNR) sequestration in ABCB1‐EGFP‐containing vesicles within 5min. In ADX/ABCB1‐EGFP loaded with DNR (20μM,2h) followed by drug washout, exogenous bacterial (b)SMase, which hydrolyzes plasma membrane sphingomyelin and initiates sphingolipid signaling, induced rapid ABCB1‐EGFP/DNR vesicle movement to the nucleus, where DNR was deposited, while ABCB1‐EGFP remained in nuclear membranes. Immunoblots of isolated nuclei and nuclear envelope membranes purified by sucrose density gradient confirmed these observations. In DNR‐exposed ADX cells, bSMase‐induced ABCB1 translocation to the nucleus peaked at 1h, and was detectable at 4h but not at 8h, suggesting transient incorporation of ABCB1 into the nuclear membrane. No nuclear ABCB1 was observed in drug sensitive DC3F cells loaded with an isotoxic DNR dose (0.2μM, 2h), indicating ABCB1 nuclear translocation by bSMase is MDR specific. Cell viability studies by MTT assay demonstrate augmented DNR toxicity in the presence of bSMase and can be enhanced by co‐incubation with the ABCB1 inhibitor, PSC833/valspodar (1μM), to prevent cellular drug efflux, in an additive manner. Intriguingly, in the postnuclear supernatant of DNR‐treated ADX cells, ABCB1 primarily sediments at 8000g, but shifts to 18000g at 4h after bSMase suggesting altered vesicular density or ABCB1 transfer to a distinct vesicular pool. In conclusion, SMase activity mobilizes ABCB1/DNR vesicles to the nucleus where DNR is deposited and reverses MDR. Support or Funding Information The work was supported by the Max Kade Foundation and Intramural Research Program at Witten/Herdecke University (IFF2016‐20) (W.K.L).