ATP7B expression confers multidrug resistance through drug sequestration
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F M Moinuddin1,2, Yoshinari Shinsato2,3, Masaharu Komatsu4, Ryoichi Mitsuo2,*, Kentaro Minami2,3, Masatatsu Yamamoto2,3, Kohich Kawahara2,3, Hirofumi Hirano1, Kazunori Arita1, Tatsuhiko Furukawa2,3
1Department of Neurosurgery, Kagoshima University Graduate School of Medical and Dental Sciences, 8-35-1 Sakuragaoka, Kagoshima 890-8544, Japan
2Department of Molecular Oncology, Kagoshima University Graduate School of Medical and Dental Sciences, 8-35-1 Sakuragaoka, Kagoshima 890-8544, Japan
3Center for the Research of Advanced Diagnosis and Therapy of Cancer, Graduate School of Medical and Dental Sciences, Kagoshima University, 8-35-1 Sakuragaoka, Kagoshima 890-8544, Japan
4Division of Food and Chemical Biology, Faculty of Fisheries, Kagoshima University, 4-50-20, Shimoarata, Kagoshima 890-0056, Japan
*Present address: Shin Nippon Biomedical Laboratories (SNBL) Ltd, 2438 Miyanoura, Kagoshima 891-1394, Japan
Tatsuhiko Furukawa, e-mail: [email protected]
Keywords: ATP7B, copper transporter, multidrug resistance, doxorubicin, SN-38
Received: November 10, 2015 Accepted: February 23, 2016 Published: March 14, 2016
We previously reported that ATP7B is involved in cisplatin resistance and ATP7A confers multidrug resistance (MDR) in cancer cells.
In this study, we show that ATP7B expressing cells also are resistant to doxorubicin, SN-38, etoposide, and paclitaxel as well as cisplatin.
In ATP7B expressing cells, doxorubicin relocated from the nuclei to the late-endosome at 4 hours after doxorubicin exposure. EGFP-ATP7B mainly colocalized with doxorubicin.
ATP7B has six metal binding sites (MBSs) in the N-terminal cytoplasmic region. To investigate the role of the MBSs of ATP7B in doxorubicin resistance, we used three mutant ATP7B (Cu0, Cu6 and M6C/S) expressing cells. Cu0 has no MBSs, Cu6 has only the sixth MBS and M6C/S carries CXXC to SXXS mutation in the sixth MBS. Cu6 expressing cells were less resistance to the anticancer agents than wild type ATP7B expressing cells, and had doxorubicin sequestration in the late-endosome. Cu0- and M6C/S-expressing cells were sensitive to doxorubicin. In these cells, doxorubicin did not relocalize to the late-endosome. EGFP-M6C/S mainly localized to the trans-Golgi network (TGN) even in the presence of copper. Thus the cysteine residues in the sixth MBS of ATP7B are essential for MDR phenotype.
Finally, we found that ammonium chloride and tamoxifen suppressed late endosomal sequestration of doxorubicin, thereby attenuating drug resistance. These results suggest that the sequestration depends on the acidity of the vesicles partly.
We here demonstrate that ATP7B confers MDR by facilitating nuclear drug efflux and late endosomal drug sequestration.
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