Cigarette smoke extract affects functional activity of MRP1 in bronchial epithelial cells.
Cigarette smoke is the principal risk factor for development of chronic obstructive pulmonary disease (COPD). Multidrug resistance-associated protein 1 (MRP1) is a member of the ATP-binding cassette (ABC) superfamily of transporters, which transport physiologic and toxic substrates across cell membranes. MRP1 is highly expressed in lung epithelium. This study aims to analyze the effect of cigarette smoke extract (CSE) on MRP1 activity. In the human bronchial epithelial cell line 16HBE14o-, MRP1 function was studied flow cytometrically by cellular retention of carboxyfluorescein (CF) after CSE incubation and MRP1 downregulation by RNA interference (siRNA). Cell survival was measured by the MTT assay. Immunocytochemically, it was shown that 16HBE14o(-) expressed MRP1 and breast cancer resistance protein. Coincubation of CSE IC50 (1.53% +/- 0.22%) with MK571 further decreased cell survival 31% (p, = 0.018). CSE increased cellular CF retention dose dependently from 1.7-fold at 5% CSE to 10.3-fold at 40% CSE (both p < 0.05). siRNA reduced MRP1 RNA expression with 49% and increased CF accumulation 67% versus control transfected cells. CSE exposure further increased CF retention 24% (p = 0.031). A linear positive relation between MRP1 function and CSE-modulating effects (r = 0.99, p =0.089) was shown in untransfected, control transfected, and MRP1 downregulated 16HBE14o- cells analogous to blocking effects with MRP1 inhibitor MK571 (r = 0.99, p = 0.034). In conclusion, cigarette smoke extract affects MRP1 activity probably competitively in bronchial epithelial cells. Inhibition of MRP1 in turn results in higher CSE toxicity. We propose that MRP1 may be a protective protein for COPD development.
van der Deen
Margaretha
M
Department of Medical Oncology, University Medical Center Groningen, University of Groningen, 9713 GZ Groningen, The Netherlands.
de Vries
Elisabeth G E
EG
Visserman
Hylke
H
Zandbergen
Wouter
W
Postma
Dirkje S
DS
Timens
Wim
W
Timmer-Bosscha
Hetty
H
D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters / Q000378:metabolism; D000951:Antigens, Neoplasm / Q000378:metabolism; D001980:Bronchi / Q000166:cytology* / Q000187:drug effects* / Q000378:metabolism; D016923:Cell Death / Q000187:drug effects; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D004847:Epithelial Cells / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism*; D005434:Flow Cytometry; D005452:Fluoresceins / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000378:metabolism*; D009363:Neoplasm Proteins / Q000378:metabolism; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D012906:Smoke / Q000009:adverse effects*
D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters; D000951:Antigens, Neoplasm; D005452:Fluoresceins; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D009363:Neoplasm Proteins; D034741:RNA, Small Interfering; D012906:Smoke; C116614:ABCG2 protein, human; C020163:blocking factor; C024098:6-carboxyfluorescein; C421109:multidrug resistance-associated protein 1