Functional study of intracellular P-gp- and MRP1-mediated pumping of free cytosolic pirarubicin into acidic organelles in intrinsic resistant SiHa cells.
We sought to determine the efficiency of the intracellular functional P-gp- and MRP1-mediated pumping of THP into acidic organelles in SiHa cells and etoposide-resistant SiHa/VP16 cells. The expression of both MDR1 and MRP1 genes of SiHa and SiHa/VP16 cells was clearly shown by using RT-PCR. The functional studies of both intracellular functional P-gp- and MRP1-mediated pumping were performed by using THP in a conventional spectrofluorometer, and they demonstrated that SiHa and SiHa/VP16 cells are good models to illustrate the functional role of intracellular P-gp and MRP1 in the transport of free cytosolic drug into acidic organelles. The functional P-gp and MRP1 proteins were identified both on plasma membranes and on intracellular vesicle membranes. Within the limit of experimental error, similar efficiencies in THP transport were observed in the two proteins at both locations in SiHa and SiHa/VP16 cells. The P-gp- and MRP1-mediated pump coefficient (k v a), Michealis-Menten's constant (K V m), and maximal pumping rate (V V max) values of those located on vesicular membranes were 1.87 +/- 0.30 pL x cell-1 x s-1, 1.63 +/- 0.21 microM, and 4.95 +/- 0.45 nM x s-1</sup>, respectively. Drug retention inside acidic organelles (C mon V) of SiHa cells was significantly higher than that of SiHa/VP16 cells, perhaps a consequence of slower movement of recycling endosomes and (or) lysosomes to the cell membrane of SiHa cells, leading to distended organelles and cell death. Our results suggest that intracellular P-gp and MRP1 proteins play an important role in the transport of free drug from cytosol to cytoplasmic acidic organelles.
Laochariyakul
Ponpun
P
Laboratory of Physical Chemistry, Molecular and Cellular Biology, Faculty of Science, Burapha University, Bangsaen, Chonburi 20131 Thailand.
Ponglikitmongkol
Mathurose
M
Mankhetkorn
Samlee
S
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000235:genetics / Q000502:physiology*; D000143:Acids; D000903:Antibiotics, Antineoplastic / Q000493:pharmacokinetics / Q000633:toxicity; D001692:Biological Transport / Q000187:drug effects / Q000502:physiology; D002294:Carcinoma, Squamous Cell / Q000378:metabolism / Q000473:pathology; D016923:Cell Death / Q000187:drug effects; D045744:Cell Line, Tumor; D003600:Cytosol / Q000378:metabolism / Q000502:physiology*; D004317:Doxorubicin / Q000031:analogs & derivatives* / Q000493:pharmacokinetics* / Q000633:toxicity; D019008:Drug Resistance, Neoplasm; D006801:Humans; D007424:Intracellular Fluid / Q000378:metabolism / Q000502:physiology; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000502:physiology*; D015388:Organelles / Q000187:drug effects / Q000502:physiology*
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000143:Acids; D000903:Antibiotics, Antineoplastic; D004317:Doxorubicin; D027425:ATP-Binding Cassette, Sub-Family C Proteins; C027260:pirarubicin; C421109:multidrug resistance-associated protein 1