Article Figures & Data
Figures
- Fig. 1.
Expression of BCRP, P-glycoprotein, and MRP1 in the transfectants. Protein samples (20 μg protein/lane) were resolved by 4–20% SDS-PAGE and transferred onto nitrocellulose membranes. A, blots were treated with anti-BCRP antibody 3488. B, blots were treated with anti-P-glycoprotein antibody C219. C, blots were treated with anti-MRP1 antibody MRPm6. The blots were incubated with the appropriate peroxidase-conjugated secondary antibodies. Membrane-bound peroxidase was visualized using ECL Plus chemiluminescence detection kit. Lane 1: KB-3-1; Lane 2: KB/MRP; Lane 3: K562; Lane 4: K562/MDR; and Lane 5: K562/BCRP.
- Fig. 2.
Chemical structures of estrone, estrogen antagonists, and estrogen agonists.
- Fig. 3.
Effect of estrone, estrogen antagonists, and estrogen agonists on the intracellular accumulation of topotecan in K562 and K562/BCRP cells. Cells were incubated with or without 20 μm topotecan in the presence or absence of 30 μm test compound. Cellular content of topotecan was measured by FACS. Bold lines, with topotecan; dotted lines, without topotecan. In K562/BCRP cells with topotecan (bold line), a fluorescence peak shift to the right indicates cellular uptake of topotecan in the presence of estrone or other test compounds, whereas a slight shift occurred in the absence of test compounds. In contrast, fluorescence peak shifts to the right were observed in K562 cells, irrespective of the type of test compounds.
- Fig. 4.
Reversal of BCRP-mediated drug resistance by estrone, estrogen antagonists, and agonists. K562 (open symbols) and K562/BCRP (closed symbols) cells were cultured for 5 days with increasing concentrations of SN-38 or mitoxantrone in the absence or presence of fixed doses of estrone, estrogen antagonists, or agonists. Cell numbers were counted with a Coulter counter. Each point is an average of triplicate determinations. SDs are <10% of mean values. A, effect of estrone on the sensitivity to SN-38 (A-1) and mitoxantrone (A-2). B, effect of tamoxifen on the sensitivity to SN-38 (B-1) and mitoxantrone (B-2). C, effect of toremifene on the sensitivity to SN-38 (C-1) and mitoxantrone (C-2). D, effect of diethylstilbestrol on the sensitivity to SN-38 (D-1) and mitoxantrone (D-2). E, effect of diethylstilbestrol dipropionate on the sensitivity to SN-38 (E-1) and mitoxantrone (E-2).
- Fig. 5.
Chemical structures of TAG-1–TAG-139 used in the screening of anti-BCRP activity.
- Fig. 6.
Effects of TAG-1–TAG-139 on the intracellular accumulation of topotecan in K562/BCRP cells. Cells were incubated with or without 20 μm topotecan in the presence or absence of 30 μm TAG-compounds. Cellular content of topotecan was measured by FACS. Bold lines, with topotecan; dotted lines, without topotecan. With topotecan (bold line), a fluorescence peak shift to the right indicates cellular uptake of topotecan in the presence of TAG-compounds, although a slight shift occurred in the absence of compounds. In K562 cells, TAG-compounds did not affect topotecan uptake (data not shown).
- Fig. 7
Reversal of BCRP-mediated drug resistance by TAG-11 and TAG-139. K562 (open symbols) and K562/BCRP (closed symbols) cells were cultured for 5 days with increasing concentrations of SN-38 or mitoxantrone in the absence or presence of TAG-11 (0.5, 1, or 2 μm). Cell numbers were counted with a Coulter counter. Each point is an average of triplicate determinations. SDs are <10% of mean values. A, effect of TAG-11 on the sensitivity to SN-38 (A-1) and mitoxantrone (A-2). B, effect of TAG-139 on the sensitivity to SN-38 (B-1) and mitoxantrone (B-2).
- Fig. 8
Antiestrogen activity of TAG-compounds. Antiestrogen activity of TAG-compounds was estimated by the inhibition of estradiol binding to ER-α and ER-β using enzyme immunoassay. Degree of binding inhibition was calculated as the ratio of decrease in estradiol binding in the presence of 37.5 nm test compound divided by that in the presence of 300 nm diethylstilbestrol. Data are represented as mean values ± SD. A, inhibition of estradiol binding to ER-α. B, inhibition of estradiol binding to ER-β.
- Fig. 9.
Effect of TAG-139 on P-glycoprotein- and MRP1-mediated drug resistance. Cells were cultured for 5 days with increasing concentrations of anticancer agents in the absence or presence of TAG-139 (1 or 2 μm). Cell numbers were counted with a Coulter counter. Each point is an average of triplicate determinations. SDs are <10% of mean values. A, effect of TAG-139 on the doxorubicin sensitivity (A-1) and vincristine sensitivity (A-2) of K562 (open symbols) and K562/MDR (closed symbols) cells. B, effect of TAG-139 on the doxorubicin sensitivity (B-1) and VP-16 sensitivity (B-2) of KB-3–1 (open symbols) and KB/MDR (closed symbols) cells.
Tables
- Table 1
Drug resistance of K562/BCRP cellsa
Drug IC50 (ng/ml) Degree of resistance K562 K562/BCRP SN-38 0.42 ± 0.02 10 ± 0.3 24 Mitoxantrone 0.33 ± 0.02 3.6 ± 0.2 11 Topotecan 3.1 ± 0.1 30 ± 2 10 -
↵a K562 or K562/BCRP cells were cultured for 5 days with increasing concentrations of SN-38, mitoxantrone, or topotecan. Cell numbers were counted with a Coulter counter, and IC50 was determined. Degree of resistance is the ratio of IC50 for K562/BCRP cells divided by that for K562 cells. Data are represented as mean values ± SD from triplicate determinations.
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- Table 2
Reversal of BCRP-mediated SN-38 resistance by TAG-compoundsa
Compound Reversal index 1 μm 3 μm Tamoxifen NT 1.02 ± 0.02 TAG-1 NT 1.11 ± 0.06 TAG-2 NT 1.26 ± 0.02 TAG-3 NT 1.08 ± 0.08 TAG-5 NT 1.17 ± 0.11 TAG-7 NT 1.15 ± 0.14 TAG-8 NT 1.67 ± 0.02 TAG-11 1.36 ± 0.04 1.91 ± 0.02 TAG-12 NT 1.07 ± 0.04 TAG-13 NT 1.09 ± 0.01 TAG-72 1.12 ± 0.12 NT TAG-126 1.31 ± 0.14 NT TAG-139 2.32 ± 0.08 4.02 ± 0.12 -
a K562/BCRP cells were cultured for 5 days with increasing concentrations of SN-38 in the absence or presence of reversing agents (1 or 3 μm, as indicated). Cell numbers were counted with a Coulter counter, and concentration of drug required for IC50 was determined. Reversal index is the ratio of IC50 in the absence of reversing agent divided by that in the presence of reversing agent. Data are represented as mean values ± SD from triplicate determinations. NT, not tested.
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Volume 2, Issue 1
