Article Figures & Data
Figures
- Figure 1
Structure of TER117. The structure of TER117 resembles that of GSH, with the addition of a benzyl group to the sulfur atom and the substitution of glycine with phenylglycine.
- Figure 2
Cooled charge-coupled device analysis of daunorubicin fluorescence in NIH3T3/MSV and NIH3T3/MRP1 cells. Cells were treated with saturating doses of daunorubicin before drug removal and replacement with DMEM containing vehicle or TER199. NIH3T3/MSV cells (A–C), NIH3T3/MRP1 cells (G–I), 50 μM TER199 + NIH3T3/MSV cells (D–F), TER199 + NIH3T3/MRP1 cells (J–L). Images were captured immediately following daunorubicin removal (A, D, G, J) or at time periods of 30 (B, E, H, K), or 120 min after daunorubicin removal (C, F, I, L). The cells were maintained on an incubated stage at 37°C during image acquisition. All images were 60× magnification.
- Figure 3
Confocal images (60×) showing immunofluorescence of MRP in NIH3T3/MSV cells (A) and NIH3T3/MRP1 cells (B). A monoclonal anti-MRP antibody was obtained from Kamiya Biomedical Company; secondary antibody was antimouse tagged with biotin; tertiary antibody, antibiotin tagged with fluorescein isothiocyanate.
- Figure 4
Accumulation of [3H]daunorubicin in NIH3T3/MSV and NIH3T3/MRP1 cells. Cells were treated with [3H]daunorubicin (1 μM) for the indicated time points in the presence (▵) or absence of 50 μM TER199 (▪, ▴) before lysis and scintillation counting to determine intracellular levels of [3H]daunorubicin. Sham-transfected NIH3T3/MSV cells are indicated by ▪ and MRP1-transfected NIH3T3/MRP1 cells by ▵ and ▴. Data are represented as means ± S.D. of three independent experiments.
- Figure 5
ATP-dependent transport of GS-DNP into NIH3T3/MRP1 membrane vesicles in the presence or absence of TER117. A, vesicle solutions (30 μg) were incubated with [3H]GS-DNP (50 nM) at 37°C in the presence of 4 mM ATP (●, ○) and an ATP-regenerating system. In control experiments, ATP was replaced by 5′-AMP (♦, ⋄; top panel, bottom line of graph). ATP-dependent transport was calculated by subtraction of the 5′-AMP values from ATP values (▪, ■). Data are represented as means ± S.D. of three independent experiments. Closed symbols represent transport in the presence of 1 mM and open in its absence. B, double reciprocal plots according to Lineweaver and Burk ▪ is absence of inhibitor and ▾ is presence of 1 mM.
- Figure 6
Altered mRNA transcript and protein levels in chronically exposed HL-60/TER199 cells. Quantitative RT-PCR (A) and Western blot analysis (B) of GSH-associated and transporter genes associated with multidrug resistance. The molecular weight of MRP1 is 190 kDa; γ-GCS heavy 73 kDa; γ-GCS light 30 kDa; and actin 42 kDa. Lanes 1 are parental HL-60 cells, and lanes 2 are HL-60/TER199 cells. The images shown are representative of three independent experiments.
- Figure 7
GSH levels in HL-60/TER199 cells. GSH concentrations were measured according to the method of Tietze. Data are represented as means ± S.D. of three independent experiments.
Tables
Drug IC50NIH3T3 IC50 3T3/ MRP IC50 3T3/MRP + TER199 Sensitization Factor Doxorubicin (nM) 13.3 ± 1.13 1301 ± 176 6.04 ± 0.071 2.1 Daunorubicin (nM) 23.1 ± 17.1 647 ± 177 317 ± 174 2.0 Etopside (μM) 0.610 ± 0.442 19.5 ± 8.43 4.40 ± 1.25 4.4 Vincristine (nM) 3.40 ± 0.720 21.4 ± 6.40 2.90 ± 1.07 7.4 Mitoxantrone (nM) 4.85 ± 2.89 108 ± 42.8 80.2 ± 46.1 1.3 5-FU (μM) 2.68 ± 0.76 2.67 ± 1.90 3.02 ± 1.77 -
Cytotoxicity studies were performed in the presence or absence of 50 μM TER199. Cells were exposed to drug for 24 h prior to its removal and replacement with normal growth media. MTS analysis was performed after 24 h. Data are represented as means ± S.D. of three independent experiments.
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Drug HL-60 HL-60/TER199 Resistance Factor Doxorubicin (nM) 59.0 ± 12 240.0 ± 55.0 4.1 Daunorubicin (nM) 16.0 ± 14 48.0 ± 22.0 3.0 Etoposide (μM) 0.4 ± 0.1 2.2 ± 0.3 5.0 Vincristine (nM) 0.3 ± 0.2 2.5 ± 1.1 8.0 5-FU (μM) 5.0 ± 0.4 9.3 ± 2.3 1.9 Mitoxantrone (nM) 10.2 ± 9.0 36.7 ± 17.0 3.6 Mitomycin C (nM) 170.0 ± 26.0 156.0 ± 45.0 0.9 Cisplatin (μM) 0.8 ± 0.5 0.7 ± 0.4 0.9 Melphalan (μM) 1.0 ± 0.2 0.8 ± 0.2 0.8 Paclitaxel (nM) 1.6 ± 0.6 3.8 ± 2.0 2.4 -
Cells were exposed to the indicated drugs for a period of 72 h before MTS analysis. Data are represented means ± S.D. of three independent experiments.
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