Article Figures & Data
Figures
- Figure 1.
Comparison of RLIP76 levels in cultured malignant cells versus nonmalignant cells. Aliquots of crude detergent extracts of the membrane fractions of malignant cells (H1618, H358, OVCAR-3, PC-3, MCF-7, B16, HepG2, and DG-1) and nonmalignant cells (HAVSM, HUVEC, HLMVEC, and HLBEC), containing 200 μg protein were used for SDS-PAGE and Western blotting against anti-RLIP76 IgG as primary antibody and horseradish peroxidase–conjugated goat anti-rabbit IgG as secondary antibody and developed with 4-chloro-1-napthol as chromogenic substrate. Results were quantified by scanning densitometry of the full-length RLIP76 protein band near 109 kDa. β-Actin was used as an internal control.
- Figure 2.
Comparison of cytotoxicity effects of anti-RLIP76 IgG and RLIP76 siRNA between malignant and nonmalignant cells. A, effect of preimmune IgG (gray columns) and anti-RLIP76 IgG (white columns; 37 μg/mL final concentration) on cell survival was determined by MTT assay ( 38). B, depletion of RLIP76 expression in cells by RLIP76 siRNA was done, using Transmessenger Transfection Reagent Kit, according to the manufacturer's (Qiagen) instructions. Briefly, B16 melanoma (malignant) and HLMVEC (nonmalignant) cells were incubated for 3 hours with various concentrations of siRNA (ranging from 0 to 100 μg/mL final concentration) in Transmessenger Transfection Reagent, washed with PBS, followed by 48 hours of incubation at 37°C in medium before Western blotting with anti-RLIP76 IgG as primary antibody. C, the time-dependent effect of RLIP76 siRNA (fixed at 20 μg/mL final concentration) were also evaluated using eight malignant and four nonmalignant cells by determining RLIP76 protein levels by Western blot analyses at 0, 6, 12, 24, and 48 hours, after treatment of RLIP76 siRNA, using anti-RLIP76 IgG as primary antibody. D, MTT assay in eight malignant and four nonmalignant cells was also done 48 hours after treatment of siRNA: scrambled siRNA (gray columns) and RLIP76 siRNA (white columns; 20 μg/mL final concentration), using Transmessenger Transfection Reagent Kit (Qiagen). B and C, internal control (β-actin) is shown below the respective Western blots from each cell line. A and D, columns, mean from three separate determinations with eight replicates each; bars, ± SD (n = 24).
- Figure 3.
Effect of anti-RLIP76 IgG on apoptosis as determined by TUNEL assay. Malignant cells (H358, OVCAR-3, PC-3, and B16 melanoma) and nonmalignant cells (HAVSMC, HUVEC) were treated with either preimmune serum or anti-RLIP76 IgG (37 μg/mL final concentration) for 24 hours and then washed off with PBS. Approximately 1 × 106 cells were fixed onto poly-l-lysine-coated slides, and the TUNEL apoptosis assay was done using the Promega Apoptosis Detection Kit according to the protocol provided by the manufacturer. Slides were analyzed by laser scanning fluorescence microscope (Zeiss LSM510 META). Photographs taken at identical exposures (×400 magnification). Apoptotic cells show green fluorescence.
- Figure 4.
Antineoplastic effects of RLIP76 inhibition or depletion in mouse melanoma. C57B mice were injected on their flanks with 2 × 106 B16 melanoma cells and tumors were measured by calipers. When the surface area of the tumor (product of bidimensional measurements) exceeded 40 mm2 (day 11), animals were injected i.p. with 100 μL diluent alone (PBS), or the same volume of diluent containing 200 μg of anti-RLIP76 IgG, RLIP76 siRNA508-528, or RLIP76 phosphorothioate antisense508-528. Additional control animals were injected with preimmune IgG, scrambled siRNA, or antisense. Left, tumor measurements for the index lesion are presented with all control groups (⧫), preimmune IgG (▪), scrambled siRNA (▴) or antisense (×) versus all treated groups (*), anti-RLIP76 IgG (•), RLIP76 siRNA (+), or RLIP76 phosphorothioate antisense (−). Arrows, days on which treatment was given and repeated. Right, photographs of animals taken at 8 days after treatment are shown for all treatment groups.
Tables
- Table 1.
RLIP76 protein and transport activity in malignant and nonmalignant cell lines
RLIP76 protein Transport activity (pmol/min/mg) μg/108 cells Total protein (%) Doxorubicin DNP-SG Malignant B16 (mouse melanoma) 71 ± 6 0.8 443 ± 34 1,796 ± 145 DG-1 (human melanoma) 63 ± 5 0.8 384 ± 28 1,562 ± 112 OVCAR-3 (human ovary) 53 ± 3 0.7 298 ± 23 1,194 ± 122 PC-3 (human prostate) 46 ± 3 0.6 211 ± 26 893 ± 66 H358 (human lung, NSCLC) 36 ± 3 0.6 180 ± 15 695 ± 56 H1618 (human lung, SCLC) 32 ± 3 0.5 96 ± 12 361 ± 41 MCF-7 (human breast) 15 ± 1 0.2 36 ± 3 105 ± 7 HepG2 (human liver) 17 ± 1 0.3 55 ± 5 167 ± 14 Nonmalignant HLMVEC (human lung endothelium) 19 ± 2 0.3 40 ± 5 136 ± 14 HLBEC (human lung epithelium) 22 ± 2 0.4 46 ± 4 150 ± 20 HAVSM (human aorta smooth muscle) 15 ± 1 0.3 40 ± 6 125 ± 10 HUVEC (human umbilical endothelial) 14 ± 1 0.2 36 ± 4 113 ± 12 -
NOTE: Cell lines were cultured in respective medium as described in Materials and Methods, and homogenate was prepared from 1 × 108 cells. RLIP76 was purified from total membrane fraction by DNP-SG affinity chromatography ( 28, 35), and quantified by ELISA. Purification table and SDS-PAGE of purified RLIP76 from different cells are presented in the Supplemental Data (Table A and Fig. A). Total membrane proteins were quantified by dye-binding method ( 39). For transport studies, plasma membrane fraction obtained from 2 × 107 cells was enriched for IOVs by wheat germ agglutinin affinity chromatography ( 32). Transport activity was calculated from measurements of uptake of 14C-doxorubicin (specific activity, 8.2 × 104 cpm/nmol) or 3H-DNP-SG (specific activity, 3.6 × 103 cpm/nmol) into the IOVs in the absence or presence of 4 mmol/L ATP after 10 minutes of incubation at 37°C as previously described ( 32). Each transport study was done with three replicates in three independent experiments (n = 9).
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Supplementary Data Singhal, et al.
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