Glutathione-conjugate transport by RLIP76 is required for clathrin-dependent endocytosis and chemical carcinogenesis

Abstract

Targeted depletion of the RALBP1-encoded 76-kDa splice variant, RLIP76, causes marked and sustained regression of human xenografts of lung, colon, prostate, and kidney cancers without toxicity in nude mouse models. We proposed that the remarkable efficacy and broad spectrum of RLIP76-targeted therapy is because its glutathione-conjugate (GS-E) transport activity is required for clathrin-dependent endocytosis (CDE), which regulates all ligand-receptor signaling, and that RLIP76 is required not only for survival of cancer cells but also for their very existence. We studied RLIP76 mutant proteins and the functional consequences of their expression into RLIP76(-/-) MEFs, identified key residues for GS-E binding in RLIP76, established the requirement of RLIP76-mediated GS-E transport for CDE, and showed a direct correlation between GS-E transport activities with CDE. Depletion of RLIP76 nearly completely blocked signaling downstream of EGF in a CDE-dependent manner and Wnt5a signaling in a CDE-independent manner. The seminal prediction of this hypothesis-RLIP76(-/-) mice will be deficient in chemical neoplasia-was confirmed. Benzo[a]pyrene, dimethylbenzanthracene, and phorbol esters are ineffective in causing neoplasia in RLIP76(-/-). PMA-induced skin carcinogenesis in RLIP76(+/+) mouse was suppressed completely by depletion of either PKCα or RLIP76 by siRNA or antisense and could be restored by topical application of RLIP76 protein in RLIP76(-/-) mouse skin. Likewise, chemical pulmonary carcinogenesis was absent in female and nearly absent in male RLIP76(-/-) mice. In RLIP76(-/-) mice, p53, p38, and JNK activation did not occur in response to either carcinogen. Our findings show a fundamental role of RLIP76 in chemical carcinogenesis.

Figure 1. EGF-rhodamine endocytosis is deficient in RLIP76^−/− MEFs, and is restored completely by transient transfection with wild-type RLIP76

RLIP76^−/− MEFs were treated with pre-immune IgG (A and B), anti-RLIP76 IgG (C and D) and EGF-rhodamine endocytosis (at 10 min) (E and F). Nuclei are stained with DAPI (panel A-F). Transfection of RLIP76 restores IHC-detectable RLIP76 in the RLIP76^−/− cells (panel G), and EGF-rhodamine endocytosis is fully restored (panel H). GFP-RLIP76 construct in pcDNA3.1 was used to transfect RLIP76^−/− MEFs to show that the entry of EGF-rhodamine into the cells is deficient in RLIP76^−/−, that empty-vector GFP does not restore this, and that its entry into RLIP76^−/− cells is restored by transfection with GFP-RLIP76 (two representative micrographs are presented for each of four experimental groups) (panels I-P). Quant Image-J software was used for image analyzing and quantifying fluorescence per area by blinded observers. Photographs were taken at identical exposures using 400 × magnifications.

Figure 2. Effect of mutations on GS-E affinity, ATPase activity

Equal amounts of the purified protein (15 μg) was added to DNPSG-Sepharose-4B affinity-resin for 1h, and un-bound RLIP76 was measured in the supernatant by ELISA assay (standard curve r2 0.99) and 1/unbound was used as for DNPSG-affinity. DNPSG-stimulated ATPase-activity of each mutant was measured as previously described (19). The ATP-dependent DNP-SG transport-activity was determined after reconstituting the purified recombinant proteins into artificial asolectin-cholesterol liposomes as described previously (19). All results were normalized to control, wild-type RLIP76. Percent residual-activity is shown (panel A). A plot of DNPSG-affinity vs. either ATPase or transport-activity normalized to wild-type is presented (panel B). The effect of these mutations on GS-E binding was studied using the GSH-monochlorobimane (GSH-MCB) fluorescent-conjugate. RLIP76 MEF^−/− cells transfected with RLIP76-GFP vector were treated with 50 μM monochlorobimane (which gives rise to a fluorescent GSH-conjugate in cells) for 20 min at 37°C prior to 4 % paraformaldehyde fixation, followed by molecular interaction analysis using FRET using time-resolved confocal microscope MT 200 (from Picoquant GmbH) with pulsed diode laser excitations at 405 nm (for MCB donor) and 475 nm (for GFP donor). For MCB observation, 465 nm (10 nm band width) interference filter crossed with 430 nm long path cut-off and for GFP observation 490–530 nm interference filter crossed with 500 nm long path filter was used. The histogram graph presents observed fluorescence lifetime measured for the respective images, MCB alone (green) RLIP76-GFP (red) (panel C, left). There is no significant change in the fluorescence life time cells transfected with the mutant of RLIP76 lacking GSH-binding site (ΔS234A/K237A) (blue) (panel C, right). The mutant RLIP76 cDNAs were cloned into pcDNA3.1 eukaryotic expression vector, and successful transient transfection of RLIP76^−/− MEFs was demonstrated by IHC against RLIP76 with FITC-labeled secondary antibody (panel D, top row), and representative photomicrographs of RLIP76-mutant transfected cells showing the consequent partial restoration of EGF-rhodamine endocytosis are shown (panel D, bottom row).

Figure 3. Functional-activity of RLIP76 mutants in MEFs

The IC50 of the glutathione-conjugate metabolized drug melphalan was determined for each cell line by MTT assay, the cellular accumulation of ¹⁴C-melphalan at 30 min after exposure was determined as described previously (20, 22), and results of separate studies of endocytosis quantified by Image-J software are presented. All studies were done three times with triplicate determinations each time (panel A). A plot showing endocytosis activity of each mutant plotted against either the ATPase-activity (round symbols) or transport-activity (square symbols) is shown with a logistical regression curve demonstrating a direct and saturable relationship of CDE with these measures of activity (panel B).

Figure 4. RLIP76 and PKCα are both necessary for DMBA/PMA skin-carcinogenesis

The experiment was carried out in wild-type (RLIP76^+/+) and RLIP76 knockout (RLIP76^−/−) C57B male mice. The skin of the back was shaved and 25 nmol DMBA or vehicle control (acetone) was applied to a 1 cm² patch of shaved skin (36, 37). After two weeks, on day 14, RLIP76^+/+ animals were injected i.p. with 200 μL PBS with or without either PKCα-siRNA or RLIP76-antisense (R508). Also on day 14, RLIP76-liposomes were applied to the skin of one group of RLIP76^−/− mice. On day 15, 10 nmol PMA in acetone or acetone alone were applied to the skin. The animals were sacrificed 4 days later and skin was harvested for histological and biochemical analyses. Animals in 11 treatment groups were treated as indicated in the figure. Levels of phosphorylated JNK, p38, p53 (Active-Motif) and total caspase (Immunochemistry Technologies) were determined using ELISA assays, and normalized to the values found in vehicle-control skin. Values presented are mean and s.d. of three determinations of fold-activation from n = 8 animals per group.

Figure 5. RLIP76 and PKCα are both necessary for chloroform/PMA skin-carcinogenesis

The experiment was carried out in RLIP76^+/+ and RLIP76^−/− C57B male mice. On day 1, RLIP76^+/+ animals were injected i.p. with 200 μL PBS with or without either PKCα-siRNA, or RLIP76-antisense (R508). Also on day 1, RLIP76-liposomes were applied to the skin of one group of RLIP76^−/− mice. On day 2, 10 nmol PMA in chloroform or chloroform alone were applied to the skin. The animals were sacrificed on day 6 and skin was harvested for histological and biochemical analyses. Photographs of representative animals from each group and H&E stained histological sections as well as levels of phosphorylated JNK, p38, p53 (Active-Motif) and total caspase (Immunochemistry Technologies) are presented. Skin from three animals from each of 7 groups was analyzed for p53, JNK, p38, and total caspases. Values presented are mean and s.d. of triplicate determinations of fold-activation with respect to the vehicle-control group. RLIP76 and PKCα protein in 28,000 × g supernatants of skin homogenate was determined by Western-blot analyses using anti-RLIP76, and anti-PKCα antibodies (inset).

Figure 6. Absence of RLIP76 reduces B[a]P-mediated lung and stomach adenocarcinomas

These experiments 15 animals per group, twelve-weeks-old, 30 male and 30 female each RLIP76^+/+ and RLIP76^−/− mice, divided into two groups (control and BaP treatment). Animals were treated with 3 mg BaP in 0.2 mL corn oil, twice 4-weeks apart, or an equal volume of corn oil. Surviving animals were sacrificed at 9 months by CO2 inhalation followed by cervical dislocation after ~36 weeks from the beginning of the experiment and the lungs and stomach were harvested for histopathological examination. Percent surviving in each group (panel A), cancer incidence determined by complete autopsy (panel B) and representative photomicrographs of H stained histological slides (panel C) are presented. Fold change in p53, p38, JNK and caspase-activation in lung (panel D) and stomach (panel E) tissues of male and female RLIP76^+/+ and RLIP76^−/− as compared with respective vehicle control treated group are presented for n = 3 animals per group. The numbers of carcinomas in different groups were compared by ANOVA.