UV irradiation resistance-associated gene suppresses apoptosis by interfering with BAX activation

Abstract

Ultraviolet irradiation resistance-associated gene (UVRAG) is a well-known regulator of autophagy by promoting autophagosome formation and maturation. However, little is known about the non-autophagic functions of UVRAG. Here, we present evidence that UVRAG functions as an unusual BCL2-associated X protein (Bax) suppressor to regulate apoptosis. Chemotherapy and radiation induces UVRAG expression and subsequently upregulates autophagy and apoptosis in tumour cells. Depletion of UVRAG expression by RNA interference renders tumour cells more sensitive to chemotherapy- and radiation-induced apoptosis in vitro and in vivo. Moreover, UVRAG interacts with Bax, which inhibits apoptotic stimuli-induced mitochondrial translocation of Bax, reduction of mitochondrial membrane potential, cytochrome c release and activation of caspase-9 and -3. Our findings show that UVRAG has an essential role in the intrinsic mitochondrial pathway of apoptosis by regulating the localization of Bax. This pathway represents a target for clinical intervention against tumours.

Figure 1

Genotoxic or metabolic stress-induced UVRAG expression in tumour cells. (A,B) Expression of UVRAG under cell stress. HL60 and HCT116 cells were treated with doxorubicin (1 μg/ml), cisplatin (10 μm) and UV irradiation (5 min after 50 mJ/cm²) for the indicated time, and then apoptosis, autophagy and fluorescence intensity of UVRAG was assayed. Scale bar, 10 μm. (C) In parallel, the total protein extracts were used for western blot analysis. (D,E) Pharmacological inhibition (for example, 3-methyladenine (3-MA), 10 mM) or genetic deletion (for example, ATG5^−/−) of the autophagy pathway decreases the protein and mRNA expression of UVRAG and increases apoptosis at 12 h in HL60 cells treated with doxorubicin (Doxo) and UV irradiation (n=3, ^*P<0.05). AU, arbitrary units; C-PARP, cleaved poly-ADP ribose polymerase; LC3, light chain 3; mRNA, messenger RNA; PI, propidium iodide; UV, ultraviolet; UVRAG, ultraviolet irradiation resistance-associated gene.

Figure 2

Inhibition of UVRAG expression increases tumour-cell sensitivity to chemotherapy and radiation therapy in vivo and in vitro. (A–C) After transfection with UVRAG shRNA or control shRNA for 48 h, HL60 (A), HCT116 (B) and HeLa (C) cells were treated with doxorubicin (Doxo, 1 μg/ml), cisplatin (Cis, 10 μm) and UV irradiation (5 min after 50 mJ/cm²) for 12 h or starvation (HBSS) for 3 h, and then apoptosis and autophagy were assayed (n=3, ^*P<0.05). (D) Analysis of LC3 processing by autophagy in the presence or absence of lysosomal protease inhibitors pepstatin A (10 μg/ml) and E64D (10 μg/ml) after doxorubicin (1 μg/ml) treatment for 12 h. ^*P<0.05 compared with control shRNA group. (E) After transfection with UVRAG shRNA or control shRNA for 48 h, ATG5^+/+ and ATG5^−/− murine embryonic fibroblasts (MEFs) were treated with doxorubicin (1 μg/ml), with or without Z-VAD-FMK (20 μM) for 12 h, and then apoptosis was assayed (n=3, ^*P<0.05). (F) Nude mice were inoculated with 4 × 10⁶ HL60 tumour cells following transfection of control (shControl) or UVRAG (shUVRAG)-specific shRNA and treated with doxorubicin (10 mg/kg) beginning at day 7. Tumours were measured twice weekly, and volumes were calculated for 21 days (n=10, ^*P<0.05; shControl+Doxo compared with shUVRAG+Doxo). (G) On day 21 in the experiments described in (F), apoptosis and UVRAG expression in tumour samples were assayed by TUNEL or western blotting, respectively (n=10, ^*P<0.05). BAX, BCL2-associated X protein; HBSS, Hank's Buffered Salt Solution; LC3, light chain 3; shRNA, short-hairpin RNA; TUNEL, terminal deoxytransferase uridine triphosphate nick end labelling; UV, ultraviolet; UVRAG, ultraviolet irradiation resistance associated gene.

Figure 3

UVRAG inhibits BAX-induced apoptosis. (A) Control shRNA or UVRAG shRNA cells were transfected with BAX plasmid, and apoptosis was assayed (n=3, ^*P<0.05). (B) In parallel, the mitochondrial membrane potential, cytochrome c release from mitochondria, and caspase-3 and -9 activation in HL60 cells were analysed (n=3, ^*P<0.05). (C) After transfection with UVRAG vector or control vector for 48 h, cells were transected or treated with BAX (1 μg), BAD (1 μg), BID (1 μg), FAS antibody (10 μg/ml) or TRAIL (100 ng/ml) for 24 h, then cell apoptosis was analysed (n=3, ^*P<0.05). (D) Knockdown of UVRAG in BAX^−/− HCT116 cells restored the sensitivity to UV treatment-induced apoptosis (n=3, ^*P<0.05). AU, arbitrary units; BAX, BCL2-associated X protein; shRNA, short-hairpin RNA; UV, ultraviolet; UVRAG, ultraviolet irradiation resistance associated gene.

Figure 4

UVRAG interacts with Bax. (A,B) HL60 and HCT116 cells were treated with UV irradiation for 24 h and then assayed for protein expression levels as indicated by co-immunoprecipitation (RIPA or CHAPS buffer) or western blotting. (C) Proteins in the pellet after GST pull-down assays in HCT116 cell lysate were analysed by SDS–PAGE and immunodetected with UVRAG or GST antibodies. (D) UVRAG C2 domain deletion terminates BAX binding. HCT116 cells were transfected with Flag-UVRAG or its mutants (ΔC2, ΔCCD), and then assayed for protein expression levels as indicated by IP or western blot. (E) Apoptosis was assayed in indicated HCT116 cells after treatment with doxorubicin (Doxo, 1 μg/ml), UV irradiation (5 min after 50 mJ/cm²) or transfected with BAX (1 μg) for 24 h. BAX, BCL2-associated X protein; CCD, coiled-coil domain; CHAPS, 3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulphonate; GST, glutathione-S-transferase; IB, immunoblotting; IgG, immunoglobulin G; IP, immunoprecipitation; RIPA, radioimmunoprecipitation assay; SDS–PAGE, SDS–polyacrylamide gel electrophoresis; UV, ultraviolet; UVRAG, ultraviolet irradiation resistance associated gene.

Figure 5

UVRAG inhibits mitochondrial translocation of BAX. After transfection with indicated shRNA or cDNA for 48 h, HL60 cells were treated with doxorubicin (Doxo) and UV irradiation for 12 h, and then BAX in cytosol (Cyt) and mitochondria (Mit) was assayed by western blotting (A–C) and activation of BAX (D–F) was assayed by western blotting after IP using BAX monoclonal antibody (clone 6A7) ^*P<0.05. AU, arbitrary units; BAX, BCL2-associated X protein; CHAPS, 3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulphonate buffer; Cyt c, cytochrome c; IP, immunoprecipitation; shRNA, short-hairpin RNA; UV, ultraviolet; UVRAG, ultraviolet irradiation resistance associated gene.