NSC-640358 acts as RXRα ligand to promote TNFα-mediated apoptosis of cancer cell

Abstract

Retinoid X receptor α (RXRα) and its N-terminally truncated version tRXRα play important roles in tumorigenesis, while some RXRα ligands possess potent anti-cancer activities by targeting and modulating the tumorigenic effects of RXRα and tRXRα. Here we describe NSC-640358 (N-6), a thiazolyl-pyrazole derived compound, acts as a selective RXRα ligand to promote TNFα-mediated apoptosis of cancer cell. N-6 binds to RXRα and inhibits the transactivation of RXRα homodimer and RXRα/TR3 heterodimer. Using mutational analysis and computational study, we determine that Arg316 in RXRα, essential for 9-cis-retinoic acid binding and activating RXRα transactivation, is not required for antagonist effects of N-6, whereas Trp305 and Phe313 are crucial for N-6 binding to RXRα by forming extra π-π stacking interactions with N-6, indicating a distinct RXRα binding mode of N-6. N-6 inhibits TR3-stimulated transactivation of Gal4-DBD-RXRα-LBD by binding to the ligand binding pocket of RXRα-LBD, suggesting a strategy to regulate TR3 activity indirectly by using small molecules to target its interacting partner RXRα. For its physiological activities, we show that N-6 strongly inhibits tumor necrosis factor α (TNFα)-induced AKT activation and stimulates TNFα-mediated apoptosis in cancer cells in an RXRα/tRXRα dependent manner. The inhibition of TNFα-induced tRXRα/p85α complex formation by N-6 implies that N-6 targets tRXRα to inhibit TNFα-induced AKT activation and to induce cancer cell apoptosis. Together, our data illustrate a new RXRα ligand with a unique RXRα binding mode and the abilities to regulate TR3 activity indirectly and to induce TNFα-mediated cancer cell apoptosis by targeting RXRα/tRXRα.

Figure 1

N-6 binds to RXRα. (A) Structure of NSC-640358 (N-6). (B) RXRα-LBD protein was incubated with [³H]9-cis-RA in the presence or absence of unlabeled 9-cis-RA or N-6. Bound [³H]9-cis-RA was quantitated by liquid scintillation counting. (C) Gradient concentrations of N-6 were injected through flow cells immobilized with RXRα-LBD. The kinetic profiles are shown and the dissociation constant (K _d) of the N-6/RXRα-LBD complex was calculated to be 15.755 × 10⁻⁶ mol/L. (D) RXRα-LBD protein enhanced fluorescent intensity of N-6 (ex 278 nm, em 338 nm, cutoff 325 nm, delay 50 μs, integration 450 μs). One of three similar experiments is shown

Figure 2

N-6 is a selective antagonist of RXRα. (A–C) CV-1 cells were cotransfected with (TREpal)₂-tk-CAT and pCMV-Myc-RXRα (A), (TREpal)₂-tk-CAT and pCMV-Myc-RARγ (B), or NurRE-tk-CAT and pCMV-Myc-Nur77 (C). Cells were treated with N-6 (10 μmol/L) and 9-cis-RA (10⁻⁷ mol/L) for 18 h. CAT activities were measured and normalized to β-galactosidase activities (*P < 0.05). (D–I) MCF-7 cells were cotransfected with pG5-Gaussia-Dura and pBIND-RXRαLBD (D), pBIND-RARγLBD (E), pBIND-TR3 (F), pBIND-GRLBD (G), pBIND-LXRαLBD (H), or pBIND-PPARγLBD (I). Cells were treated with N-6 (10 μmol/L) in the presence or absence of 0.1 μmol/L 9-cis-RA (D and E), 1 μmol/L Dexamethasone (Dex) (G), 1 μmol/L T0901317 (T09) (H), or 1 μmol/L Rosiglitazone (Ros) (I) for 18 h. Reporter activities were measured and normalized (*P < 0.05). (J) CV-1 cells transfected with βRARE-tk-CAT, RXRα and TR3 expression vectors were treated with N-6 (10 μmol/L) in the presence or absence of 9-cis-RA (10⁻⁷ mol/L) for 18 h. CAT activities were measured and normalized to β-galactosidase activities (*P < 0.05). (K) GST-RXRα-LBD was incubated with different concentrations of N-6 in the presence of 9-cis-RA (10⁻⁸ mol/L) for 2 h. FRET signals were measured and normalized. The IC₅₀ of N-6 was calculated to be 3.29 × 10⁻⁵ mol/L. One of three similar experiments is shown. Data shown are mean ± SD

Figure 3

N-6 inhibits TR3 transcriptional activity by binding to RXRα. (A–D, and F) MCF-7 cells cotransfected with pG5-Gaussia-Dura reporter vector and the indicated expression vectors were treated with or without N-6 (10 μmol/L), UVI3003 (1 μmol/L), and 9-cis-RA (10⁻⁷ mol/L) for 18 h. Reporter activities were measured and normalized. Data shown are mean ± SD (*P < 0.05). (E) HEK293T cells cotransfected with pCMV-Myc-TR3 and pBIND-RXRα-LBD expression vectors were treated with UVI3003 (1 μmol/L) or N-6 (10 μmol/L) for 18 h. Cell lysate were analyzed for heterodimerization of Nur77 and Gal4-DBD-RXRα-LBD by co-immunoprecipitation with anti-myc antibody. One of three similar experiments is shown

Figure 4

Arg316 is not required for N-6 binding to RXRα. (A) MCF-7 cells cotransfected with pG5-Gaussia-Dura reporter vector and pBIND-RXRα-LBD or pBIND-RXRα-LBD/R316E expression vectors were treated with or without N-6 (10 μmol/L) in the presence or absence of CD3254 (10⁻⁷ mol/L) for 18 h. Reporter activities were measured and normalized. Data shown are mean ± SD (*P < 0.05). (B) Comparison of the docked conformation of N-6 (gray) with the crystal structure of LG100754 (green). (C) N-6 was docked into the LBP of the co-crystal structure of LG100754 and RXRα-LBD (PDB 3A9E). Salt bridges are shown as dotted yellow lines, and residues interacting with N-6 are shown in magenta. (D–E) Gradient concentrations of N-6 were injected through flow cells immobilized with RXRα-LBD/Trp305Ala (D) and RXRα-LBD/Phe313Ala (E), respectively. The kinetic profiles are shown and the dissociation constants (K _d) of the N-6/RXRα-LBD complex were calculated to be 1.0 × 10⁻³ mol/L (D) and more than 1.0 × 10⁻² mol/L (E). (F) RXRα-LBD proteins (2 mg/mL) was incubated with DMSO, 10 μmol/L 9-cis-RA, 25 μmol/L N-6 or 50 μmol/L N-6 for 3 h, and proteins were separated by 8% nondenaturing PAGE followed by Commassie Blue staining

Figure 5

N-6 inhibits TNFα-induced AKT activation in a tRXRα-dependent manner. (A) HCT116 cells were pretreated with N-6 (10 μmol/L) for 2 h in serum free medium before being exposed to TNFα (10 ng/mL) for an additional 30 min. Lysates prepared were analyzed by Western blotting for AKT activation. (B) A549 cells transfected with RXRα siRNA or control siRNA for 48 h were treated with N-6 (10 μmol/L) for 2 h in serum free medium before being exposed to TNFα (10 ng/mL) for an additional 30 min. Lysates prepared were analyzed by Western blotting for AKT activation. (C and D) H292 cells (C) or A549 cells (D) pretreated with N-6 (10 μmol/L) for 2 h in serum free medium before being exposed to TNFα (10 ng/mL) for an additional 30 min were analyzed for p85α/tRXRα interaction by co-immunoprecipitation assay using anti-RXRα antibodies of D20 or ΔN197. Immunoprecipitates were analyzed by Western blotting for the presence of p85α and tRXRα. One of three similar experiments is shown

Figure 6

Combination of TNFα and N-6 induces cancer cell apoptosis in an RXRα/tRXRα-dependent manner. (A) HeLa cells grown in 6-well plates were treated with or without N-6 (5 μmol/L) and TNFα (10 ng/mL) for 3 days. Colonies were stained with 0.1% crystal violet and counted. (B and C) HCT116 and MCF-7 cells were treated with or without N-6 (10 μmol/L) and TNFα (10 ng/ mL) for 15 h in serum free medium. Cell lysates prepared were analyzed by Western blotting for PARP cleavage. (D) MCF-7 cells grown in 24-well plates were treated with or without N-6 (10 μmol/L) and/or TNFα (10 μg/mL) for 3 days. Apoptotic cells were detected by TUNEL staining and counted. (E) HCT116 cells transfected with RXRα siRNA or control siRNA for 48 h were treated with or without N-6 (10 μmol/L) and TNFα (10 ng/mL) for 15 h. Cell lysates prepared were analyzed by Western blotting for PARP and caspase-8 cleavage. One of three similar experiments is shown