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

Chen F, Chen J, Lin J, Cheltsov AV, Xu L, Chen Y, Zeng Z, Chen L, Huang M, Hu M, Ye X, Zhou Y, Wang G, Su Y, Zhang L, Zhou F, Zhang XK, Zhou H - Protein Cell (2015)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: School of Pharmaceutical Sciences, Xiamen University, Xiamen, 361102, China.

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.

No MeSH data available.


Related in: MedlinePlus

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
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Fig5: 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

Mentions: It has been reported that RXRα antagonist K-80003 inhibits TNFα-induced AKT activation in a tRXRα-dependent pathway (Zhou et al., 2010). Here we examined whether N-6 had a similar function. Figure 5A showed that N-6 significantly inhibited TNFα-induced AKT activation in HCT116 cells, revealed by its suppression of the expression of phosphor-AKT but not total AKT (Fig. 5A). Similar results were obtained in A549 cells (Fig. 5B). We then determined the requirement of RXRα/tRXRα for the inhibition of AKT activation by N-6. Transfection of RXRα siRNA in A549 cells, which reduced both the full-length RXRα and tRXRα expression, abolished the inhibitory effect of N-6 on TNFα-induced AKT activation (Fig. 5B). Thus, the expression of RXRα/tRXRα is essential for AKT inhibition by N-6. We have reported that K-80003 inhibited AKT activation by blocking the TNFα-induced interaction of tRXRα and p85α (Zhou et al., 2010). We then investigated whether N-6 had the similar action. Two anti-RXRα antibodies, ∆N197 and D20, were used in the co-immunoprecipitation assay, of which ∆N197 but not D20 could recognize both RXRα and tRXRα (Zhou et al., 2010). Consistent with our previous reports (Zhou et al., 2010), TNFα strongly induced the interaction of tRXRα but not RXRα with p85α showed by our co-immunoprecipitation experiment (Fig. 5C). However, when cells were treated with N-6 together with TNFα, the interaction of tRXRα and p85α promoted by TNFα was completely blocked (Fig. 5C and 5D). Thus, the inhibitory effect of N-6 on TNFα-induced AKT activation might rely on its disruption of the interaction of tRXRα with p85α.Figure 5


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

Chen F, Chen J, Lin J, Cheltsov AV, Xu L, Chen Y, Zeng Z, Chen L, Huang M, Hu M, Ye X, Zhou Y, Wang G, Su Y, Zhang L, Zhou F, Zhang XK, Zhou H - Protein Cell (2015)

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
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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Fig5: 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
Mentions: It has been reported that RXRα antagonist K-80003 inhibits TNFα-induced AKT activation in a tRXRα-dependent pathway (Zhou et al., 2010). Here we examined whether N-6 had a similar function. Figure 5A showed that N-6 significantly inhibited TNFα-induced AKT activation in HCT116 cells, revealed by its suppression of the expression of phosphor-AKT but not total AKT (Fig. 5A). Similar results were obtained in A549 cells (Fig. 5B). We then determined the requirement of RXRα/tRXRα for the inhibition of AKT activation by N-6. Transfection of RXRα siRNA in A549 cells, which reduced both the full-length RXRα and tRXRα expression, abolished the inhibitory effect of N-6 on TNFα-induced AKT activation (Fig. 5B). Thus, the expression of RXRα/tRXRα is essential for AKT inhibition by N-6. We have reported that K-80003 inhibited AKT activation by blocking the TNFα-induced interaction of tRXRα and p85α (Zhou et al., 2010). We then investigated whether N-6 had the similar action. Two anti-RXRα antibodies, ∆N197 and D20, were used in the co-immunoprecipitation assay, of which ∆N197 but not D20 could recognize both RXRα and tRXRα (Zhou et al., 2010). Consistent with our previous reports (Zhou et al., 2010), TNFα strongly induced the interaction of tRXRα but not RXRα with p85α showed by our co-immunoprecipitation experiment (Fig. 5C). However, when cells were treated with N-6 together with TNFα, the interaction of tRXRα and p85α promoted by TNFα was completely blocked (Fig. 5C and 5D). Thus, the inhibitory effect of N-6 on TNFα-induced AKT activation might rely on its disruption of the interaction of tRXRα with p85α.Figure 5

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: School of Pharmaceutical Sciences, Xiamen University, Xiamen, 361102, China.

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.

No MeSH data available.


Related in: MedlinePlus