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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 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
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Fig3: 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

Mentions: In the absence of RXRα agonists, the fusion protein Gal4-DBD-RXRα-LBD only exhibited basal transactivation. However, transfection of TR3 remarkably enhanced its transactivation (Fig. 3A). The salt bridges formed by Glu453/456 in helix 12 and Arg302 in helix 4 are essential for maintaining co-activator binding site in RXRα (Egea et al., 2000; Egea et al., 2002). The fusion protein Gal4-DBD-RXRα-LBD/E453/456A, with Ala substituted with Glu, completely lost transactivation ability in response to 9-cis-RA and CD3254 (Fig. 3B). However, TR3 was still able to stimulate the reporter gene activity together with Gal4-DBD-RXRα-LBD/E453/456A (Fig. 3C). Sequence analysis indicated there were no cognate TR3 binding sites in the G5 reporter vector. Thus, the ability of TR3 to induce the transactivation was due to the interaction of Gal4-DBD-RXRα-LBD with TR3 and TR3 ligand-independent transactivation. No matter what above fusion proteins were used, TR3-induced transactivation was potently inhibited by N-6 (Fig. 3D). However, in the case of Gal4-DBD-RXRα-LBD/C432Y with disrupted RXRα-LBP due to the substitution of Cys with Tyr, N-6 completely lost its activity (Fig. 3D), suggesting the necessity of the binding of N-6 to RXRα-LBD. Similar results were observed when RXRα antagonist UVI3003 was examined (Fig. 3D). Thus, N-6 and UVI3003 bind to RXRα to indirectly inhibit TR3 transactivation. One possible mechanism by which N-6 inhibited TR3 transactivation was the disruption of the interactions of TR3 with the fusion proteins, which was analyzed by our co-immunoprecipitation experiments. As shown in Fig. 3E, Myc-TR3 interacted with the fusion proteins, as evidenced by their co-precipitation. Neither N-6 nor UVI3003 affected their interactions (Fig. 3E). GAL4-DBD-TR3-LBD had low, if there was, transactivation activity, which was due to the loss of the N-terminal TR3 (Wansa et al., 2002). When both GAL4-DBD-TR3-LBD and p16-ACT-RXRα fusion proteins existed, a dramatic induction of the reporter gene activity was observed, which reflected a typical mammalian two-hybrid assay to show the interaction of RXRα and TR3-LBD. In this context, neither N-6 nor UVI3003 was able to inhibit the induced transactivation (Fig. 3F), which also demonstrated the inability of N-6 and UVI3003 for blocking the interaction of TR3 and RXRα. Therefore, it is not through the disruption of the interaction of TR3 and RXRα for N-6 to inhibit TR3 transactivation.Figure 3


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 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
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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Fig3: 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
Mentions: In the absence of RXRα agonists, the fusion protein Gal4-DBD-RXRα-LBD only exhibited basal transactivation. However, transfection of TR3 remarkably enhanced its transactivation (Fig. 3A). The salt bridges formed by Glu453/456 in helix 12 and Arg302 in helix 4 are essential for maintaining co-activator binding site in RXRα (Egea et al., 2000; Egea et al., 2002). The fusion protein Gal4-DBD-RXRα-LBD/E453/456A, with Ala substituted with Glu, completely lost transactivation ability in response to 9-cis-RA and CD3254 (Fig. 3B). However, TR3 was still able to stimulate the reporter gene activity together with Gal4-DBD-RXRα-LBD/E453/456A (Fig. 3C). Sequence analysis indicated there were no cognate TR3 binding sites in the G5 reporter vector. Thus, the ability of TR3 to induce the transactivation was due to the interaction of Gal4-DBD-RXRα-LBD with TR3 and TR3 ligand-independent transactivation. No matter what above fusion proteins were used, TR3-induced transactivation was potently inhibited by N-6 (Fig. 3D). However, in the case of Gal4-DBD-RXRα-LBD/C432Y with disrupted RXRα-LBP due to the substitution of Cys with Tyr, N-6 completely lost its activity (Fig. 3D), suggesting the necessity of the binding of N-6 to RXRα-LBD. Similar results were observed when RXRα antagonist UVI3003 was examined (Fig. 3D). Thus, N-6 and UVI3003 bind to RXRα to indirectly inhibit TR3 transactivation. One possible mechanism by which N-6 inhibited TR3 transactivation was the disruption of the interactions of TR3 with the fusion proteins, which was analyzed by our co-immunoprecipitation experiments. As shown in Fig. 3E, Myc-TR3 interacted with the fusion proteins, as evidenced by their co-precipitation. Neither N-6 nor UVI3003 affected their interactions (Fig. 3E). GAL4-DBD-TR3-LBD had low, if there was, transactivation activity, which was due to the loss of the N-terminal TR3 (Wansa et al., 2002). When both GAL4-DBD-TR3-LBD and p16-ACT-RXRα fusion proteins existed, a dramatic induction of the reporter gene activity was observed, which reflected a typical mammalian two-hybrid assay to show the interaction of RXRα and TR3-LBD. In this context, neither N-6 nor UVI3003 was able to inhibit the induced transactivation (Fig. 3F), which also demonstrated the inability of N-6 and UVI3003 for blocking the interaction of TR3 and RXRα. Therefore, it is not through the disruption of the interaction of TR3 and RXRα for N-6 to inhibit TR3 transactivation.Figure 3

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