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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 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
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Related In: Results  -  Collection


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Fig2: 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

Mentions: To examine whether N-6 regulated RXRα transactivation, a reporter gene containing RXRα homodimer-responsive elements, (TREpal)2-tk-CAT (Dawson et al., 2001), was transfected with a RXRα expression vector into CV-1 cells that lack detectable levels of endogenous RXRα (Zhang et al., 1992a, b). Treatment of cells with 9-cis-RA potently induced the transactivation of RXRα homodimer, which was dose-dependently inhibited by N-6 (Fig. 2A). In contrast, N-6 failed to inhibit 9-cis-RA-induced transactivation of RARγ homodimer or the ligand-independent transactivation of Nur77 (Fig. 2B and 2C) (Kolluri et al., 2003). Thus, N-6 selectively inhibits RXRα transactivation, which was further confirmed by our mammalian one-hybrid technology-based assay. N-6 strongly inhibited 9-cis-RA-induced Gal4-DBD-RXRα-LBD transactivation but not the other chimeric nuclear receptors, such as RARγ, TR3, GR, LXRα and PPARγ (Fig. 2D–I). We next examined the effects of N-6 on the transcriptional activity of RXRα/TR3 heterodimer. To this end, CV-1 cells were transfected with RXRα and TR3 expression vectors together with βRARE-tk-CAT reporter. 9-cis-RA-induced transactivation of RXRα/TR3 heterodimer was strongly inhibited by N-6 (Fig. 2J). It is known that the binding of 9-cis-RA induces RXRα conformational change, leading to co-activator recruitment. We then examined whether N-6 inhibited 9-cis-RA-induced co-activator recruitment. As shown in Fig. 2K, N-6 dose-dependently inhibited 9-cis-RA-induced interaction of co-activator and RXRα-LBD with an IC50 at 32.9 μmol/L. Taken together, these findings indicate that N-6 is a selective antagonist of RXRα.


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 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
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Related In: Results  -  Collection

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Fig2: 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
Mentions: To examine whether N-6 regulated RXRα transactivation, a reporter gene containing RXRα homodimer-responsive elements, (TREpal)2-tk-CAT (Dawson et al., 2001), was transfected with a RXRα expression vector into CV-1 cells that lack detectable levels of endogenous RXRα (Zhang et al., 1992a, b). Treatment of cells with 9-cis-RA potently induced the transactivation of RXRα homodimer, which was dose-dependently inhibited by N-6 (Fig. 2A). In contrast, N-6 failed to inhibit 9-cis-RA-induced transactivation of RARγ homodimer or the ligand-independent transactivation of Nur77 (Fig. 2B and 2C) (Kolluri et al., 2003). Thus, N-6 selectively inhibits RXRα transactivation, which was further confirmed by our mammalian one-hybrid technology-based assay. N-6 strongly inhibited 9-cis-RA-induced Gal4-DBD-RXRα-LBD transactivation but not the other chimeric nuclear receptors, such as RARγ, TR3, GR, LXRα and PPARγ (Fig. 2D–I). We next examined the effects of N-6 on the transcriptional activity of RXRα/TR3 heterodimer. To this end, CV-1 cells were transfected with RXRα and TR3 expression vectors together with βRARE-tk-CAT reporter. 9-cis-RA-induced transactivation of RXRα/TR3 heterodimer was strongly inhibited by N-6 (Fig. 2J). It is known that the binding of 9-cis-RA induces RXRα conformational change, leading to co-activator recruitment. We then examined whether N-6 inhibited 9-cis-RA-induced co-activator recruitment. As shown in Fig. 2K, N-6 dose-dependently inhibited 9-cis-RA-induced interaction of co-activator and RXRα-LBD with an IC50 at 32.9 μmol/L. Taken together, these findings indicate that N-6 is a selective antagonist of RXRα.

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