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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

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
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Fig6: 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

Mentions: The ability of N-6 to inhibit AKT activation prompted us to examine the effect of N-6 on the survival of cancer cells by colonogenic survival assay. Treatment of cells with N-6 significantly inhibited colony formation of HeLa cells, which was dramatically enhanced by TNFα (Fig. 6A). The inhibition of TNFα-induced AKT activation often leads to the activation of TNFα-mediated apoptosis (Zhou et al., 2010; Wang et al., 2013). Indeed, the combination of TNFα and N-6 synergistically induced PARP cleavage in HCT116 cells (Fig. 6B). The synergistic induction of PARP cleavage was TNFα-concentration dependent in MCF-7 cells (Fig. 6C). Furthermore, our TUNEL staining assay confirmed the synergistic pro-apoptosis of the combination (Fig. 6D). We then determined the role of RXRα/tRXRα in apoptosis induction of the combination using knocking down approach. Compared to control siRNA, RXRα siRNA impaired the ability of the combination of N-6 and TNFα for the induction of PARP cleavage (Fig. 6E). Accompanying with PARP cleavage, the combined treatment also induced caspase-8 activation, which was also inhibited by the suppression of RXRα/tRXRα expression (Fig. 6E). Thus, RXRα/tRXRα is involved in the apoptotic induction of the combination of N-6 and TNFα.Figure 6


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)

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
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Fig6: 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
Mentions: The ability of N-6 to inhibit AKT activation prompted us to examine the effect of N-6 on the survival of cancer cells by colonogenic survival assay. Treatment of cells with N-6 significantly inhibited colony formation of HeLa cells, which was dramatically enhanced by TNFα (Fig. 6A). The inhibition of TNFα-induced AKT activation often leads to the activation of TNFα-mediated apoptosis (Zhou et al., 2010; Wang et al., 2013). Indeed, the combination of TNFα and N-6 synergistically induced PARP cleavage in HCT116 cells (Fig. 6B). The synergistic induction of PARP cleavage was TNFα-concentration dependent in MCF-7 cells (Fig. 6C). Furthermore, our TUNEL staining assay confirmed the synergistic pro-apoptosis of the combination (Fig. 6D). We then determined the role of RXRα/tRXRα in apoptosis induction of the combination using knocking down approach. Compared to control siRNA, RXRα siRNA impaired the ability of the combination of N-6 and TNFα for the induction of PARP cleavage (Fig. 6E). Accompanying with PARP cleavage, the combined treatment also induced caspase-8 activation, which was also inhibited by the suppression of RXRα/tRXRα expression (Fig. 6E). Thus, RXRα/tRXRα is involved in the apoptotic induction of the combination of N-6 and TNFα.Figure 6

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