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Novel β -carbolines against colorectal cancer cell growth via inhibition of Wnt/ β -catenin signaling

View Article: PubMed Central - PubMed

ABSTRACT

Wnt signaling pathway is aberrantly activated in a variety of cancers, especially in colorectal cancer (CRC), because of mutations in the genes encoding adenomatous polyposis coli (APC), β-catenin and Axin. Small-molecule antagonists of Wnt/β-catenin signaling are attractive candidates for developing effective therapeutics for CRC. In this study, we have identified a novel Wnt signaling inhibitor, isopropyl 9-ethyl-1- (naphthalen-1-yl)-9H-pyrido[3,4-b]indole-3- carboxylate (Z86). Z86 inhibited Wnt reporter activities and the expression of endogenous Wnt signaling target genes in mammalian cells and antagonized the second axis formation of Xenopus embryos induced by Wnt8. We showed that Z86 treatment inhibits GSK3β (Ser9) phosphorylation, leading to its overactivation and promoting the phosphorylation and degradation of β-catenin. In vitro, Z86 selectively inhibited the growth of CRC cells with constitutive Wnt signaling and caused obvious G1-phase arrest of the cell cycle. Notably, in a nude mouse model, Z86 inhibited dramatically the xenografted tumor growth of CRC. Daily intraperitoneal injection of Z86 at 5 mg/kg resulted in >70% reduction in the tumor weight of HCT116 cell origin that was associated with decreased GSK3β (Ser9) phosphorylation and increased β-catenin phosphorylation. Taken together, our findings provide a novel promising chemotype for CRC therapeutics development targeting the canonical Wnt signaling.

No MeSH data available.


Identification of Z86 as a potent antagonist of Wnt signaling. (a) Structures of the β-carboline compounds. (b) Effects of Z64, Z80 and Z86 on the Topflash reporter activity. The HEK293W cells were treated with different doses of Z64, Z80 and Z86 respectively for 24 h. Relative luciferase activity (Topflash/Renilla) measured represents the level of activated Wnt signaling. (c) The calculated IC50 values of Wnt signaling inhibition of the compounds are displayed in the table. (d) Z86 preferentially inhibits Wnt signaling (ST-Luc) over the NF-κB signaling pathway. NF-κB signaling was stimulated with 25 ng/ml TNFα treatment for 24 h in HEK293T cells that were subsequently treated with Z86 (20 μM) for 24 h and the NF-κB-Luc luciferase activity was measured. Each bar is the mean±S.D. from three independent experiments. NS, not significant, relative to the vehicle control.
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fig1: Identification of Z86 as a potent antagonist of Wnt signaling. (a) Structures of the β-carboline compounds. (b) Effects of Z64, Z80 and Z86 on the Topflash reporter activity. The HEK293W cells were treated with different doses of Z64, Z80 and Z86 respectively for 24 h. Relative luciferase activity (Topflash/Renilla) measured represents the level of activated Wnt signaling. (c) The calculated IC50 values of Wnt signaling inhibition of the compounds are displayed in the table. (d) Z86 preferentially inhibits Wnt signaling (ST-Luc) over the NF-κB signaling pathway. NF-κB signaling was stimulated with 25 ng/ml TNFα treatment for 24 h in HEK293T cells that were subsequently treated with Z86 (20 μM) for 24 h and the NF-κB-Luc luciferase activity was measured. Each bar is the mean±S.D. from three independent experiments. NS, not significant, relative to the vehicle control.

Mentions: To identify compounds that inhibit the canonical Wnt signaling pathway, a chemical library consisting of 4000 chemically diverse compounds was screened. Our screening led to the identification of β-carboline structure type as a new class of inhibitors of Wnt signaling. Followed by this discovery, a library of β-carboline derivatives (∼150 compounds) were synthesized (Supplementary Figure S1). The inhibitory activities of the derivatives on Wnt signaling were investigated and the structure–activity relationship was characterized. As shown in Figures 1a–c, compounds Z64, Z80 and Z86, which bear a methyl, ethyl and isopropyl ester substituent at C-3 position and with substituents at indole N-9 position, exhibited strong activities to inhibit the canonical Wnt signaling in the SuperTopflash reporter activity assay with median inhibitory concentration (IC50) values of 6.0, 7.5 and 2.8 μM, respectively. However, Z83 without substituents at indole N-9 position only exhibited weak activity with IC50 values of >20 μM. These results suggested that the N-9 substituents of β-carboline played an important role in the Wnt inhibition activity. Furthermore, compounds with a bulky group possess a better activity than with a smaller group like methyl or ethyl ester substituent at C-3 position. To test the specificity of the inhibitory activity of Z86 on Wnt signaling, we performed nuclear factor (NF)-κB reporter (NF-κB-Luc) activity assays. As shown in Figure 1d, Z86 had little effects on the reporter activity of NF-κB signaling at the concentration that significantly inhibited the SuperTopflash activity in HEK293T cells.


Novel β -carbolines against colorectal cancer cell growth via inhibition of Wnt/ β -catenin signaling
Identification of Z86 as a potent antagonist of Wnt signaling. (a) Structures of the β-carboline compounds. (b) Effects of Z64, Z80 and Z86 on the Topflash reporter activity. The HEK293W cells were treated with different doses of Z64, Z80 and Z86 respectively for 24 h. Relative luciferase activity (Topflash/Renilla) measured represents the level of activated Wnt signaling. (c) The calculated IC50 values of Wnt signaling inhibition of the compounds are displayed in the table. (d) Z86 preferentially inhibits Wnt signaling (ST-Luc) over the NF-κB signaling pathway. NF-κB signaling was stimulated with 25 ng/ml TNFα treatment for 24 h in HEK293T cells that were subsequently treated with Z86 (20 μM) for 24 h and the NF-κB-Luc luciferase activity was measured. Each bar is the mean±S.D. from three independent experiments. NS, not significant, relative to the vehicle control.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4979417&req=5

fig1: Identification of Z86 as a potent antagonist of Wnt signaling. (a) Structures of the β-carboline compounds. (b) Effects of Z64, Z80 and Z86 on the Topflash reporter activity. The HEK293W cells were treated with different doses of Z64, Z80 and Z86 respectively for 24 h. Relative luciferase activity (Topflash/Renilla) measured represents the level of activated Wnt signaling. (c) The calculated IC50 values of Wnt signaling inhibition of the compounds are displayed in the table. (d) Z86 preferentially inhibits Wnt signaling (ST-Luc) over the NF-κB signaling pathway. NF-κB signaling was stimulated with 25 ng/ml TNFα treatment for 24 h in HEK293T cells that were subsequently treated with Z86 (20 μM) for 24 h and the NF-κB-Luc luciferase activity was measured. Each bar is the mean±S.D. from three independent experiments. NS, not significant, relative to the vehicle control.
Mentions: To identify compounds that inhibit the canonical Wnt signaling pathway, a chemical library consisting of 4000 chemically diverse compounds was screened. Our screening led to the identification of β-carboline structure type as a new class of inhibitors of Wnt signaling. Followed by this discovery, a library of β-carboline derivatives (∼150 compounds) were synthesized (Supplementary Figure S1). The inhibitory activities of the derivatives on Wnt signaling were investigated and the structure–activity relationship was characterized. As shown in Figures 1a–c, compounds Z64, Z80 and Z86, which bear a methyl, ethyl and isopropyl ester substituent at C-3 position and with substituents at indole N-9 position, exhibited strong activities to inhibit the canonical Wnt signaling in the SuperTopflash reporter activity assay with median inhibitory concentration (IC50) values of 6.0, 7.5 and 2.8 μM, respectively. However, Z83 without substituents at indole N-9 position only exhibited weak activity with IC50 values of >20 μM. These results suggested that the N-9 substituents of β-carboline played an important role in the Wnt inhibition activity. Furthermore, compounds with a bulky group possess a better activity than with a smaller group like methyl or ethyl ester substituent at C-3 position. To test the specificity of the inhibitory activity of Z86 on Wnt signaling, we performed nuclear factor (NF)-κB reporter (NF-κB-Luc) activity assays. As shown in Figure 1d, Z86 had little effects on the reporter activity of NF-κB signaling at the concentration that significantly inhibited the SuperTopflash activity in HEK293T cells.

View Article: PubMed Central - PubMed

ABSTRACT

Wnt signaling pathway is aberrantly activated in a variety of cancers, especially in colorectal cancer (CRC), because of mutations in the genes encoding adenomatous polyposis coli (APC), β-catenin and Axin. Small-molecule antagonists of Wnt/β-catenin signaling are attractive candidates for developing effective therapeutics for CRC. In this study, we have identified a novel Wnt signaling inhibitor, isopropyl 9-ethyl-1- (naphthalen-1-yl)-9H-pyrido[3,4-b]indole-3- carboxylate (Z86). Z86 inhibited Wnt reporter activities and the expression of endogenous Wnt signaling target genes in mammalian cells and antagonized the second axis formation of Xenopus embryos induced by Wnt8. We showed that Z86 treatment inhibits GSK3β (Ser9) phosphorylation, leading to its overactivation and promoting the phosphorylation and degradation of β-catenin. In vitro, Z86 selectively inhibited the growth of CRC cells with constitutive Wnt signaling and caused obvious G1-phase arrest of the cell cycle. Notably, in a nude mouse model, Z86 inhibited dramatically the xenografted tumor growth of CRC. Daily intraperitoneal injection of Z86 at 5 mg/kg resulted in >70% reduction in the tumor weight of HCT116 cell origin that was associated with decreased GSK3β (Ser9) phosphorylation and increased β-catenin phosphorylation. Taken together, our findings provide a novel promising chemotype for CRC therapeutics development targeting the canonical Wnt signaling.

No MeSH data available.