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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.


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Z86 inhibits Wnt signaling through suppression of GSK3β phosphorylation. (a) Z86 reduces the phosphorylation of GSK3β (Ser9) in both Wnt3a stably transfected HEK293W cells and CRC cells. Cells were incubated with 20 μM of Z86, and cell extracts were subjected to western blot analysis to detect phosphorylated GSK3β (Ser9) and the total GSK3β. β-Actin was used as the loading control. (b) HEK293T cells were transfected with luciferase reporters (Topflash and Renilla) together with Wnt1 (64 ng per well), DNGSK3β (30 ng per well) in 96-well plates, or incubated 20 mM of LiCl respectively. After transfection or treatment for 3 h, cells were treated with Z86 at indicated dosage for additional 24 h and luciferase activities were then measured. (c, upper panel) Schematic drawing showing the procedure of Xenopus animal cap RT-PCR assays. (c, lower panel) Z86 inhibits Wnt8, but not DNGSK3β-induced expression of Siamois and Xnr3 in Xenopus animal cap cells. The experiment was repeated three times and representative results are shown. Amplification of ornithine decarboxylase (ODC) confirmed RNA integrity. NC, negative control with the reverse transcriptase omitted in the RT reaction; Con, control.
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fig4: Z86 inhibits Wnt signaling through suppression of GSK3β phosphorylation. (a) Z86 reduces the phosphorylation of GSK3β (Ser9) in both Wnt3a stably transfected HEK293W cells and CRC cells. Cells were incubated with 20 μM of Z86, and cell extracts were subjected to western blot analysis to detect phosphorylated GSK3β (Ser9) and the total GSK3β. β-Actin was used as the loading control. (b) HEK293T cells were transfected with luciferase reporters (Topflash and Renilla) together with Wnt1 (64 ng per well), DNGSK3β (30 ng per well) in 96-well plates, or incubated 20 mM of LiCl respectively. After transfection or treatment for 3 h, cells were treated with Z86 at indicated dosage for additional 24 h and luciferase activities were then measured. (c, upper panel) Schematic drawing showing the procedure of Xenopus animal cap RT-PCR assays. (c, lower panel) Z86 inhibits Wnt8, but not DNGSK3β-induced expression of Siamois and Xnr3 in Xenopus animal cap cells. The experiment was repeated three times and representative results are shown. Amplification of ornithine decarboxylase (ODC) confirmed RNA integrity. NC, negative control with the reverse transcriptase omitted in the RT reaction; Con, control.

Mentions: GSK3β is the central component in the multiprotein destruction complex consisting of GSK3β/Axin/APC/casein kinase 1 (CK1) that controls the phosphorylation and degradation of β-catenin, and functions as a negative regulator of Wnt signaling pathway.30–32 Therefore, we further investigated whether GSK3β is involved in Z86-promoted β-catenin phosphorylation and degradation in colon cancer cell lines. Interestingly, phosphorylated GSK3β (Ser9), the inactive status, significantly reduced upon Z86 treatment in HEK293W cells as well as in HCT116 and SW480 cells, with slight increase of the total level of GSK3β protein (Figure 4a). In contrast, LiCl, an inhibitor of GSK3β, increased the phosphorylation of GSK3β (Ser9) in our study (data not shown), consistent with previous study.33 We then tested whether LiCl or dominant-negative kinase-inactive GSK3β (DNGSK3β)30 could rescue the inhibitory activity of Z86 on the Wnt signaling. HEK293T cells were transiently transfected with Wnt1 and DNGSK3β or pretreated with LiCl, respectively, before the cells were treated with Z86, and then Wnt reporter activities were monitored. Z86 efficiently antagonized Wnt1-induced activation of the canonical Wnt signaling but exhibited much weaker inhibitory activity on the Topflash activity stimulated by LiCl and, furthermore, lost the inhibitory activity completely when the Wnt signaling was stimulated with DNGSK3β (Figure 4b).


Novel β -carbolines against colorectal cancer cell growth via inhibition of Wnt/ β -catenin signaling
Z86 inhibits Wnt signaling through suppression of GSK3β phosphorylation. (a) Z86 reduces the phosphorylation of GSK3β (Ser9) in both Wnt3a stably transfected HEK293W cells and CRC cells. Cells were incubated with 20 μM of Z86, and cell extracts were subjected to western blot analysis to detect phosphorylated GSK3β (Ser9) and the total GSK3β. β-Actin was used as the loading control. (b) HEK293T cells were transfected with luciferase reporters (Topflash and Renilla) together with Wnt1 (64 ng per well), DNGSK3β (30 ng per well) in 96-well plates, or incubated 20 mM of LiCl respectively. After transfection or treatment for 3 h, cells were treated with Z86 at indicated dosage for additional 24 h and luciferase activities were then measured. (c, upper panel) Schematic drawing showing the procedure of Xenopus animal cap RT-PCR assays. (c, lower panel) Z86 inhibits Wnt8, but not DNGSK3β-induced expression of Siamois and Xnr3 in Xenopus animal cap cells. The experiment was repeated three times and representative results are shown. Amplification of ornithine decarboxylase (ODC) confirmed RNA integrity. NC, negative control with the reverse transcriptase omitted in the RT reaction; Con, control.
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fig4: Z86 inhibits Wnt signaling through suppression of GSK3β phosphorylation. (a) Z86 reduces the phosphorylation of GSK3β (Ser9) in both Wnt3a stably transfected HEK293W cells and CRC cells. Cells were incubated with 20 μM of Z86, and cell extracts were subjected to western blot analysis to detect phosphorylated GSK3β (Ser9) and the total GSK3β. β-Actin was used as the loading control. (b) HEK293T cells were transfected with luciferase reporters (Topflash and Renilla) together with Wnt1 (64 ng per well), DNGSK3β (30 ng per well) in 96-well plates, or incubated 20 mM of LiCl respectively. After transfection or treatment for 3 h, cells were treated with Z86 at indicated dosage for additional 24 h and luciferase activities were then measured. (c, upper panel) Schematic drawing showing the procedure of Xenopus animal cap RT-PCR assays. (c, lower panel) Z86 inhibits Wnt8, but not DNGSK3β-induced expression of Siamois and Xnr3 in Xenopus animal cap cells. The experiment was repeated three times and representative results are shown. Amplification of ornithine decarboxylase (ODC) confirmed RNA integrity. NC, negative control with the reverse transcriptase omitted in the RT reaction; Con, control.
Mentions: GSK3β is the central component in the multiprotein destruction complex consisting of GSK3β/Axin/APC/casein kinase 1 (CK1) that controls the phosphorylation and degradation of β-catenin, and functions as a negative regulator of Wnt signaling pathway.30–32 Therefore, we further investigated whether GSK3β is involved in Z86-promoted β-catenin phosphorylation and degradation in colon cancer cell lines. Interestingly, phosphorylated GSK3β (Ser9), the inactive status, significantly reduced upon Z86 treatment in HEK293W cells as well as in HCT116 and SW480 cells, with slight increase of the total level of GSK3β protein (Figure 4a). In contrast, LiCl, an inhibitor of GSK3β, increased the phosphorylation of GSK3β (Ser9) in our study (data not shown), consistent with previous study.33 We then tested whether LiCl or dominant-negative kinase-inactive GSK3β (DNGSK3β)30 could rescue the inhibitory activity of Z86 on the Wnt signaling. HEK293T cells were transiently transfected with Wnt1 and DNGSK3β or pretreated with LiCl, respectively, before the cells were treated with Z86, and then Wnt reporter activities were monitored. Z86 efficiently antagonized Wnt1-induced activation of the canonical Wnt signaling but exhibited much weaker inhibitory activity on the Topflash activity stimulated by LiCl and, furthermore, lost the inhibitory activity completely when the Wnt signaling was stimulated with DNGSK3β (Figure 4b).

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.


Related in: MedlinePlus