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Cancers (Basel)
2019 Dec 07;1112:. doi: 10.3390/cancers11121968.
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The Chalcone Lonchocarpin Inhibits Wnt/β-Catenin Signaling and Suppresses Colorectal Cancer Proliferation.
Predes D
,
Oliveira LFS
,
Ferreira LSS
,
Maia LA
,
Delou JMA
,
Faletti A
,
Oliveira I
,
Amado NG
,
Reis AH
,
Fraga CAM
,
Kuster R
,
Mendes FA
,
Borges HL
,
Abreu JG
.
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The deregulation of the Wnt/β-catenin signaling pathway is a central event in colorectal cancer progression, thus a promising target for drug development. Many natural compounds, such as flavonoids, have been described as Wnt/β-catenin inhibitors and consequently modulate important biological processes like inflammation, redox balance, cancer promotion and progress, as well as cancer cell death. In this context, we identified the chalcone lonchocarpin isolated from Lonchocarpus sericeus as a Wnt/β-catenin pathway inhibitor, both in vitro and in vivo. Lonchocarpin impairs β-catenin nuclear localization and also inhibits the constitutively active form of TCF4, dnTCF4-VP16. Xenopus laevis embryology assays suggest that lonchocarpin acts at the transcriptional level. Additionally, we described lonchocarpin inhibitory effects on cell migration and cell proliferation on HCT116, SW480, and DLD-1 colorectal cancer cell lines, without any detectable effects on the non-tumoral intestinal cell line IEC-6. Moreover, lonchocarpin reduces tumor proliferation on the colorectal cancer AOM/DSS mice model. Taken together, our results support lonchocarpin as a novel Wnt/β-catenin inhibitor compound that impairs colorectal cancer cell growth in vitro and in vivo.
433522/2018-6 - 18/02/2019 Conselho Nacional de Desenvolvimento Científico e Tecnológico, E-26/203.009/2018 (239212) Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro
Figure 1. Lonchocarpin inhibits the Wnt/β-catenin pathway. Treatment with lonchocarpin inhibits Wnt reporter activity. (A) Lonchocarpin chemical structure, (B) RKO-pBAR/Renilla, and (C) SW480-pBAR/Renila gene reporter luciferase assay. (D) Lonchocarpin half maximal inhibitory concentration is 4 µM in the SW480-pBAR/Renilla cell lineage. Graph bars represent mean and SD. (E–I) SW480 β-catenin and DAPI immunocytochemistry staining showed that lonchocarpin decreases β-catenin translocation after lonchocarpin treatment. RKO cells were treated with (E–Eâ€) L-cell control conditioned medium or Wnt3a conditioned medium with (F–Fâ€) DMSO, (G–Gâ€) 10 µM or (H–Hâ€) 20 µM lonchocarpin. (I–Iâ€) Immunocytochemistry negative control. (J) Quantification of nuclear β-catenin positive cell count ratio. Graph bars represent mean and SEM. (K) Immunoblotting depicting β-catenin and β-actin total levels of RKO cells treated with Wnt3a CM (conditioned medium) for 7 h. Densitometry is shown as the ratio of β-catenin/β-actin. (K) Immunoblot of cell lysate of RKO cells treated with 1-L-cell CM, 2-Wnt3a CM + DMSO, 3-Wnt3a CM + lonchocarpin 20 µM, 4-BIO 1 µM + DMSO, 5-BIO 1 µM + lonchocarpin 20 µM. (L) Immunoblot of cytosolic and nuclear fractions of RKO cells treated with 1,6-L-cell CM, 2,7-Wnt3a CM + DMSO, 3,8-Wnt3a CM + lonchocarpin 20 µM, 4,9-BIO 1 µM + DMSO, 5,10-BIO 1 µM + lonchocarpin 20 µM. Cytosolic densitometry was calculated considering α-Tubulin as the loading control, while nuclear densitometry considered Lamin A/C as the loading control. * p < 0.05, ** p < 0.01, *** p < 0.001. Scale bar represents 20 μm.
Figure 2. Lonchocarpin inhibits Wnt/β-catenin pathway downstream to TCF4. Lonchocarpin suppresses Wnt/β-catenin pathway induced by Wnt3a CM treatment (A) or by transfection with (B) β-catenin, (C) β-catenin S33A, or (D) dnTCF4VP16 in HEK293T cells. (E) Proposed lonchocarpin mechanism of action. Graph bars represent mean and SD. *** p < 0.001.
Figure 3. Lonchocarpin alters axial patterning in Xenopus laevis embryos and inhibits Wnt-8 induced axis and S01234-luciferase reporter. The 4-cell stage injected embryos display a smaller head compared uninjected and DMSO-injected embryos (A–D). Stage 9 blastulae injected embryos display a larger head (arrow) and cement gland (arrow) compared to uninjected or DMSO-injected embryos (E–H). Injection of xWnt8 mRNA into ventralblastomere induced ectopic axis (arrowhead) compared to uninjected embryos (I–K,P). Lonchocarpin inhibited Wnt8-induced secondary axis (M,P), but not DMSO (L,P). Lonchocarpin injection inhibits Wnt8-induced S01234-luciferase specific reporter activation in Xenopus laevis embryo (N,O) *** p < 0.001. Graph bars represent mean and SD (see also Figure S1).
Figure 4. Lonchocarpin inhibits HCT116, SW480, and DLD-1 colorectal cancer cell lines proliferation. Proliferation assay shows that lonchocarpin suppresses proliferation of HCT116, SW480, and DLD-1 colorectal cancer cell lines, while not affecting IEC-6 non-tumor small intestine cell line proliferation. DAPI and EdU stained cells micrographs acquired 24 h post 5, 10, and 20 µM lonchocarpin treatment. (A–E) HCT116, (F–J) SW480, (K–O) DLD-1, (P–T) IEC-6. Graphs show the percentage of EdU positive cells. Scale bar represents 10 μm. Graph bars represent mean and SD. MTT assay shows that lower lonchocarpin concentrations are less cytotoxic to the non-tumor cell line IEC-6 compared to the tumor cell lines. MTT assay performed after treatment with 10, 20, 30, 40, and 50 µM lonchocarpin of (U) HCT116, (V) SW480, (W) DLD-1, and (X) IEC-6 cells during 24, 48, and 72 h. R.U. (Relative Units). * p < 0.1, ** p < 0.01. Graphs show mean and SEM.
Figure 5. Lonchocarpin inhibits cell migration in HCT116, SW480, and DLD-1 colorectal cancer cell lines. Scratch assay shows that lonchocarpin impairs migration of HCT116, SW480, and DLD-1 colorectal cancer cell lines while not interfering with the IEC-6 non-tumor intestine cell line migration. Images show cell migration through the scratch healing 24 h post treatment with 5, 10, and 20 µM lonchocarpin (A–E) HCT116, (F–J) SW480, (K–O) DLD-1, (P–T) IEC-6. Graph shows relative wound area closure relative to time 0 h. All conditions were normalized to DMSO. * p < 0.1, *** p < 0.001. Scale bar represents 100 μm. Graph bars represent mean and SD.
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