Piperine suppresses the Wnt/β-catenin pathway and has anti-cancer effects on colorectal cancer cells

Abstract

More than 94% of colorectal cancer cases have mutations in one or more Wnt/β-catenin signaling pathway components. Inactivating mutations in APC or activating mutations in β-catenin (CTNNB1) lead to signaling overactivation and subsequent intestinal hyperplasia. Numerous classes of medicines derived from synthetic or natural small molecules, including alkaloids, have benefited the treatment of different diseases, including cancer, Piperine is a true alkaloid, derived from lysine, responsible for the spicy taste of black pepper (Piper nigrum) and long pepper (Piper longum). Studies have shown that piperine has a wide range of pharmacological properties; however, piperine molecular mechanisms of action are still not fully understood. By using Wnt/β-catenin pathway epistasis experiment we show that piperine inhibits the canonical Wnt pathway induced by overexpression of β-catenin, β-catenin S33A or dnTCF4 VP16, while also suppressing β-catenin nuclear localization in HCT116 cell line. Additionally, piperine impairs cell proliferation and migration in HCT116, SW480 and DLD-1 colorectal tumor cell lines, while not affecting the non-tumoral cell line IEC-6. In summary, piperine inhibits the canonical Wnt signaling pathway and displays anti-cancer effects on colorectal cancer cell lines.

Figure 1

Piperine inhibits TCF/LEF induced transcription. (A) Molecular structure of piperine. (B) Relative luciferase activity of RKO pBAR/Renilla cells treated or not with different concentrations of piperine and L-Wnt3a conditioned medium. (C) Relative luciferase activity of SW480 pBAR/Renilla cells treated or not with different concentrations of piperine. Piperine inhibits Wnt signaling on both cells that have normal (RKO) or overexpressed (SW480) Wnt signaling. (D) Relative luciferase activity of HEK293T cells transfected with (E) pCS2, (F) β-catenin WT, (G) β-catenin S33A or (H) dnTCF4 VP16 and treated or not with different concentrations of piperine. ***p < 0.001, ****p < 0.0001.

Figure 2

Piperine reduces β-catenin nuclear localization. Immunostainings of β-catenin of RKO cells treated with (A–A″) L-cell conditioned medium, with (B–B″) L-Wnt3a conditioned medium co-treated with DMSO or with (C–C″) piperine 100 µM. (D–D″) XAV939 was used as a positive control for Wnt signaling inhibition. (E) Graph of β-catenin positive nuclei percentage quantification. (F) Immunoblot for β-catenin of HCT116 cells untreated or treated with DMSO or 50, 100 μM piperine for 24 h. The raw immunoblot data is shown in Supplementary Figure S4. Scale bar = 38 μm. *p < 0.05, **p < 0.01.

Figure 3

Piperine does not affect viability of non-tumoral intestine cell line IEC-6. Graphs show MTT assay absorbance levels in (A) HCT116, (B) SW480, (C) DLD-1 and (D) IEC-6 cell lines, after 24, 48 or 72 h of treatment with 20, 30, 50, 100 or 200 μM piperine. *p < 0.05, ***p < 0.001.

Figure 4

Piperine decreases colorectal cancer cell lines proliferation. Immunocytochemistry showing DAPI staining of (A–E) HCT116, (G–H) SW480, (J–N) DLD-1 and (P–T) IEC-6, and EdU staining of (A′–E′) HCT116, (G′–H′) SW480, (J′–N′) DLD-1 and (P′–T′) IEC-6. Cells were treated with DMSO, 50, 100, 200 μM piperine, or untreated according to label. Quantification of the percentage of EdU positive nuclei of (F) HCT116 cells, (I) SW480, (O) DLD-1, (U) IEC-6 cells treated or not with 50, 100 or 200 µM piperine. *p < 0.05, **p < 0.01, ***p < 0.001. Scale bar = 50 μm.

Figure 5

Piperine impairs cell cycle progression of SW480 and RKO cell lines. (A–D) Histograms represent DNA content of cells in G0/G1 (gate M1), S (gate M2) and G2/M phase (gate M3) of the cell cycle of SW480 cells untreated or treated with DMSO, 50 and 100 μM piperine for 48 h. (E) G0/G1 (F) S and (G) G2/M phase contents graphs of treated SW480 cells. (H) Western blot for analysis of c-myc and pH3 protein contents of SW480 cells treated with DMSO, 50 or 100 μM piperine, or 10 μM XAV939 for 24 h. β-actin was stained as a loading control. (I–L) Histograms of RKO cells untreated or treated with the described conditions. (M) G0/G1 (N) S and (O) G2/M phase contents graphs of treated RKO cells. (P) Western blot of treated RKO cells. These data are representative of three independent experiments. The raw immunoblot data is shown in Supplementary Figure S4. *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 6

Piperine impairs colorectal tumor cell lines migration. Phase-contrast images of the scratch wound immediately after scratch of (A–D) HCT116, (F–J) SW480, (L–O) DLD-1 and (P–S) IEC-6 cell lines. Phase contrast images of scratch wound 24 h after scratch of (A′–D′) HCT116, (F′–J′) SW480, (L′–O′) DLD-1 and (P′–S′) IEC-6 cell lines. Cells were treated with DMSO, 50, 100 μM piperine, or untreated according to label. Quantification of relative scratch wound closure of (E) HCT116, (K) SW480, (P) DLD-1, (S) IEC-6 treated or not with 50, 100 µM piperine. *p < 0.05, **p < 0.01, ***p < 0.001. Scale bar = 140 μm.