Cepharanthine suppresses APC-mutant colorectal cancers by down-regulating the expression of β-catenin

Abstract

The aberrant activation of the Wnt/β-catenin signaling pathway is closely associated with the development of various carcinomas, especially colorectal cancers (CRCs), where adenomatous colorectal polyposis (APC) mutations are the most frequently observed, which limits the anti-tumor efficiency of inhibitors targeting the upstream of Wnt/β-catenin pathway. The anti-tumor activity of the naturally occurring alkaloid cepharanthine (CEP) extracted from the plant Stephania cepharantha Hayata has been reported in various types of tumors. We previously observed that its derivatives inhibited the Wnt/β-catenin signaling in liver cancer; however, the specific mechanism remains unknown. In this study, we confirmed CEP can effectively inhibit APC-mutant CRC cell lines (SW480, SW620, LoVo) through disturbing of the Wnt/β-catenin signaling and elucidated the underlying mechanisms. Here, we demonstrate that CEP attenuates the Wnt/β-catenin signaling by decreasing the β-catenin, subsequently impeding the proliferation of APC-mutant CRCs. Moreover, CEP induced β-catenin transcription inhibition rather than the instability of β-catenin protein and mRNA contributes to reduction of β-catenin. Taken together, our findings identify CEP as the first β-catenin transcriptional inhibitor in the modulation of Wnt/β-catenin signaling and indicate CEP as a potential therapeutic option for the treatment of APC-mutated CRCs.

Keywords: APC; CEP; Colorectal cancer; Transcriptional inhibitor; Wnt/β-catenin.

Fig. 1

CEP suppresses the growth of APC-mutant CRCs. A Chemical structure of CEP. B Cell viability of NCM460, SW480, SW620 and LoVo cells exposed to CEP (0–16 μM) for 24, 48 h was determined by MTS assay. C IC₅₀ concentrations were estimated based on dose–response curves using the GraphPad software. D Representative images of the cell clone formation assay for SW480 and SW620 cells exposed to CEP at indicated concentrations in plates for 2 weeks. E Flow cytometry analysis of cell cycle distribution in APC-mutant CRCs treated with CEP. F Quantification data of cells distributed at each stage of cell cycle

Fig. 2

Inhibition of CEP on the transcriptional activity of β‐catenin/TCF4 in APC-mutant CRCs. A APC-mutant CRCs were transfected with TOPflash luciferase reporter and Renilla plamids and then exposed to increasing concentrations of CEP, with HEK293W cells treated with indicated concentrations CEP directly for 24 h, followed by the measurement of the luciferase activity. B Western blot analysis of proteins of β-catenin, Axin2 and CyclinD1 in APC-mutant CRCs. Quantification of the protein expression was performed using ImageJ software

Fig. 3

CEP decreases the nuclear β‐catenin in APC-mutant CRCs. A The Western blotting was performed to detect the β-catenin protein in cytosol and nucleus with CEP treated cells. The cytoplasm marker applied was β-actin, whereas the nucleus marker used was Lamin A/C. B Distribution analysis of β-catenin (green) and nucleus (blue) in SW480 and LoVo cells with Immunofluorescence analysis. Scale bar, 100 µM

Fig. 4

β-catenin mediates inhibition of the proliferation of APC-mutant CRCs by CEP. A, B β-catenin was measured by Western blotting after CTNNB1 siRNA infection in SW480 and LoVo cells. C, D MTS assay was conducted in SW480 and LoVo cells transfected with siRNAs targeting CTNNB1. Relative cell viability was normalized to the CEP-free group

Fig. 5

CEP reduces β-catenin protein synthesis. A, B Western blotting was applied to detect p-β-catenin, active β-catenin in SW480 and SW620 cells exposed to CEP for 48 h. C, D SW480, SW620 cells were subjected to designated dosages of CEP with or without 20 μM MG132 for 16 h, and then analyzed using western blotting. E, F Representative Western blot and quantification of β-catenin within cell extracts of CRC (SW480, SW620) cells pretreated with or without CHX (100 μg/ml) for 2 h followed by 12 h indicated concentrations of CEP exposure. The quantification of β-catenin protein expression was performed using ImageJ software. The Western blot results were quantified and standardized using β-actin as a reference

Fig. 6

CEP decreases the transcription of β-catenin. A–D SW480 and SW620 cells administrated with CEP for indicated doses and times were subjected to RT-PCR assay, and GAPDH was used to normalize the relative expression level. E The mRNA levels of β-catenin were analyzed in SW480 cells exposed to 9 μM CEP for different times in the presence of ACTD. F SW480 cells were treated with given doses of CEP for 24 h, the luciferase activity of the CTNNB1 promoter was determined by a luciferase reporter assay