Dihydrocapsaicin Inhibits Cell Proliferation and Metastasis in Melanoma via Down-regulating β-Catenin Pathway

Abstract

Dihydrocapsaicin (DHC) is one of the main components of capsaicinoids in Capsicum. It has been reported that DHC exerts anti-cancer effects on diverse malignant tumors, such as colorectal cancer, breast cancer, and glioma. However, studies focused on the effect of DHC upon melanoma have rarely been done. In the present study, melanoma A375 and MV3 cell lines were treated with DHC and the cell proliferation, migration, and invasion were significantly suppressed. Furthermore, DHC effectively inhibited xenograft tumor growth and pulmonary metastasis of melanoma cells in NOD/SCID mice model. It was identified that β-catenin, which plays significant roles in cell proliferation and epithelial-mesenchymal transition, was down-regulated after DHC treatment. In addition, cyclin D1, c-Myc, MMP2, and MMP7, which are critical in diverse cellular process regulation as downstream proteins of β-catenin, were all decreased. Mechanistically, DHC accelerates ubiquitination of β-catenin and up-regulates the beta-transducin repeat containing E3 ubiquitin protein ligase (BTRC) in melanoma cells. The DHC induced suppression of cell proliferation, migration, and invasion were partly rescued by exogenous β-catenin overexpression, both in vitro and in vivo. Taken together, DHC may serve as a candidate natural compound for human melanoma treatment through β-catenin pathway.

Keywords: cell proliferation; dihydrocapsaicin; melanoma; metastasis; ubiquitination; β-catenin.

Figure 1

DHC shows anti-proliferation activity in melanoma cells. (A) The IC50 of DHC for A375 and MV3 cells was measured. (B) Cell viabilities of A375 and MV3 cells treated with DHC (0, 50, 100, and 200 μM) for 48 h. (C) EdU staining of A375 and MV3 cells treated with 100 μM DHC for 48 h. Scale bar, 50 μm. (D) Cell cycle analysis of A375 and MV3 cells treated with 100 μM DHC for 48 h. (E, G) The protein and mRNA levels of cyclin D1, c-Myc, and cyclin A2 in A375 and MV3 cells treated with 0, 50, 100, and 200 μM DHC for 48 h. (F, H) The protein and mRNA levels of cyclin D1, c-Myc, and cyclin A2 in A375 and MV3 cells treated with 100 μM DHC for 0, 12, 24, and 48 h. *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant.

Figure 2

DHC inhibits the migration and invasion of melanoma cells. (A) Wound closure proportion of A375 and MV3 cells under the treatment of 100 μM DHC. Scale bar, 100 μm. (B, C) Migration and invasion transwell assays were performed in A375 and MV3 cells treated with 100 μM DHC. Scale bar, 100 μm. (D, F) The protein and mRNA levels of β-catenin, MMP2, and MMP7 in A375 and MV3 cells treated with 0, 50, 100, and 200 μM DHC for 48 h. (E, G) The protein and mRNA levels of β-catenin, MMP2, and MMP7 in A375 and MV3 cells treated with 100 μM DHC for 0, 12, 24, and 48 h. *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant.

Figure 3

DHC suppresses tumorigenesis and pulmonary metastasis of melanoma cells. (A) Colonies generated by A375 and MV3 cells after treatment with 100 μM DHC for 3 weeks. Scale bar, 1 mm. (B) Tumor volume of A375 xenograft tumors in mice treated with DHC (20 mg/kg/day for 28 days) and DMSO. (C, D) The tumors in mice were excised and weighed. (E) IHC of Ki67 in the xenograft tumors. Scale bar, 100 μm. (F) H&E staining of the lungs from A375 metastasis mice model after treatment with DHC (20 mg/kg/day for 45 days) and DMSO. **P < 0.01; ***P < 0.001.

Figure 4

Overexpression of β-catenin retrieves DHC-induced cell proliferation inhibition. (A) The protein expression of β-catenin in β-catenin/vector overexpressed A375 and MV3 cells treated with 100 μM DHC for 48 h. (B) Cell viabilities of β-catenin/vector overexpressed A375 and MV3 cells treated with 100 μM DHC for 48 h. (C) EdU staining of β-catenin/vector overexpressed A375 and MV3 cells treated with 100 μM DHC for 48 h. (D, E) Cell cycle analysis of β-catenin/vector overexpressed A375 and MV3 cells treated with 100 μM DHC for 48 h. (F, G) The protein and mRNA levels of cyclin D1, c-Myc, and cyclin A2 in β-catenin/vector overexpressed A375 and MV3 cells treated with 100 μM DHC for 48 h. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 5

Overexpression of β-catenin retrieves DHC-induced cell migration and invasion inhibition. (A, B) Migration and invasion transwell assays were performed in β-catenin/vector overexpressed A375 and MV3 cells treated with 100 μM DHC. Scale bar, 100 μm. (C, D) The protein expression of β-catenin, MMP2, and MMP7 in β-catenin/vector overexpressed A375 and MV3 cells treated with 100 μM DHC for 48 h. (E, F) The mRNA expression of MMP2 and MMP7 in β-catenin/vector overexpressed A375 and MV3 cells treated with 100 μM DHC for 48 h. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 6

Overexpression of β-catenin retrieves DHC-induced inhibition of tumor growth and pulmonary metastasis of melanoma cells. (A) Colonies generated by β-catenin/vector overexpressed A375 and MV3 cells after treatment with 100 μM DHC for 3 weeks. Scale bar, 1 mm. (B) Tumor volume of β-catenin/vector overexpressed A375 xenograft tumors in mice after treatment with DHC (20 mg/kg/day for 28 days) and DMSO. (C, D) The tumors in mice were excised and weighed. (E) IHC of β-catenin and Ki67 in the xenograft tumors. Scale bar, 100 μm. (F) H&E staining of the lungs from β-catenin/vector overexpressed A375 metastasis mice model after treatment with DHC (20 mg/kg/day for 45 days) and DMSO. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 7

DHC down-regulates β-catenin by increasing its ubiquitination. (A, B) The protein turnover rate of β-catenin in A375 cells under the treatment of 100 μM DHC. (C) The protein expression of β-catenin in 100 μM DHC or DMSO treated cells in the presents or absents of MG132. (D) Ubiquitination levels of β-catenin was detected in HA-Ub transfected 293FT cells after treatment with DHC. (E) The expression of BTRC and FBXW7 in A375 and MV3 cells treated with 0, 50, 100, and 200 μM of DHC for 48 h. (F) Model of the impact of DHC on melanoma cells by the regulation of β-catenin signaling pathway was shown.