Dietary delphinidin inhibits human colorectal cancer metastasis associating with upregulation of miR-204-3p and suppression of the integrin/FAK axis

Abstract

Delphinidin is a flavonoid belonging to dietary anthocyanidin family that has been reported to possess diverse anti-tumoral activities. However, the effects of delphinidin on colorectal cancer (CRC) cells and the underlying mechanisms are not fully understood. Thus, we aimed to investigate the anti-cancer activity of delphinidin in CRC cells and the underlying molecular mechanisms. The effects of delphinidin on the viability, metastatic characteristics, signaling, and microRNA (miR) profile of human CRC cell lines used were analyzed. In vivo metastasis was also evaluated using xenograft animal models. Our findings showed that delphinidin (<100 μM) inhibited the colony formation of DLD-1, SW480, and SW620 cells, but non-significantly affected cell viability. Delphinidin also suppressed the migratory ability and invasiveness of the tested CRC cell lines, downregulated integrin αV/β3 expression, inhibited focal adhesion kinase (FAK)/Src/paxillin signaling, and interfered with cytoskeletal construction. Analysis of the miR expression profile revealed a number of miRs, particularly miR-204-3p, that were significantly upregulated and downregulated by delphinidin. Abolishing the expression of one upregulated miR, miR-204-3p, with an antagomir restored delphinidin-mediated inhibition of cell migration and invasiveness in DLD-1 cells as well as the αV/β3-integrin/FAK/Src axis. Delphinidin also inhibited the lung metastasis of DLD-1 cells in the xenograft animal model. Collectively, these results indicate that the migration and invasion of CRC cells are inhibited by delphinidin, and the mechanism may involve the upregulation of miR-204-3p and consequent suppression of the αV/β3-integrin/FAK axis. These findings suggest that delphinidin exerts anti-metastatic effects in CRC cells by inhibiting integrin/FAK signaling and indicate that miR-204-3p may play an important role in CRC metastasis.

Figure 1

Effects of delphinidin on the viability of human CRC cells. Cells were treated with delphinidin at serial concentrations for 24 h, and then the cell viability was assessed by MTT assay. Cell viability was presented as percentage of the DMSO control (0 μM) in mean ± standard deviation. No significant difference was observed among each treatment.

Figure 2

Delphinidin inhibited colony formation and cell adhesion of human CRC cells. Cells were cultured in soft agar or collagen-coated plates, treated with delphinidin (DEL) at serial concentrations for 24 h, and then subject to (A) colony formation assay or (B) cell adhesion assessment, respectively. Quantitative data was acquired from three independent experiments and presented as mean ± standard deviation. * and **, P < 0.05 and P < 0.01 as compared to the DMSO control (0 μM).

Figure 3

Delphinidin reduced cell migration and invasion of human CRC cells. Cells were cultured on membrane without or with Matrigel, treated with delphinidin (DEL) at serial concentrations for 24 h, and then subject to (A) transmigration assay or (B) invasion analysis. Quantitative data was acquired from three independent experiments and presented as mean ± standard deviation. **P < 0.01 as compared to the DMSO control (0 μM).

Figure 4

Delphinidin suppressed epithelial-to-mesenchymal transition in human CRC cells. Cells were treated with delphinidin (DEL) at serial concentrations for 24 h, then the cells were collected and lysed for the detection of epithelial and mesenchymal markers by using Western blotting. Quantitative data was acquired by using densitometric analysis from three independent experiments and presented as mean ± standard deviation. * and **P < 0.05 and P < 0.01 as compared to the DMSO control (0 μM).

Figure 5

Delphinidin reduced integrin expression and inhibited FAK cascade in DLD-1 cells. Cells were treated with delphinidin (DEL) at serial concentrations for 24 h, then the cells were collected and lysed for the detection of (A) integrins, (B) FAK signaling components, and (C) integrin-associated adaptor proteins by using Western blotting. Quantitative data was acquired by using densitometric analysis from three independent experiments and presented as mean ± standard deviation. * and **P < 0.05 and P < 0.01 as compared to the DMSO control (0 μM).

Figure 6

Involvement of miR-204-3p in the inhibited integrin/FAK cascade and the suppressed cell migration and invasion of human CRC cell DLD-1 in response to delphinidin. (A) Cells were treated with delphinidin (DEL) at 100 μM for 24 h, and then the cells were lysed for RNA extraction and microRNA expression profiling by using microarray analysis. MicroRNAs with up-regulation and down-regulation in response to delphinidin were labeled with red and green color, respectively. (B) Cells were transfected with control vector (NC) or anti-miR-204-3p, treated with delphinidin for 24 h, and then lysed for the assessment of integrin expression and FAK activation by Western blotting. (C) Cells were transfected with control vector or anti-miR-204-3p, treated with delphinidin for 24 h, and then subject to the transmigration and invasion assay. Quantitative data was acquired from three independent experiments and presented as mean ± standard deviation. * and **P < 0.05 and P < 0.01 as compared to the DMSO control (0 μM).

Figure 7

Delphinidin in vivo attenuated the metastasis of human CRC cell DLD-1 in xenograft mice. DLD-1 cells stably expressing luciferase were intraperitoneally injected into mice, and (A) the metastasized DLD-1 cells were detected by using IVIS image system after two weeks, then (B) the mice were sacrificed to acquire liver samples for phenotyping and weighing. Quantitative data was acquired by using photodensitometric analysis from three independent experiments and presented as mean ± standard deviation. P values as compared to normal or sham control were indicated.