X H Pan1, W W Quan2, J L Wu2, W D Xiao1, Z J Sun2, D Li2. 1. School of Biomedical Sciences, Huaqiao University, Quanzhou 362021, China. 2. Department of Clinical Laboratory, Tongji Hospital, Tongji University School of Medicine, Shanghai 200065, China.
Abstract
Objective: To investigate the effect and molecular mechanism of antimicrobial peptide LL-37 secreted by stromal cells on the growth of colorectal cancer cells. Methods: Colorectal cancer cells SW480 or HCT116 were co-cultured with human macrophages using Transwell(®) maxicell inserts to mimic the tumor microenvironment. The effect of macrophages on the proliferation of colorectal cancer cells was detected by Bromodeoxyuridine and enzyme-linked immunosorbent assay (BrdU-ELISA). The expression of LL-37 mRNA and protein in macrophages and colorectal cancer cells was evaluated by reverse transcription-real-time quantitative PCR (RT-qPCR) and Western blot. LL-37 neutralizing antibody was added to abrogate the LL-37 activation. Additionally, macrophages were transfected with LL-37 shRNA plasmids to inhibit LL-37 expression. And then, the proliferation of colorectal cancer cells was observed. Furthermore, the growth-related signaling pathways were detected by Western blot. Results: The BrdU-ELISA results showed that the absorbance of SW480 cells increased from 1.072±0.097 to 5.121±0.407 after co-culture (P<0.001), and that of HCT116 cells increased from 1.229±0.073 to 3.495±0.228 (P<0.001). RT-qPCR results showed that LL-37 mRNA expression in macrophages significantly increased from 2.682±0.191 to 6.117±0.768 after co-incubation (P<0.05), whereas that in SW480 had no significant difference. Consistently the protein expression of LL-37 in macrophages was significantly increased by Western blot, while it did not change in SW480. The proliferation rate of SW480 cells was repressed by adding LL-37 neutralizing antibody or LL-37 shRNA plasmid. Furthermore, Western blot analysis showed that the expression of non-phosphorylated (activated) β-catenin and its target genes cyclin D1 as well as c-myc were distinctly increased in co-cultured SW480 cells, which could be reversed by anti-LL-37 antibodies. Conclusion: Macrophages promote the in vitro proliferation of colorectal cancer cells by enhancing the expression and secretion of antimicrobial peptides LL-37, and it seems that LL-37 activates colorectal cancer cells via Wnt/β-catenin pathway.
Objective: To investigate the effect and molecular mechanism of antimicrobial peptide LL-37 secreted by stromal cells on the growth of colorectal cancer cells. Methods:Colorectal cancer cells SW480 or HCT116 were co-cultured with human macrophages using Transwell(®) maxicell inserts to mimic the tumor microenvironment. The effect of macrophages on the proliferation of colorectal cancer cells was detected by Bromodeoxyuridine and enzyme-linked immunosorbent assay (BrdU-ELISA). The expression of LL-37 mRNA and protein in macrophages and colorectal cancer cells was evaluated by reverse transcription-real-time quantitative PCR (RT-qPCR) and Western blot. LL-37 neutralizing antibody was added to abrogate the LL-37 activation. Additionally, macrophages were transfected with LL-37 shRNA plasmids to inhibit LL-37 expression. And then, the proliferation of colorectal cancer cells was observed. Furthermore, the growth-related signaling pathways were detected by Western blot. Results: The BrdU-ELISA results showed that the absorbance of SW480 cells increased from 1.072±0.097 to 5.121±0.407 after co-culture (P<0.001), and that of HCT116 cells increased from 1.229±0.073 to 3.495±0.228 (P<0.001). RT-qPCR results showed that LL-37 mRNA expression in macrophages significantly increased from 2.682±0.191 to 6.117±0.768 after co-incubation (P<0.05), whereas that in SW480 had no significant difference. Consistently the protein expression of LL-37 in macrophages was significantly increased by Western blot, while it did not change in SW480. The proliferation rate of SW480 cells was repressed by adding LL-37 neutralizing antibody or LL-37 shRNA plasmid. Furthermore, Western blot analysis showed that the expression of non-phosphorylated (activated) β-catenin and its target genes cyclin D1 as well as c-myc were distinctly increased in co-cultured SW480 cells, which could be reversed by anti-LL-37 antibodies. Conclusion: Macrophages promote the in vitro proliferation of colorectal cancer cells by enhancing the expression and secretion of antimicrobial peptides LL-37, and it seems that LL-37 activates colorectal cancer cells via Wnt/β-catenin pathway.