ABSTRACT
Objective@#To isolate and identify exosomes from human serum, explore the feasibility of exosomal CEA for the diagnosis of colorectal cancer.@*Methods@#Retrospective study.64 cases with colorectal cancer patients(41 cases with normal CEA results and 23cases with high CEA results), 20 cases with benign colorectal diseases patients and 40 cases with healthy people of department of physical examination from October 2015 to December 2016 in Tongji Hospital of Tongji University. Exosomes were isolated from these serum using ExoQuick, and then identified by using transmission electron microscopy, and Western Blot for morphology and molecular phenotype.The serum level of CEA and exosomal CEA was measureed by enzyme linked immunosorbent assay (ELISA). The diagnostic efficacy of serum Exosomal CEA concentration in the colorectal cancer by using U test and the subject work characteristic curve (ROC). @*Results@#Exosomes in human serum was successfully extracted with ExoQuick kit.Exosomal CEA concentration in 64 cases with colorectal cancer patients (1 056.36-28 637.78)pg/ml was significantly higher than in cases with benign colorectal diseases patients (394.61-2 437.83)pg/ml and healthy controls(610.89-2 076.70)pg/ml(U=124.000, U=119.000, P<0.01). Exosomal CEA concentration in 41 colorectal cancer patients with normal serum CEA concentration(1 056.36-5 832.07)pg/ml was significantly higher than in benign colorectal diseases patients and healthy controls(U=113.000, U=119.000; P<0.01). ROC analysis of exosomal CEA yielded an AUC(area under the ROC curve)of 0.954(95% CI=0.919-0.988), which was higher than the serum CEA.The area under the ROC curve of serum Exosomal CEA concentration for colorectal cancer diagnosis is 0.954(95% CI=0.919-0.988), which is superior to the serum CEA concentration[0.636 (95% CI=0.531-0.742)]. @*Conclusions@#Isolation and detection of serum Exosomal CEA concentrations have a good diagnostic efficacy in colorectal cancer. It′s possible to be a marker for early diagnosis of colorectal cancer.(Chin J Lab Med, 2018, 41: 503-508)
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.
ABSTRACT
Objective@#To investigate the effect and mechanism of the antibacterial peptide cathelicidin secreted by tumor associated macrophages on the growth of colorectal cancer in mice.@*Methods@#Azoxymethane (AOM)/ dextran sodium sulfate (DSS) method was used to establish a mouse model of colitis associated colon cancer. To induce tumor formation, cathelicidin antibody, IgG antibody (positive control) or PBS (negative control) was respectively injected into mice once every 3 days and lasted one month. Then the pictures of mice colon were taken, and the numbers of tumor were counted and evaluated. Expressions of cathelicidin in tumor associated macrophages isolated from tumor and adjacent normal tissues of mice were examined by quantitative RT-PCR (qRT-PCR) and Western blot. Expressions of the tumor proliferating antigen Ki-67, macrophage marker CD68 and cathelicidin in tumor and non-tumor tissues were determined by immunohistochemistry analysis. Apoptosis of cells from tumor tissues was analyzed by using TdT-mediated dUTP nick-end labeling (TUNEL).@*Results@#In colon tumor tissues, cathlicidin strongly expressed in inflammatory cells (macrophages), but weakly expressed in tumor cells. The tumor number and size in mice injected with cathelicidin neutralizing antibody were 4.50±1.18 and (1.74±0.18) mm, respectively, significantly lower than 13.88±1.98 and (3.74±0.38) mm of mice injected with PBS (t=4.07, t=4.72; P< 0.01) and 15.25±1.82 and (3.40±0.36) mm of mice injected with IgG antibody (t=4.96, t=4.08; P<0.01). The Ki-67 positive rate of cells in tumor tissues of mice injected with cathelicidin neutralizing antibody was (28.20±3.44) %, significantly lower than (68.20±3.51) % of mice injected with PBS (t=8.135, P<0.01) and (69.20±3.41) % of mice injected with IgG antibody (t=8.461, P<0.01). Immunohistochemistry analyses showed that the expression of CD68 in tumor tissues of mice injected with cathelicidin antibody was significantly lower than that of mice injected with IgG antibody or PBS. TUNEL result showed that treatment with cathelicidin neutralizing antibody had negligible effect on the apoptosis of tumor cells.@*Conclusions@#Cathelicidin secreted by tumor associated macrophages can promote the growth of colorectal cancer in mice, and neutralizing cathelicidin activity can inhibit the growth and proliferation of colorectal cancer. Cathelicidin mediated promotion of colon cancer proliferation may mainly be exerted by recruiting inflammatory cells such as macrophages into the tumor microenvironment.