Original articleDownregulation of the CXCR4/CXCL12 axis blocks the activation of the Wnt/β-catenin pathway in human colon cancer cells
Introduction
CXCL12, also as stromal cell-derived factor 1 (SDF-1), is known to function as a chemotactic factor for lymphocytes, dendritic cells and monocytes [1], [2]. Since Kin et al. [3] reported an increased level of chemokine CXCL12 and its receptor in patients with colon cancers, CXCL12/CXCR4 axis has been considered as a valuable marker of cancer metastasis [4], [5], [6]. In human body, CXCL12 is expressed by stroma cells in different tissues, such as liver, lung and heart [7]. High levels of CXCL12 were found in colon carcinoma-associated fibroblasts (CAF) cells in 36.8% of patients [8], [9]. It is hypothesized that elevated CXCL12 secrete from stroma cells, where it work as a telecrine or paracrine interaction factor between stroma cells and cancer cells [10]. CXCR4 is a 352-amino acid seven-span trans-membrane G protein coupled receptor that selectively binds ligand CXCL12. Overexpression of CXCR4 has been considered to associate with cancer invasion and metastasis [11]. In colorectal cancer patients with stage IV, the overall median survival with CXCR4 expressing tumors was 9 months compared to 23 months for CXCR4 negative cases [3]. CXCR4 expression was also found to associate with larger tumor size, advanced tumor-node-metastasis (TNM) stage and poor histological grade [12], [13], [14].
Although the CXCR4/CXCL12 axis was found aberrant expression in cancers, its roles in the development of cancers has not been completely defined and the mechanism underlying the processes of activation has largely remained unknown. We hypothesized that activation of the CXCR4/CXCL12 axis might play crucial roles in cancer invasion and metastasis. High expression of the CXCR4/CXCL12 axis might induce cancer growth and metastasis through activation of the Wnt/β-catenin pathways.
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Cell line and cell culture
Human colon cancer cell lines HT-29, LOVO, HCT-116, SW620 and SW480 were purchased from American Type Culture Collection (ATCC; Manassas, USA). All cell lines were maintained in RPMI-1640 medium, supplemented with 10% (v/v) heat-inactivated newborn calf serum, penicillin-streptomycin (100 IU/mL–100 μg/mL), 10 mM HEPES buffer and 2 mM glutamine, at 37 °C in a humid atmosphere containing 5% CO2 and 95% air.
RNA interference assay
Small interfering RNA (siRNA) against CXCR4 was designed by GenePharma (Shanghai, China). The
CXCR4 expression in human colon cancer cell lines
To select CXCR4 with highest expression, we examined five colon cancer cell lines (LOVO, HT-29, HCT-116, SW620, SW480) by the RT–PCR and western blotting assays. CXCR4 at basic levels were significant different in these cell lines. HT-29 was found highest expression of CXCR4 as determined both in the levels of mRNA and protein (Fig. 1A and B). We thus selected HT-29 cells for the subsequent experiments.
We performed the RNA interference assay to knockdown CXCR4 in HT-29 cells. The efficacy of
Discussion
Colon cancer is a multi-factorial disease characterized by excessive adenoma growth, invasion and metastasis. Although the morbidity is decreasing, it is still the second leading cause of deaths due to cancers worldwide [20]. Since most patients are diagnosed at advanced stage, they generally have a poor prognosis with median survival times of less than 1 year. Moreover, the recurrence rate is high even successful initial treatment [21]. Molecular mechanisms of how this cancer develop and spread
Acknowledgment
This project was supported by the grants from Natural Science Foundation of China (grant nos. 81173090, 91229113, 81373435) and from Beijing Science Foundation (7142017). We behold genuine thankfulness to Research Institute McGill University Health Centre for financial support.
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Zu-Hua Gao and Zhi-Yu Song contributed equally to this work.