| BackgroundsColorectal cancer (CRC), as one of the most common gastrointestinal diseases, has become a major threat to human health with its morbidity stays in the second place and mortality in the third place. The mean reason leading to death is the uncontrolled metastasis and local recurrence; therefore people have conducted a lot of research on the mechanism of colorectal cancer metastasis. The major process of metastasis include excessive cell growth and angiogenesis in the primary tumor, the secretion collagenase which degrades extracellular matrix (ECM) and basement membrane, the detachment of tumor cells from the primary site, entering into the blood or lymph vessels, forming cancer embolus and migrating along with blood stream, migration of tumor cells into secondary sites and forming a new tumor through proliferation and angiogenesis. Cancer metastasis is a complex dynamic process which is composed of many relatively independent steps and each step involves a variety of molecular events.There are a lot of explanation for the mechanism of invasion and metastasis of tumor cells, and the tumor "homing" theory claim that some tumor cells highly express chemokine receptors, and target organs highly express chemokines, tumor cells can migrate to target organs under the induction of chemokines just as white blood cells. CXCL12 is an extracellular chemokine which binds to its cell surface G protein-coupled receptor CXCR4. CXCL12 and CXCR4 can form an coupled molecular and promote migration of white blood cells and hematopoietic stem cells. Recently, some study found that CXCR4 was highly expressed in colorectal cancer tissue and CXCL12 was highly expressed in its most common metastasis target organ such as lymph node and liver. Substance extracted from liver tissue has a strong chemotaxis on CXCR4 high-expressed CRC cells. This suggests that CXCR4/CXCL12 may participate in the process of invasion and metastasis of CRC, but the specific mechanism is still unclear. Through experiment in vitro, this study mainly explore the effects and mechanisms of CXCR4/CXCL12 axis on human CRC cell invasion and metastasis that may provide theoretical basis for the development of efficient anti-invasion and metastasis drugs.MethodsThe expression of CXCR4 in LOVO, HT-29, HCT-116, SW620, and SW480 CRC cells was detected by western blotting and Real time PCR assay. HT-29 cell was performed by Lipofectamine 2000 siRNA interference assay to down regulate the expression of CXCR4. The efficacy of CXCR4 siRNA in different time was evaluated by western blotting assays. Cells were exposed to CXCL12 and their growth activity were examined by CCK-8 and colonal forming assay. Cell migration and invasive ability was estimated by using wound healing and transwell chamber. Matrix metalloproteinase (MMPs) activity were analyzed by the gelatin zymography assay. The expression of molecules relevant to the PI3K/AKT/mTOR, MEK/ERK, and Wnt/β-catenin pathway were analyzed by the Western blotting assay. Nuclear and cytoplasmic β-catenin expressions were examined by using the NE-PER nuclear and cytoplasmic extraction reagents.ResultsAmong LOVO, HT-29, HCT-116, SW620, and SW480 cell lines, HT-29 was found highest expression of CXCR4 as determined both in the levels of mRNA and protein level. We thus selected HT-29 cells for the subsequent experiments. The CXCR4 protein levels were strongly reduced by 61.1%,74.8%, and 82.4%, respectively after 24,48 and 72 h siRNA transfection (P<0.05). HT-29 cells highly responded to CXCL12 stimulation, showing the increase of cell proliferation, invasion and migration through the Matrigeal. When the CXCR4 was knockdown, the effects of CXCL12 on proliferation, invasion and migration were lost. The secretion and activity of MMP-2 and MMP-9 were also stimulated in HT-29 cells exposure to CXCL12. However, HT-29 cells did not response to CXCL12 stimulation after the CXCR4 knockdown. CXCL12 can stimulate the transient phosphorylation of AKT, mTOR, and ERK which suggest that CXCR4/CXCL12 axis activated the PI3K/AKT/mTOR and MAPK/ERK signaling pathway. Significant changes of β-catenin expression were not seen as compared with their individual control bands in whole cell lysate and cystol lysate (P>0.05), while β-catenin was recruited significantly in nuclei by 76.9% compared with the vehicle-treated cell. In the CXCR4 knockdown cells, down-regulation of CXCR4 could block the CXCL12-induced β-catenin recruit in nuclei (P>0.05 vs. wild type HT-29 cells). CXCL12 can also promote the expression of β-catenin downstream protein such as CyclinD1, MMP-7 and survivin.ConclusionsCXCR4/CXCL12 axis promotes the proliferation, invasion and metastasis of CRC cells. It is mainly through promoting MMP-2, MMP-9 secretion and PI3K/AKT/mTOR, MEK/ERK and Wnt/β-catenin signaling pathway. So CXCR4/CXCL12 axis can be used as a potential anti-invasion and metastasis drug targets. Targeted therapy utilizing the CXCR4/CXCL12 axis could be an effective strategy for treatment of CRC. |
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