The Role And Mechanism Of CXCR1/2in Proliferation,Migration And Invasion Of Gastric Cancer Cells

BackgroundGastric cancer is one of the most common causes of cancer-related deaths worldwide, and the data from International Agency for Research on Cancer show that the morbidity and mortality of gastric cancer ranges the forefront of the malignant tumors. Uncontrolled proliferation, invasion, metastasis and drug resistance are the major poor prognostic factors for gastric cancer. Therefore, improvement in the therapy of gastric cancer now depends on improving our knowledge of the complicated molecular mechanisms governing the progression and aggressiveness of the disease. Non-resolving inflammation plays a critical role in the development and process of gastric cancer. In the dialectical relationship between inflammation and tumor dynamic network scalability and robustness, chemokine receptors and their ligands are an important class of non-controlled inflammatory factors, and they express in some tumors and involve in tumor carcinogenesis, proliferation, growth, invasion, metastasis, and drug-resistance process.C-X-C chemokine receptor1/2(CXCR1/2), including CXCR1and CXCR2, belong to chemokine receptor families. Initial studies indicated that CXCR1/2express mainly on neutrophils and are originally characterized by their ability to regulate downstream protein targets (such as NF-kB, AP-1, MMP9, etc.) of signaling pathways and then induce chemotaxis of leukocytes, calcium ion flow, cell proliferation, movement, and regulation of angiogenesis. Recently, it was found that CXCR1/2are overexpressed in numerous solid tumors, and the studies revealed a close correlation with proliferation, angiogenesis, invasion, metastasis and drug resistance of the tumor. Although there have been some studies on CXCR1/2in several cancer types and there have been a few reports on expression of CXCR1/2in gastric carcinoma, the expression and functional roles of CXCR1/2in gastric adenocarcinoma progression remain controversial and unclear. ObjectiveThis study focused on an important class of non-resolving inflammation factors CXCR1/2to detect the expression of CXCR1/2in gastric cancer, explore its relationship with clinical and pathological features and study the changes of biological behavior of gastric cancer cells and their mechanisms after targeted blocking CXCR1/2and CXCR1gene silencing and over-expression. The project may provide some valuable experimental evidence for designing targeted drugs of CXCR1and/or CXCR2to treat gastric cancer.Methods1. Studying the correlation between the expression of CXCR1/2and clinicopathological factors, and and relevant indicators expression of regulating gastric cancer cell biological behaviors in patients with gastric carcinoma.(1)69primary and sporadic gastric adenocarcinoma tissue samples and their corresponding non-neoplastic mucosa specimens was retrieved, and we detected CXCR1/2expression by IHC and discussed its correlation with clinicopathological factors.(2) we analyzed the correlation between the expression of CXCR1/2and ERK1/2and AKT phosphorylation, and relevant indicators expression of the proliferation, growth and apoptosis (Bcl-2and Bax, CyclinDl, EGFR and Ki-67), angiogenesis(VEGF and CD34), invasion and metastasis (MMP-9, MMP-2, TIMP-2and E-cadherin), which are involved in the regulation of tumor proliferation, growth, angiogenesis, invasion and metastasis.2. Investigating the changes of gastric cancer cell biological behaviors and the mechanism of inhibiting CXCR1/2. The system of Repertaxin (a small molecule inhibitor of CXCR1/2activation) blocking CXCR1/2was established in vivo and in vitro. Repertaxin alone,5-FU alone, Repertaxin and5-FU in combination, and a control group were applied to gastric cancer cells and nude mice model.(1) Cell proliferation, cycle and apoptosis of gastric cancer cells were investigated by MTT assay, colony forming assay, cell cycle analysis and apoptosis assay. (2) Migration and invasion of gastric cancer cells were detected by wound-healing assay, cell migration and invasion assay.(3) It was detected that the mRNA level of the proliferation, growth, apoptosis(bcl-2, bax, CyclinDl, epidermal growth factor receptor EGFR and ki-67), angiogenesis(vascular endothelial growth factor VEGF), invasion and metastasis (matrix metallopeptidase MMP9, MMP2, tissue inhibitor of metalloproteinases TIMP2and E-cadherin) related indicators were by real-time-PCR.(4) It was assayed that the extracellular signal regulated protein kinase ERK1/2and AKT phosphorylation and expression of proliferation, growth, apoptosis(bcl-2, bax, CyclinDl, EGFR and ki-67), angiogenesis(VEGF), invasion and metastasis(MMP9, MMP2, TIMP2and E-cadherin) related indicators by Western-blotting.(5) Nude mice model:The growth of transplanted tumors was observated. After three weeks of the therapy, all mice were euthanized according to institutional guidelines, and local tumors were resected and analyzed. A part of every tumor tissue was fixed and processed for immunohistochemistry and TUNEL assay.3. Exploring the changes of gastric cancer cell biological behaviors and the mechanism of CXCR1gene silencing and over-expression.(1) The CXCR1expression was investigated by RT-PCR and Western-blotting in normal gastric mucosa cell line GES-1and gastric cancer cell lines(MKN28, MKN45, BGC823, SGC7901and AGS) to identify the cell lines of CXCR1over expression and that of CXCR1low expression.(2) Constructing interference vector (pYr-1.1-CXCR1-shRNA), over-expression plasmid vector (pIRES2-ZsGreenl-CXCR1) and negative control plasmid vector, and establishing the stable transfected cell lines of CXCR1silencing and over-expression.(3) Investigating off-target effects, for CXCR1interference plasmid vector, designing and constructing interference reverted plasmid vector (pIRES2-ZsGreenl-CXCR1-Mut), gastric cancer cells were doubly transfected to carry out functional rescue experiment.(4) Cell proliferation, cycle and apoptosis were investigated by MTT assay, colony forming assay, cell cycle analysis and apoptosis assay.(5) Migration and invasion of gastric cancer cells were detected by wound-healing assay, cell migration and invasion assay.(6) It was detected that the mRNA level of the proliferation, growth, apoptosis(bcl-2, bax, CyclinD1, EGFR and ki-67), angiogenesis (VEGF), invasion and metastasis(MMP9, MMP2, TIMP2and E-cadherin) related indicators were by real-time-PCR.(7) It was assayed that CXCR1, ERK1/2and AKT phosphorylation and expression of the proliferation, growth, apoptosis(bcl-2and bax, CyclinD1, EGFR and ki-67), angiogenesis(VEGF), invasion and metastasis(MMP9, MMP2, TIMP2and E-cadherin) related indicators by Western-blotting.(8) Nude mice model:The growth of transplanted tumors was observed. After three weeks of the therapy, all mice were euthanized according to institutional guidelines, and local tumors were resected and analyzed. A part of every tumor tissue was fixed and processed for HE and immunohistochemistry assay.Results4. CXCR1/2expression was associated with clinicopathological parameters of gastric cancer.(1) The CXCR1/2expression was significantly higher in gastric carcinoma compared to corresponding non-neoplastic mucosa tissues and was significantly associated with invasion, lymph node metastasis and TNM staging. However, no correlation was observed between CXCR1/2expression and gender, age and tumor differentiation.(2) Correlation analysis between CXCR1/2expression and the indicators expression of the phosphorylation(AKT, ERK, pAKT and pERK), proliferation, growth and apoptosis(Bcl-2, Bax, Cyclin Dl, EGFR and Ki-67), angiogenesis(VEGF and MVD), invasion and metastasis (MMP-9, MMP-2, TIMP-2and E-cadherin) by using the Spearman correlation test showed that CXCR1/2expression significantly correlated with pAKT, pERK, Cyclin D1, EGFR, Bcl-2, MVD, MMP-9and MMP-2, but CXCR1/2and AKT, ERK, Ki-67, Bax, VEGF, TIMP-2and E-cadherin expression had no significant correlation in gastric carcinoma.5. Repertaxin blocking CXCR1/2could change gastric cancer cell biological behaviors and enhance the effects of5-FU on gastric cancer in vitro and xenografts.(1) Repertaxin alone effectively induced gastric cancer cell cycle arrest, apoptosis, retarded proliferation and growth, and inhibited gastric cancer cell migration and invasion. The efficacy Repertaxin and5-FU in combination was much better than that of Repertaxin alone or5-FU alone.(2) Repertaxin repressed the phosphorylation of AKT and ERK1/2. The efficacy Repertaxin and5-FU in combination was much better than that of Repertaxin alone or5-FU alone.(3) Repertaxin decreased the expression of bcl-2, cyclin D1, EGFR, VEGF, MMP2and MMP9, and increased the level of bax, TIMP2and E-cadherin. The efficacy Repertaxin and5-FU in combination was much better than that of Repertaxin alone or5-FU alone.6. CXCR1silencing and over-expression could change gastric cancer cell biological behaviors.(1) The off-target effects didn’t appear in the CXCR1interference.(2) CXCR1silencing induced gastric cancer cell cycle arrest and apoptosis, retarded proliferation and growth, and inhibited gastric cancer cell migration and invasion. CXCR1over-expression promoted gastric cancer cell cycle, proliferation, growth, migration and invasion, and inhibited gastric cancer cell apoptosis.(3) CXCR1silencing decreased the phosphorylation level of AKT and ERK1/2.(4) CXCR1silencing inhibited the expression of bcl-2, CyclinDl, EGFR, VEGF, MMP2and MMP9, and promoted the expression of bax and E-cadherin.Conclusion:1. CXCR1/2was overexpressed in gastric cancer tissue and gastric cancer cells, and for the first time it was found that CXCR1/2expression was correlated positively with the proliferation, invasion, development and progression of gastric cancer.2. For the first time, it was found that CXCR1/2binding its ligands promoted the proliferation, invasion and metastasis of gastric cancer by inhibiting ERK1/2and AKT signaling pathway, and hereafter significantly decreasing the expression of bcl-2, cyclin D1, EGFR, VEGF, MMP2and MMP9, and increasing the level of bax, TIMP2and E-cadherin.