| Gastric cancer(GC)is the third leading cause of cancer-related death in China.The incidence of GC was 30.77/100,000 in 2010,accounting for 13.08% of all cancers in China.Although targeted therapy and immunotherapy have developed rapidly,chemotherapy is still the most effective treatment for patients of advanced gastric cancer.Angiogenesis plays an important role in the development of cancer by promoting the survival and invasion of cancer cells.Vascular endothelial growth factor(VEGF)is one of the key factors in the angiogenesis process.VEGF and its receptors are targets for anti-angiogenic drugs.The addition of antiangiogenic drugs to chemotherapy is a promising approach,even though the improvement in survival is only 2 month.However,identifying anti-angiogenic therapy-sensitive patients remains challenging for oncologists.By binding to granulocyte colony-stimulating factor(G-CSF)receptor(GCSFR),G-CSF stimulates neutrophil production,differentiation and mobilization.Recombinant human G-CSF(rhG-CSF)is widely used for chemotherapy or radiotherapy-induced leukopenia.Recent studies in vitro have demonstrated that G-CSF can be produced by carcinoma cells and tumor stromal myofibroblasts and induce tumor proliferation and migration.In many cases,G-CSF-producing tumors are detected at an advanced stage and are associated with a poor prognosis.G-CSF expression has shown a strong correlation with resistance to anti-VEGF treatment.Treatment with anti-G-CSF monoclonal antibody results in reduced tumor angiogenesis and growth.Thus,G-CSF might be a potential biomarker for prognosis and anti-angiogenic drug efficacy.However,the expression of G-CSF and G-CSFR in GC and their effects remain to be elucidated.With serum and cancer tissue of GC patients,cultured GC cells and vascular endothelial cells selected as the research objects,the study was conducted through various methods including immunohistochemistry(IHC),enzyme-linked immunosorbent assay(ELISA),Western blotting,quantitative real-time polymerase chain reaction PCR(q RT-PCR),CCK8 cell proliferation assay,wound healing assay and transwell invasion assay et al.To explore the role of G-CSF and G-CSFR in the development of GC,the diagnosis and prognosis significance of serum G-CSF,the relationship between G-CSF/GCSFR expression and tumor biological behavior,as well as the effects of GCSF on GC cells and vascular endothelial cells were analyzed.The study aims to provide new methods for the diagnosis and prognostic evaluation of GC,and for the identifying patients who would benefit from anti-angiogenic treatment.Part one Serum G-CSF Level and Clinical Significance in Patients with Gastric CancerObjective: Aiming to explore the role of serum G-CSF in the diagnosis and prognosis of GC patients through detecting the level of serum G-CSF in untreated GC patients and analyzing its relationships with clinical pathologies and overall survival(OS).Methods:1.Serum were collected from 40 healthy controls and 60 GC patients,who did not receive any anti-tumor therapy.All patients were followed for survival.2.The level of G-CSF in serum was detected by enzyme linked immunosorbent assay(ELISA).3.The diagnostic sensitivity and specificity of serum G-CSF were defined,followed by the analysis on correlations between serum G-CSF and clinical characteristics and OS.Results:1.The levels of serum G-CSF in GC patients were significantly higher than those in healthy people.2.The increase of TNM stages leaded to the gradual decrease of serum G-CSF levels in GC patients.The level of serum G-CSF in stage I patients was significantly higher than that in stage IV patients.Serum G-CSF level was not correlated with other pathological factors such as age,sex,Lauren classification,tumor differentiation,depth of tumor invasion and lymph node metastasis.3.The sensitivity of G-CSF(58.3%)in the diagnosis of GC was similar to that of CA724(40.0%),and superior to that of CEA(36.7%)and CA199(26.7%).4.There was no correlation between serum G-CSF level and peripheral neutrophil count.5.There was no correlation between serum G-CSF level and the survival time of GC patients.Summary:1.The levels of serum G-CSF in GC patients were significantly higher than those in healthy people.2.The level of serum G-CSF in GC patients was gradually decreased from stage I to IV,indicating that serum G-CSF elevated in the early stage of GC.Part two The Expression and Clinical Significance of G-CSF and G-CSFR in Gastric Cancer TissuesObjective: To figure out the role of G-CSF and G-CSFR in GC development,we detected their expression in GC tissues and normal gastric mucosa,along with their correlations with clinical parameters and OS.Methods:1.Seventy GC tissue samples and thirty normal gastric mucosa tissues were collected and detected the expression of G-CSF and G-CSFR by immunohistochemistry(IHC).GC tissues were simultaneously detected VEGFA expression,and CD34 expression which used to calculate the microvascular density(MVD).2.A total of 40 paired GC tissues and matched normal mucosa were used to measure the G-CSF,G-CSFR and VEGF-A levels by ELISA.3.The difference in G-CSF/G-CSFR expression levels between GC tissues and normal gastric mucosa tissues were analyzed.4.Correlations between G-CSF/G-CSFR expression and clinical characteristics,VEGF-A levels,MVD and OS were analyzed.Results:1.IHC results showed that the high expression rate of G-CSFR in GC tissue was significantly higher than that in normal gastric mucosa tissue,but there was no significant difference in the high expression rate of G-CSF.There was significantly positive correlation between G-CSF expression and TNM stages,and between G-CSFR expression and lymph node metastasis.There was no relationship between G-CSF/G-CSFR expression and other clinical pathologies,such as gender,age,tumor differentiation,Lauren typing,and tumot invasion depth.The expression of G-CSF and G-CSFR in GC tissues showed weak correlation.2.The ELISA results showed that the level of G-CSFR in GC tissues was significantly higher than that in adjacent normal gastric mucosa tissues.G-CSF and G-CSFR were significantly correlated to each other in GC tissue.3.In GC tissue,G-CSFR expression,not G-CSF,was significantly correlated to VEGF-A expression.4.Patients with high G-CSFR expression had significantly higher MVD than those with low G-CSFR expression,but the MVD difference did not distinguished between patients with high and low G-CSF expression.5.IHC results showed that the patients with high G-CSF expression in their GC tissue had significantly shorter OS than the those with low G-CSF expression [median survival(95% CI): 25.6 months(18.7 months-32.5 months)vs.46.5 months(36.9 months-44.4 months,P=0.006].There was no significant difference in OS between high-and low-G-CSFR expression patients [median survival(95% CI): 34.7 months(30.7 months-38.6 months)vs 46.3 months(39.6 months-53.1 months),P = 0.053].6.In ELISA assay,patients were divided into 2 groups according to the cutoff value of 115.0pg/ml for G-CSF level or 469.0pg/ml for G-CSFR.The OS of patients with G-CSF level >115.0pg/ml group was significantly shorter than that of patients with ?115.0pg/ml group [median survival(95% CI): 26.3 months(17.1 months-35.5 months)vs.37.1 months(34.1 months-40.1 months),P=0.004].The OS of patients with G-CSFR level >469.0 pg/ml was significantly shorter than that of patients with ?469.0 pg/ml [median survival(95% CI)): 30.3 months(24.6 months-36.0 months)vs.39.1 months(38.1 months-39.8 months),P = 0.010].Summary:1.Compared to normal gastric mucosa,G-CSFR expression in GC tissue was significantly elevated while the G-CSF expression didn't change dramatically.There was a positive correlation between G-CSF and G-CSFR expression in GC tissues.2.G-CSF expression intensity was significantly correlated with TNM stages,and G-CSFR expression was significantly correlated with lymph node metastasis,suggesting that G-CSF and G-CSFR promote the progression of GC.3.G-CSFR expression was positively correlated to VEGF-A and MVD,suggesting that elevated G-CSFR expression should promote angiogenesis in GC.4.The expression of G-CSF and G-CSFR in GC were related to the survival time of patients,in which the survival time of patients was shortened when G-CSF or G-CSFR are highly expressed in GC tissue.Part three G-CSF Effects on the Biological Behavior of SGC-7901 and AGS Gastric Cancer Cells and Their MechanismObjective: To investigate the effects of G-CSF on the biological behavior of SGC-7901 and AGS GC cells and their mechanism.Methods:1.The expression of G-CSF and G-CSFR in four cell lines of GC and one normal gastric mucosa cell line was detected by q RT-PCR and Western Blotting.SGC-7901 and AGS cell lines were chosen for the following researches because of the high expression of G-CSFR.2.CCK-8 assay was used to study the proliferation effect of G-CSF on SGC-7901 and AGS cells.3.The expression of PCNA was detected by qRT-PCR and Western Blotting to explore the influence of G-CSF on its expression in SGC-7901 and AGS cells.4.The effect of G-CSF on migration and invasion of SGC-7901 and AGS cells were studied through wound healing assay and Transwell assay.5.The effect of G-CSF on G-CSFR expression was detected by qRT-PCR and Western Blotting.6.The expression of JAK2,STAT3 and phosphorylated STAT3 protein were detected by Western Blotting,to figure out the G-CSF effects on JAK2/ STAT3 signaling pathways in GC cells.Results:1.Cell proliferation and PCNA expression were markedly increased in SGC-7901 and AGS cells after G-CSF treatment.2.The migration and invasion of SGC-7901 and AGS cells were increased significantly after G-CSF treatment.3.G-CSF up-regulated the expression of G-CSFR in GC cells.4.G-CSF could up-regulate protein level of phospho-STAT3 and JAK2 in SGC-7901 and AGS cells,which could be inhibited by the anti-G-CSFR antibody.Summary:1.G-CSF could up-regulate the expression of G-CSFR in GC cells.2.Through binding to G-CSFR,G-CSF promoted proliferation,migration,and invasion of GC cells by activating the JAK2/STAT3 signaling pathway.Part four G-CSF Effects on the Biological behavior of Human Umbilical Vein Endothelial Cells(HUVEC)Objective: To investigate the effects of G-CSF on the biological behavior of HUVEC.Methods:1.The proliferation effect of G-CSF on HUVEC cells was studied through CCK-8 assay.2.The expression of PCNA was detected by qRT-PCR and Western Blotting,to explore the influence of G-CSF on its expression in HUVEC.3.The effects of G-CSF on migration and invasion of HUVEC cells were studied through wound healing assay and Transwell assay.4.G-CSF effects on the tube formation of HUVEC cells were studied by the tube formation assay.Results:1.Compared with the control group,cell proliferation and PCNA expression didn't change significantly in the HUVEC cells after G-CSF treatment.2.The migration and invasion of HUVEC cells were increased significantly after G-CSF treatment.3.After incubating of HUVECs with G-CSF,tube formation was gradually enhanced in a concentration-dependent manner.Summary:G-CSF can promote angiogenesis by promoting the migration,invasion and tube formation of HUVEC.Conclusions:1.The levels of serum G-CSF in GC patients are significantly higher than those in healthy people.The sensitivity of G-CSF in the diagnosis of GC is similar to that of CA724,and superior to that of CEA and CA199.From stage I to IV,the level of serum G-CSF in GC patients is gradually decreased.2.Compared to normal gastric mucosa,G-CSFR expression in GC tissue is significantly elevated,but G-CSF expression does not change significantly.The survival time of patients is shortened when G-CSF or G-CSFR is highly expressed in GC tissue.3.G-CSF expression is significantly correlated with TNM stages,and GCSFR expression is significantly associated with lymph node metastasis,VEGF-A and MVD.4.Through binding to G-CSFR,G-CSF promotes proliferation,migration,and invasion of GC cells by activating the JAK2/STAT3 signaling pathway.5.G-CSF can promote vascular endothelial cell migration,invasion,and tube formation. |
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