| BackgroundColon cancer is one of the most common human cancers,with approximately 600,000 new cases diagnosed worldwide each year,accounting for 9%of all cancers.Surgery is the only universally accepted treatment offering any hope of cure for colon cancer.About 85% of patients diagnosed with colon cancer can undergo surgical resection.Metastases to the regional lymph nodes are found in 40-70%of cases at the time of resection.About 85%of all recurrences that are designed to occur in colon cancer are evident within 3 years after surgical resection.The use of all currently available therapies(such as surgery,radiation, chemotherapy,or biotherapy) has only modest impact on overall survival of patients with advanced-stage,metastatic disease.Metastasis is still the major cause of death from cancer.The spread of cancer cells to regional lymph nodes through the lymphatic system is the first step in the dissemination of many solid cancers.Experiments in animal models demonstrated that tumor-associated lymphangiogenesis can be induced by overexpression of vascular endothelial growth factor C(VEGF-C),and that may be involved in subsequent tumor lymphatic metastasis.Moreover,the intervene targeting VEGF-C and its receptor VEGFR-3 signaling resulted in decreased tumor lymphangiogenesis and reduction of lymph node metastases.These data suggested the important roles of VEGF-C and its receptor VEGFR-3 in tumor lymphangiogenesis and lymph node metastasis.Although a number of molecules have been implicated in the lymphatic metastasis of cancer cells,the precise mechanisms determining the directional migration and invasion of colon cancer cells into specific organs remain to be demonstrated.It was recently reported that the migration and metastasis of tumor cells,similar to the recruitment of leukocytes,are strictly regulated by chemokines and their receptors. Different tumor cells express different CC and CXC chemokine receptors,and their corresponding ligands are often expressed at the sites of tumor spread.CXC chemokine receptor-4(CXCR4) is expressed on most tumor cell surface including colon carcinoma cell. On the other hand,Stromal-derived factor-1(SDF-1) is a unique ligand of the CXCR4, which promotes the chemotaxis of tumor cells and can be expressed in lymph nodes. Chemokine receptors are coupled to heterotrimeric G proteins and induce cancer cell movement towards a concentration gradient of the cognate chemokine.New evidence indicates that Chemokine receptors play a critical role in the homing of metastatic tumor cells.Lymphatic endothelial cells can produce the chemokine SDF-1α,which is a CXC chemokine receptor-4(CXCR4) specific ligand.CXCR4/SDF-1αinduced the chemotaxis effect between tumor cells and lymphatic endothelial cells.Thus,tumor cells were anchored in lymph nodes,and became invasive and aggressive,as lymph node tissues expressed highly SDF-1α.A recent study demonstrated that when breast cancer cell lines and primary breast cancer tissue expressed high levels of CXCR4,its ligand SDF-1αwas expressed correspondingly at high levels in the lung,liver and bone of the patients,which were usually metastatic sites.Neutralization of SDF-1/CXCR4 interactions leads to a marked inhibition of lymph node and lung metastases.The involvement of CXCR4 in metastasis is not limited to breast cancer,as CXCR4 is expressed in many other tumor cell lines that also respond to SDF-1.These findings support strongly the chemical chemotaxis effect of homing theory and suggest that CXCR4/SDF-1αplays a key role in the process of organ-selective metastasis.Therefore,chemokine receptors CXCR4 expressed on tumor cells represent potential targets for therapeutic interventions.Objective1.To investigate the expressional characteristics of SDF-1,CXCR4,VEGF-C, VEGFR-3,Podoplanin in human colon carcinomas and the relationship between which and clinical pathological parameters,to explore the significance of SDF-1,CXCR4,VEGF-C, VEGFR-3,Podoplanin in lymph mode metastasis of colon carcinomas.2.To investigate the effects of Chemokine receptor CXCR4 antisense-RNA transfected into HT-29 and HCT-116 cell line to the functional expression of VEGF-C mRNA and VEGF-C protein,and to observe the inhibitive effect of CXCR4 antisense-RNA on the cell growth kinetics and the invasive ability in colon cancer HT-29 as well as HCT-116 cells in vitro.3.To study the inhibitory effect of CXCR4 RNA interference(RNAi) on VEGF-C mRNA and VEGF-C protein expression in HT-29 and HCT-116 cell,and to assess the impacts on the Biological Behavior of HT-29 and HCT-116 cell in vitro.Methods1.Immuohistochemistry(SP) was used to detect the expression of SDF-1,CXCR4, VEGF-C,VEGFR-3 and Podoplanin in 70 samples and 12 adjacent noncancerous specimens of human colon cancer,the relationship between which and clinicpathological diameters was analyzed statistically.2.The eukaryotic expression vector pcDNA3.1(+) for CXCR4 Antisense-RNA was constructed to transfect colon cancer HT-29 and HCT-116 cell line.The expression of VEGF-C mRNA was detected by RT-PCR.The expression of VEGF-C protein was determined by Western blot.At the same time,cell growth kinetics was assessed by MTT assay,and in vitro invasive ability was assessed with Boyden chamber.3.Expression vector of CXCR4 gene targeting small interference RNA(siRNA) was constructed(pGenesil-CXCR4) and transfected into HT-29 and HCT-116 cells by lipofectamine,and the same cells transfected with unloaded vector were used as negative control,untransfected cells were used as empty control.The expression of VEGF-C mRNA and protein was identified by RT-PCR and Western blot.Cell growth kinetics was assessed by MTT method,and in vitro invasive ability was evaluated with Boyden chamber. Expression of CXCR4 gene labeled green fluorescent protein in HT-29 and HCT-116 cell was observed by fluorescent microscope.Results1.SDF-1 was overexpressed in 50 of 70 carcinoma tissues(71.4%),CXCR4 was overexpressed in 42 of 70 carcinoma tissues(60.0%),VEGF-C was overexpressed in 45 of 70(64.3%),VEGFR-3 was overexpressed in 34 of 70 carcinoma tissues(48.6%), Podoplanin was overexpressed in 38 of 70 carcinoma tissues(54.3%).The positive rates of SDF-1,CXCR4,VEGF-C,VEGFR-3 and Podoplanin expression in lymph node metastasis group was higher significantly than that in negative lymph node group respectively (88.6%vs.42.3%,75.0%vs.31.0%,79.5%vs.385%,63.4%vs.23.1%,72.7 vs.23.1%),(P＜0.01or P＜0.05). 2.The expression of SDF-1,CXCR4,VEGF-C,VEGFR-3 and Podoplanin were correlated with depth of invasion,lymph node metastasis(P＜0.01 or P＜0.05),and the expression of SDF-1 was correlated with TNM classification(P＜0.05).However,they were not related to age,sex,tumor size,histological type,cellular differentiation and distant metastasis(P＞0.05).3.There was positive correlation between the expression of SDF-1 and VEGF-C(r = 0.608,P＜0.05),between the expression of CXCR4 and VEGF-C(r = 0.750,P＜0.05), between the expression of VEGF-C and VEGFR-3(r = 0.312,P＜0.01),between the expression of Podoplanin and VEGF-C(r = 0.425,P＜0.01).4.CXCR4 Antisense-RNA recombinant was successfully constructed,after transfected into HT-29 and HCT-116 cells by lipofectamine,The expression of HT-29 VEGF-C mRNA in transfected group was decreased with the inhibition rate of 64.8%campared with control group,the difference was very remarkable(P＜0.01);The expression of HCT-116 VEGF-C protein in transfected group was decreased with the inhibition rate of 59.1%,the difference was very remarkable(P<0.01);The expression of HCT-116 VEGF-C mRNA in transfected group was decreased with the inhibition rate of 57.2%campared with control group,the difference was very remarkable(P＜0.01);The expression of HCT-116 VEGF-C protein in transfected group was decreased with the inhibition rate of 52.0%,the difference was very remarkable(P＜0.01).The mean invasive cell percentage of HT-29 cells in CXCR4 Antisense-RNA transfected group was 61.3±5.8%,which was far less than that of negative control group(93.7±7.8%) and empty control group(95.4±6.9%),(P＜0.05);The mean invasive cell percentage of HCT-116 cells in CXCR4 Antisense-RNA transfected group was 64.6±5.5%,which was far less than that of negative control group(96.4±7.8%) and empty control group(97.3±8.1%),(P＜0.05).5.After colon carcinoma cells were transfected with pGenesil-CXCR4,the expression of HT-29 cells VEGF-C mRNA and protein was suppressed with an inhibition rate of 86.6 %and 79.2%respectively,the difference was significantly compared with the empty vector transfected and untransfected HT-29 cells(P<0.01);the expression of HCT-116 cells VEGF-C mRNA and protein was suppressed with an inhibition rate of 77.4%and 71.9% respectively,the difference was significantly compared with the empty vector transfected and untransfected HCT-116 cells(P＜0.01).The growth rate of HT-29 and HCT-116 cell transfected with CXCR4 siRNA were obviously lower than those of untransfected control cell and empty vector transfected control cell.The mean invasive cell percentage of HT-29 cells in CXCR4 siRNA transfected group was 34.6±5.3%,which was far less than that of negative control group(93.9±6.4%) and empty control group(95.2±5.8%),(P＜0.05);The mean invasive cell percentage of HCT-116 cells in CXCR4 siRNA transfected group was 38.8±4.9%,which was far less than that of negative control group(97.2±6.5%) and empty control group(97.8±7.4%),(P＜0.05).The expression of CXCR4 gene labeled green fluorescent protein in HT-29 and HCT-116 cell was decreased significantly after RNA Interference.Conclusion1.The abnormal expression of SDF-1,CXCR4,VEGF-C,VEGFR-3,Podoplanin have an important significance in lymph node metastasis of colon carcinoma.We speculate that the mechanism of lymphatic invasion and metastasis in colon carcinoma are possiblely related with the CXCR4 activating induced by SDF-1,which could be promoted by VEGF-C,VEGFR-3 or Podoplanin.The combined examination of SDF-1,CXCR4, VEGF-C,VEGFR-3 and Podoplanin expression may be useful for predicting of the clinical stage,metastasis and prognosis in the patients with colon carcinoma.2.The expression of VEGF-C mRNA and protein in colon cancer HT-29 and HCT-116 cells was inhibited effectively by CXCR4 Antisense-RNA transfer,the growth and invasion of colon cancer cells in vitro were also declined significantly.These results suggest that the interaction of chemokine receptor CXCR4 and Vascular Endothelial Growth Factor-C(VEGF-C)may accelerate the abilities of proliferation,chemotaxis and invasion in colon cancer cells.The therapeutically research of CXCR4 antisense RNA by interfering in the interaction between CXCR4 and VEGF-C may be beneficial for colon carcinoma patients,especially for the patients with tendency of lymph node metastasis.3.The expression of VEGF-C mRNA and protein in HT-29 and HCT-116 cells transfected with CXCR4 siRNA were markedly inhibited,the growth rate and invasive ability of HT-29 and HCT-116 cell transfected with siRNA were also significantly decreased.Experiment results suggest that the inhibitory role of CXCR4 siRNA on the growth,proliferation,chemotaxis and invasion of colon cancer cell are specific,but the mechanism of CXCR4 and VEGF-C interaction needs further research. 4.Owing to there was a positive correlation between axillary lymphatic metastasis and the expression of CXCR4 and VEGF - C mRNA as well as VEGF - C protein in colon carcinomas.The functional status of CXCR4 could effect the chemotaxis of colon cancer cells,so it may be a new target to block lymphatic metastasis by interfering in the interaction between CXCR4 and VEGF-C.Our study would provide new theoretic base and experimental evidence for therapy of metastatic colon cancer.
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