| Objective:To investigatethe relationship of CXCR4,SDF-1 and VEGF-C in human laryngeal carcinoma and discuss the significance of the SDF-1/CXCR4 signalling Pathway in lymph node metastasis,in vitro to investigate the expression of CXCR4 in laryngeal carcinoma cell line Hep-2 and the interaction to advance the migration,invasion and proliferation of Hep-2,and to explore the crosstalk and possible mechanism between VEGF-C and SDF-1/CXCR4 single axis in the progression and lymphatic metastasis of laryngeal carcinoma,and further Investigate SDF-1/CXCR4 role and related mechanism in inducing target metastasis of laryngeal carcinoma cells to regional lymph nodes and homing.Methods1.The expression of CXCR4,VEGF-C and SDF-1 was detected by reverse transcription polymerase chain reaction(RT-PCR) and immunohistochemical SP in 10 cases of adjacent normal tissue,45 cases of laryngeal carcinoma tissue and cervical lymph node tissue.2.The expressions of CXCR4 mRNA and protein in human laryngeal carcinoma cell line Hep-2 was detected by RT-PCR and immunocytochemistry,the expression of VEGF-C was also evaluated before and after incubated with SDF-1 or CXCR4 antagonist(AMD3100).3.Methythiazolyltetrazolium(MTT) was used to analyze the effect of different concentrations of SDF-1 on the proliferation of Hep-2 cells. Transwell invasion chamber and matrigel were used to evaluate the effect of various concentrations of SDF-1 and CXCR4 antagonist(AMD3100) on the migration and invasion of Hep-2 cells.4.Transwell invasion chamber and matrigel were used to evaluate the effect of various concentrations of SDF-1 and AMD3100 on the migration and invasion of Hep-2 cells.Results1.Both in the mRNA and the level of proteins,The positive rate of the expression of CXCR4 and VEGF-C were 78%,67%and 78%,71%in primary tumor cells,were altogether higher than that in adjacent normal tissue(P＜0.05);The expreession of SDF-1 in metastasis lymph node tissue (100%) was higher than that in nonmetastasis lymph node tissue(33.3%,44.4%).There was significant correlation between CXCR4 and VEGF-C expression in the laryngeal carcinoma(P＜0.05);The expression of CXCR4 and VEGF-C in laryngeal carcinoma was significantly related to clinical TNM stage,tumor cell differentiation and lymph nodes metastasis;The expression of SDF-1 in lymph node showed significant relationship only with the state of lymph nodes metastasis(P＜0.05);2.Both of the mRNA and proteins level,The primary tumor cells and Hep-2 cells were overexpressed VEGF-C,and positive related to expression of CXCR4 and lymph node metastasis of laryngeal carcinoma(P＜0.05).VEGF-C expression in Hep-2 cells can be enhanced after incubated with SDF-1 for 24h and was inhibited by CXCR4 antagonist(AMD3100)(P＜0.05).3.Incubated for 24h,SDF-1(100 ng/ml) significantly enhanced the proliferation of Hep-2 cells compared with the control,1 ng/ml SDF-1 group,10 ng/ml SDF-1 group(0.585±0.051 vs 425±0.045,0.426±0.028,0.428±0.039,P＜0.05);Incubated for 48h,100ng/ml SDF-1 group(0.631±0.062) compared with the control(0.497±0.067),the proliferation was elevated significantly.,compared with the 100ng/ml SDF-1+1ug/ml AMD3100 group(0.459±0.048)与1ug/ml AMD3100 group(0.346±0.005),This enhancing effect of SDF-1 was significantly inhibited(P=0.0001)4.The levels of migration and invasion of the Hep-2 cells treated with 100 ng/ml SDF-1 were significantly higher than those in control,1ng/ml,10 ng/ml SDF-1(migration:268.7±24.7vs 48±7.2,65.7±30.4,147.7±37;invasion:40.5±4.5vs.0±1.0,10.7±2.7,17.7±2.0,P＜0.05),and was strongly inhibited by 1μg/ml AMD3100(3.0±1.0)(P＜0.05).Conclusion1.SDF-1 expressed Preferentially in nodes,common expression of CXCR4 in Primary tumor cells of the laryngeal carcinoma,and CXCR4 is the unique receptor to SDF-1,The CXCR4/SDF-1 signal pathway in laryngeal carcinoma was significantly related to lymph nodes metastasis, indicate that CXCR4 probably plays an important role in inducing target metastasis of cancer cells to regional lymph nodes.2.Expression of CXCR4 and VEGF-C were positive related in laryngeal carcinoma.SDF-1 can enhance its secretion of VEGF-C,and the effect can be inhibited by CXCR4 antagonist(AMD3 100).The SDF-1 /CXCR4 and VEGF-C may play cooperation role in target lymphatic metastasis of laryngeal carcinoma.3.SDF-1/CXCR4 singal aix can directly enhance Hep-2 cells proliferation,directional migration and invasion with a concentration-dependent model,which were inhibited by CXCR4 specific antagonists AMD3100。SDF-1/CXCR4 is in close relation to the occuring and developing of laryngeal carcinoma,Blocking of the SDF-1/CXCR4 will be a promising effective method to interfere the metastasis of laryngeal carcinoma and improve the survival rate.
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