| Background and ObjectiveEsophageal cancer is one of the most common malignant tumors of digestive tract in the world. China is one of the major regions which have the higher incidence of esophageal cancer.The incidence of it is among the top of the five nationwide various malignant tumors [1]. Since early signs of esophageal cancer is not obvious, the time of diagnosis has mostly entered the middle and late stage of it. Comprehensive treatment based on surgery has become a consensus throughout the world[2].Among the cases that the patients who were treated by surgical treatment, the overall survival rates were about 30% at 5 years[3,4].Early metastasis in therapy and recurrence after operation are important causes of its high mortality. However, the treatment outcome of esophageal cancer at present is not satisfied.So it is necessary to find better therapeutic scheme.As a highly selective multi-targeted oral small molecule protein kinase inhibitors, sunitinib not only inhibits the normal multiple signaling pathways,but also has the biology active of resistant to tumor angiogenesis and tumor cell proliferation[5,6].At the same time,it can inhibit various tyrosine kinase receptors,which include of vascular endothelial growth factor receptor(VEGFR),fins-like tyrosine kinase( FLT 3) receptor, inhibition of stem cell factor receptor(KIT), platelet-derived growth factor receptor(PDGFR)[7],etc.Vascular mimicry(VM) is an new model[8] which is independent of tumor microcirculation traditional tumor angiogenesis, that is recently found in certain malignancies. Previous studies found that VM phenomenon exists in esophageal cancer cell lines [9 ] and it emerges highly expressed genes associated. In this study, sunitinib act two different esophageal squamous cell carcinoma cell line like KYSE70( low grade differentiation squamous cell carcinoma) and KYSE450( well-differentiated squamous carcinoma), observed the different influence and unequal between the high and low grade differentiation about esophageal squamous cell proliferation, migration and formation of VM. To give the theoretical foundation for the further Methods1.Use RPMI-1640 containing Inactivated 10% fetal bovine serum and containing penicillin and streptomycin culture esophageal cancer cells KYSE70 and KYSE450 in vitro.2.Determined by MTT method to detect different concentrations(1. 50, 3., 6. 00 00, 12. 00, 24. 00 μ mol/L) of sunitinib different duration(24, 48, 72 h) on two different effects on cell proliferation.3.Flow cytometry to detect different concentrations(3, 6, 12 μ mol/L) of sunitinib role in cell apoptosis.4.Using three dimensional culture system simulated human body environment, to develop human esophageal cancer cells KYSE70 and KYSE450, and under the fluorescent groups observed under inverted microscope pipeline structure formation form and quantity.4.RT-PCR detection the expression of vm related gene m RNA of c- Met, mek, VE- cad, and MMP- 2 of cells before and after drug. Results1.Determined by MTT method showed that the inhibiting to KYSE450 and KYSE70 cell proliferation, will increased as the concentration of sunitib and time increased, and the inhibition has the time and the concentration dependence, statistically significant difference(P < 0.05);2.Flow cytometry results showed:(1) 3, 6, 12 mu mol/Lsunitib role respectively in KYSE70 cells and KYSE450 after 48 h, apoptosis rate(33.20 + 0.91) %,(46.00 + 1.35) %,(69.00 + 1.56) % and(35.00 + 0.21) %,(47.00 + 0.98) %,(74.30 + 1.28) %, while the control group, apoptosis rate was(1.30 + 0.26) % and(1.50 + 0.34) %.After statistical analysis showed that with the increase of concentration of sunitinib cell apoptosis rate also gradually rise, the concentration dependence of remarkable, and compared with the blank control group, the difference statistically significant(P < 0.05).The difference between two group(P = 0.053)。3. Three-dimensional culture system to observe the formation of tubular structures in each experimental group number: after about 12 hours KYSE70 and KYSE450 cells will formed typical vascular network structure, a single loop or multiple rings connected to grid. Sunitinib effects 24 hours after the two kinds of cells forming ability were significantly inhibited, annular structure fracture, is not completely closed circular and linear structure more, the more a great number of structure and concentration significantly less, statistically significant difference(P < 0.05);But no difference between the two different groups of cells(P = 0.054).4.RT-PCR assay the VM-related molecules m RNA expression group: after 48 hours sunitinib effect the expression of c- met, mek, VE- cad, MMP- 2 m RNA decreased obviously, the difference statistically significant(P < 0.05), and c- met with consistent mek loss, VE- cardin consistent with MMP- 2 drops;And no statistical difference between two groups of cells contrast(P = 0.06). Conclusion1. Sunitinib through multiple pathways effectively restrain high and low differentiation of esophageal squamous cancer cells and the expression of VE-Cad,c-met, by cutting MMP-2 expression and inhibiting angiogenesis, the formation of mimicry and other ways, and inhibition of cell proliferation and promote apoptosis effect into full play.2.Multitargets targeted therapy in esophageal squamous cell carcinomas have a further research prospects in comprehensive treatment. |
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