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Archive > Volume 23 Issue 5 > 2016,23(5):633-639. DOI:10.3872/j.issn.1007-385X.2016.05.008 Prev Next
Basic Research
Inhibitory effect of recombinant adenovirus Ad5-CCL20 combined with hyperthermia on xenografts growth of colon carcinoma CT-26 cells in mice
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Abstract:
Objective:To explore the inhibitory effect of recombinant adenovirus Ad5-CCL20 combined with hyperthermia on the growth of mouse colon carcinoma CT-26 cells in mice transplanted tumor.Methods: Colon carcinoma CT-26 cells were transfected with recombinant adenovirus Ad5-CCL20, the expression of chemokine CCL20 was assessed by ELISA method and its chemotaxis to dendritic cell(DC) was detected by chemotaxis assays.Western blotting was performed to detect the expression of HSP70 and GP96 after CT-26 cells heated. DC were induced with protein exosomes from heat-shock CT-26 cells(heat group), control group and negative group as controls. Phenotypes of DC were detected by flow cytometry. The CT-26 cells were subcutaneous inoculated into BALB/c mice, then the BALB/c mice was immunotherapyed by intramuscular injection of recombinant adenovirus Ad5-CCL20 combined with treatment of heat (combined therapy group), and simultaneitily set hyperthermia alone heat group, Ad5-CCL20 alone group, Ad5-GFP group and control group. The tumor volumes and survival rate of tumor-bearing in each group was observed. ELISAPOT method was used to examine the IFN-γ production of spleen-derived T lymphocytes and the activity of cytotoxic T lymphocytes (CTL) was measured by lactate dehydrogenase (LDH) method. The infiltration of DC at the tumor site was examined with immunohistochemistry. Results:The CT-26 cells highly expressed chemokine CCL20 after the transfection of recombinant adenovirus Ad5-CCL20. CCL20 showed obviously chemotaxis activity both to iDC and mDC, especially iDC (P<0.05). Western blotting results showed that the expression of HSP70 and GP96 were detected in CT-26 cells after heat shock, but not in pure CT-26 cells. Compared with control and unheat group, flow cytometry demonstrated that the protein from heat-shocked CT-26 cells could significantly up-regulate expressions of CD80,CD86,CCR6 and MHC-Ⅱ molecules on the DC(P<0.01). Compared with all other groups, the growth of tumor in Ad5-CCL20/heat group was significantly inhibited (\[876.8±108.7\] vs \[2 862±85.52\], \[2 660±142.6\], \[2 447±100.6\], \[1 608±135.3\] mm3,P<0.01),and the survival time of tumor-bearing mice was prolonged(P<0.05). Results of ELISAPOT and LDH respectively showed that the CTL activity of spleen-derived T lymphocytes in Ad5-CCL20/heat group was higher as compared with all other groups (P<0.05).The immunohistochemical assay also showed that the infiltration proportion of CD11c+ DC was obviously increased in the Ad5-CCL20/heat group compared with Control group, Ad5-GFP group, Ad5-CCL20 group and Hyperthermia group(P<0.05). Conclusion: Recombinant adenovirus Ad5-CCL20 combined with hyperthermia significantly inhibited tumor growth and prolonged the survival time of tumor-bearing mice by recruiting and promoting DC maturation and antigen presentation, which could be a potential therapy for colon cancer.
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Project supported by the Medical Guide Program of Science and Technology Committee of Shanghai (No.124119a36 02)
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