ABSTRACT
<p><b>OBJECTIVE</b>To investigate the effect and mechanism of B16F10-ESAT-6-gpi/IL-21 tumor cell vaccine on pulmonary metastasis in mouse model of melanoma.</p><p><b>METHODS</b>Twelve 8-week old female C57BL/6 mice were used in this study. The mice were injected with wild-type B16F10 cells through tail vein after immunization with B16F10-ESAT-6-gpi/IL-21 tumor cell vaccine, and the pulmonary metastasis was observed. The CD4(+) and CD8(+) T cells were isolated by magnetic activated cell sorting, and then used for the detection of CFSE/7-AAD cytotoxicity by flow cytometry. Serum from the mice immunized with tumor-cell vaccine was used to detect IFN-γ expression by ELISA. The expression of TGF-β2, ZEB1, E-cadherin, and N-cadherin of tumor tissues was detected by RT-PCR and immunofluorescence, respectively.</p><p><b>RESULTS</b>The mice vaccinated with B16F10-ESAT-6-gpi/IL-21 had significantly fewer nodules in the lung and lower lung weight [(285.8 ± 19.01) mg vs. (406.3 ± 27.12) mg], with lower levels of TGF-β2, ZEB1 and N-cadherin proteins but higher level of E-cadherin protein within the tumor tissue, as compared with the control mice. Meanwhile, the immunized mice had significantly increased CD8(+) T cell killing activity [(42.62 ± 3.465)% vs. (22.29 ± 1.804)%] and IFN-γ expression level [(55.200 ± 7.173) pg/ml vs. (6.435 ± 1.339) pg/ml] over the control mice.</p><p><b>CONCLUSIONS</b>The B16F10-ESAT-6-gpi/IL-21 vaccine can inhibit the metastasis of melanoma in the lung in vaccinated melanoma-bearing mice. This inhibitory effect is associated with CD8(+) T cell immune response and a higher level of IFN-γ, which may influence on the mesenchymal-epithelial transition of tumor cells.</p>
Subject(s)
Animals , Female , Humans , Mice , CD8-Positive T-Lymphocytes , Allergy and Immunology , Cadherins , Metabolism , Cancer Vaccines , Allergy and Immunology , Cell Line, Tumor , Epithelial-Mesenchymal Transition , Homeodomain Proteins , Metabolism , Interferon-gamma , Metabolism , Interleukins , Allergy and Immunology , Lung , Pathology , Lung Neoplasms , Metabolism , Melanoma , Metabolism , Pathology , Mice, Inbred C57BL , Neoplasm Transplantation , Organ Size , Transcription Factors , Metabolism , Transforming Growth Factor beta2 , Metabolism , Zinc Finger E-box-Binding Homeobox 1ABSTRACT
TLR4-MD-2 complex plays a key role in LPS recognition and its signal transduction. These steps are the vital elements of the host's defensive reaction. Studying the functional domain of TLR4 and MD-2 is very important to further understand the mechanism of LPS signal transduction. It was studied the interaction domain of TLR4 and MD-2 in living cells based on gene mutation, gene transfection and fluorescence resonance energy tramsfer(FRET) which is considered as one of the best methods used for intracellular protein-protein interaction study. CY-15P which was fused by CFP and YFP through 15 neutral amino acids was used as positive control, while co-expressed CFP and YFP proteins were used as negative control. The results showed that the ability of TLR4 binding to MD-2 decreased dramatically after the deletion of Glu~(24) ～ Met~(41) in N terminal of TLR4. Aggregation of TLR4 to LPS stimulation was observed, however, TLR4 without the Glu~(24)～ Met~(41) mutation did not aggregate. All these results indicated that TLR4 Glu~(24)～ Met~(41) might be the interaction domain of TLR4 binding to MD-2 and participate in the aggregation effect of TLR4 upon LPS stimulation.