ABSTRACT
BACKGROUND & OBJECTIVE: Angiogenesis plays an important role in growth and metastasis of tumors. Vascular endothelial growth factor (VEGF) is considered as a fundamental regulator for angiogenesis. This study was to construct a recombinant T7 phage vaccine expressing xenogenic VEGF on the capsid, and test its inhibitory effect on Lewis lung cancer cells in mice. METHODS: VEGF gene was cloned by reverse transcription-polymerase chain reaction (RT-PCR) from human lung cancer tissues, and inserted into phage using T7 Select10-3b kit to construct T7 Select10-3b_VEGF vaccine. The titer of prepared phage reached 1x10(13) pfu/ml. C57BL/6J mice were randomly divided into 3 groups: T7 Select10-3b_VEGF vaccine group (T7-VEGF), T7 phage (T7) group, normal saline (NS) group (10 mice/group). Each mouse was injected with Freundos adjuvant mixed with 1x10(12) pfu/200 microl T7 Select10-3b_VEGF, or T7, or normal saline once a week for 4 weeks. Lewis lung carcinoma model (LL/2) was established in C57BL/6J mice after 4-week immunization. Tumor growth and mouse's physical status were observed during immunization. Tumor weight and serum level of specific anti-VEGF antibody were measured by enzyme-linked immunosorbent assay (ELISA). Microvessel density (MVD) of tumors was detected by immunohistochemistry 14 days after the inoculation of tumor cells. RESULTS: Tumor weight of T7-VEGF vaccine group,T7 group, and NS group were (0.543+/-0.259)g, (0.982+/-0.359)g, (1.169+/-0.460)g, respectively. Tumor weight of T7-VEGF vaccine group was significantly lower than that of NS group (P<0.01). Serum anti-VEGF antibody level in T7-VEGF vaccine group was 1:1,000. MVD was significantly lower in T7-VEGF vaccine group than in NS group (8.5+/-0.8 vs 18.5+/-1.6, P<0.05). MVD in T7 group was 16.4+/-1.3. CONCLUSION: Recombinant T7 phage vaccine expressing xenogenic VEGF can break immunologic tolerance against self-VEGF and inhibit the growth of Lewis lung cancer cells.
Subject(s)
Bacteriophage T7/genetics , Cancer Vaccines/immunology , Carcinoma, Lewis Lung/pathology , Vascular Endothelial Growth Factor A/biosynthesis , Animals , Cancer Vaccines/genetics , Carcinoma, Lewis Lung/blood supply , Cell Line, Tumor , Cloning, Molecular , Female , Humans , Immunization , Lung Neoplasms/pathology , Mice , Mice, Inbred C57BL , Neovascularization, Pathologic/pathology , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Random Allocation , Recombinant Proteins/metabolism , Vascular Endothelial Growth Factor A/geneticsABSTRACT
A recombinant phage vaccine expressing EGFR on it's capsid was constructed and used to study the anti-tumor effect. The T7 phage display system was applied to display seven xenogenic (human, chicken) epidermal growth factor receptor extracellular domain fragments. The EGFR fragment was expressed as fused protein with 10B capsid on the surface of T7 phage. The T7-EGFR phage vaccines were injected into C57BL/6J mice, and then Lewis lung cancer cells were inoculated after 4 weeks immunization. The tumor tissue was excised and weighed after 10 days to evaluate the anti-tumor effect of each experimental group. The EGFR expression of the phage vaccine was verified by western-blot analysis. The A431 cells with high expressed EGFR was used to detect the anti-EGFR antibody by flow cytometry analysis. The results showed that the A431 cell can react with the serum obtained from the mice after three-week immunization. The experimental results confirmed that special EGFR antibody could be induced by the T7-EGFR phage vaccine. The T7-EGFR phage vaccine can elicit endogenous special EGFR antibody in mice and is capable of suppressing the tumor proliferation and retarding the growth of Lewis lung cancer. This research can be used to develop an anti-tumor vaccine for the target-therapy of EGFR(+) tumor.
Subject(s)
Carcinoma, Lewis Lung/prevention & control , ErbB Receptors/metabolism , Vaccines, Synthetic/immunology , Animals , Blotting, Western , Cell Line, Tumor , Chickens , Electrophoresis, Polyacrylamide Gel , ErbB Receptors/genetics , Flow Cytometry , Male , Mice , Mice, Inbred C57BL , Vaccines, Synthetic/genetics , Vaccines, Synthetic/metabolismABSTRACT
OBJECTIVE: To determine the changes in phenotype, proliferation activity and cytotoxicity of cytokine induced killer (CIK) cells after in vitro co-culturing with dendritic cells (DC), and then to investigate the auxiliary therapeutical effect of CIK cells after chemotherapy. METHODS: DC and CIK cells were generated by culturing prepheral blood mononuclear cells (PBMC) of healthy blood donors. Then the changes in the proliferation activity and phenotype of the cells were determined after DC and CIK cell co-culture. MTT assays were used to determine the cytotoxicity in vitro. The antitumor activity of DC and CIK cells were evaluated after chemotherapy in BALB/c nude mice bearing A549 lung cancer and BEL-7404 liver cancer respectively. RESULTS: DC and CIK cells promoted the antitumor effect of chemotherapy. Co-culture of DC with CIK cells produced a new cell population, whose cytotoxicity and proliferation activity were much higher than those of CIK cells. CONCLUSION: CIK cells co-cultured with DC are more potent than CIK cells alone in the anti-tumor effect.
