Inhibitory effects of DC vaccine sensitized with MAGE-3 combined with CpG ODN on bladder cancer BIU-87 cells / 中国肿瘤生物治疗杂志

@#Objective: To investigate the role of CpG ODN (CpG oligodeoxynucleotide) adjuvant in enhancing the anti-bladder cancer response induced by MAGE-3 (melanoma antigen gene -3) antigen and its molecular mechanism. Methods: Mononuclear cells were isolated from HLA-A2 type peripheral blood of healthy donors by Ficoll method to prepare mature DC by conventional means. DC surface markers were detected by flow cytometry. MTT assay was used to detect the promotion effect of DCs sensitized by different means (MAGE-3, CpGODN, MAGE-3+CpG ODN, irrelevant control antigen) on the proliferation of T lymphocytes and the killing effect of CTL on BIU-87 tumor cells. The tumor mass of nude mice bearing BIU-87 bladder cell xenograft were examined on Day 7 and 11 after CpG ODN+MAGE-3 sensitized DC treatment. The expression of Bcl-2/Bax protein was detected by Western blotting while the proliferation level of xenograft cells was detected by MTT assay. Results: DCs sensitized by CpG ODN combined with MAGE-3 antigenic peptides could promote the proliferation of T lymphocytes and significantly enhance the killing effect of CTLon target BIU-87 cells (P< 0.05). Compared with other sensitized DCs, in vivo experiments showed that 7 and 11 days after treatment, both the tumor volume and weight were significantly reduced (all P<0.05), and the proliferation ability of xenograft tumor was decreased (P<0.05). Compared with other sensitization means, CpG ODN+MAGE-3 especially exhibited obvious inhibitive effect on tumor growth on Day 11, and significantly promoted the proliferation of splenic monocytes of tumor bearing mice (P<0.01); moreover, Bcl-2 expression in xenograft tissues significantly decreased（P<0.01）while Bax expression significantly increased（P<0.05 or P<0.01）on Day 3 after treatment. Conclusion: CpG ODN can promote the inhibitory effect of MAGE-3 sensitized DC on bladder cancer BIU-87 cells, which will provide experimental basis for clinical application of DC vaccine in bladder cancer treatment.

A novel DNA vaccine constructed by heat shock protein 70 and melanoma antigen-encoding gene 3 against tumorigenesis.

Melanoma antigen-encoding gene 3 (MAGE-3) is an ideal candidate for a tumor vaccine although its potency need to be increased. Heat shock proteins (HSPs) represents a potential approach for increasing the potency of DNA vaccines. In the present study, a fusion DNA vaccine composed of Mycobacterium tuberculosis HSP70 and MAGE-3 was constructed and used to immunize C57BL/6 mice against B16 or B16-MAGE-3 tumor cells. The results show that the HSP70-MAGE-3 fusion DNA vaccine enhanced the frequency of MAGE-3-specific cytotoxic T-cells as compared to the MAGE-3 DNA vaccine or the HSP70/MAGE-3 cocktail DNA vaccine (P<0.05). In conclusion, the results indicate that the HSP70-MAGE-3 fusion DNA vaccine can strongly activate MAGE-3 specific cellular immunological reactions and thus significantly inhibit the growth of B16-MAGE-3 tumors, improving the survival of tumor-bearing mice, and the HSP70-MAGE-3 fusion DNA vaccine has a significant therapeutic effect on the tumors that express MAGE-3 antigens.

Identification and Immune response of Murine MAGE-3 Derived MHC-Ⅰ/MHC-ⅡRestricted Peptide Epitope / 中山大学学报(医学科学版)

[Objective] To design routine MAGE-3 derived MHC-I/MHC-II restricted peptide epitope, which containing CD4~+-CD8~+ T cell epitope peptides antigen. [Methods] The epitope peptides were made through computer simulation designing, and peptide epitopes qualification tests were performed after the synthesis of peptide antigen, ELISPOT and cell-toxic analysis were used to evaluate the proliferation ability and cytokine-release ability of peptide-stimulated T cell. [Results] The sequence of obtained MAGE-3 derived restricted epitope peptide was FITC-YEEYYPLIFLDNDQETMETSEEEEYEEYYPLIF, of which the purity ≥ 90% tested by high performance liquid chromatography. MAGE-3 epitope peptide antigen could induce T lymphocyte proliferation, and induce T lymphocyte to secret IFN-γ, which higher than that of the control group (49 vs. 6 spots/10~6, P≤0.05 ). MAGE-3 epitope peptide could induce cytotoxic T lymphocytes to cause 42% of MFC cell lysis rupture, higher than control group (P≤0.05). [Conclusion] CD4~+-CD8~+ T cell epitope MAGE-3 peptide antigen showed considerable immunological effect in vitro, and such a peptide antigen can work as therapeutic polypeptide vaccine for H-2K~K mice gastric cancer which express MAGE-3 antigen.

Expression of MAGE 3 Gene Products in Uterine Cervical Carcinoma / 대한산부인과학회잡지

OBJECTIVE: The human MAGE 3 gene encodes tumor specific antigens that are recognized by autologue cytotoxic T lymphocytes (CTL). The MAGE 3 gene is expressed not only in melanoma but in the other malignant tumors as well. There is, however, little information on the expression of the gene in uterine cervical carcinomas. The author thus studied the expression of the MAGE 3 gene products in uterine cervical carcinoma and discuss the possibility of specific immunologic diagnosis using MAGE 3 gene products. METHODS: The expression of MAGE 3 gene product in 17 normal tissues of the cervix, 32 cervical intraepithelial neoplasia (8 CIN I, 10 CIN II, 14 CIS), and 43 invasive cervical carcinomas was studied by immunohistochemistry using anti-MAGE 3 mAb 57B in paraffin sections RESULTS: No expression of MAGE 3 gene product was detected in normal cervical tissues and in cervical intraepithelial neoplasias. The expression of MAGE 3 gene product was detected in 30.2% (13/43) of invasive cervical carcinomas. The MAGE 3 gene product was stained as a cytoplasmic protein in cancer cells. No statistically significant differences were observed between MAGE 3 gene product expression status and clinicopathologic parameters. CONCLUSIONS: The MAGE 3 gene products was expressed in invasive cervical carcinoma tissues.

Expressions of MAGE-3, PCNA, p21, and p53 Proteins in the Hypopharyngeal Squamous Cell Carcinoma Cell Line (PNUH-12) Analysed by Bivariate Flow Cytometry

MAGE (melanoma antigen gene) is a tumor specific shared antigen, presented by HLA class I molecules, which is recognized by cytotoxic T lymphocytes. MAGE proteins are expressed in malignant tumor cells, in contrast to no expression in normal or benign tissues except for testis and placenta. MAGE might be a potential target for immunotherapy of malignant tumors. However, its biological aspects associated with cell cycle are not yet described. The flow cytometry is a useful tool for objective and quantitative analyses of heterogenous tumor cell population. To understand the status of MAGE related to cell cycle and its relationship with p53 as the G1 checkpoint regulator, p21, and PCNA as a proliferative index, we investigated expression of MAGE-3 protein, mutant p53, p21, and PCNA by flow cytometry and immunohistochemical stain. In addition, double stains for MAGE-3/p53, p53/PCNA, and p53/p21 were analysed with bivariate flow cytometry. DNA histograms using MAGE-3/PI (DNA) and p53/PI (DNA) were also analysed. The cell line (PNUH- 12) used for this study originated from a hypopharyngeal squamous cell carcinoma, which has point mutation (exon 7, C-->G) of p53. The expression rate of MAGE-3 was 83%, PCNA 85%, and p53 81%. No expression for p21 was identified. MAGE-3 was expressed in cytoplasm, while both PCNA and p53 were expressed in nuclei of tumor cells. With bivariate analyses, coexpression rates of MAGE-3/p53 and p53/PCNA were 0.96 and 0.97, respectively. Both MAGE-3 and p53 showed constantly high level throughout the cell cycle. These results suggest that expression of MAGE-3 and mutant p53 is not dependent on the cell cycle. p21 seems to be inactivated.

Relationship between Expressions of Tumor - Associated Antigen MAGE-3 and p53 Proteins during Cell Cycle by Bivariate Analysis of Flow Cytometry / 대한암학회지

PURPOSE: MAGE (melanoma antigen gene) is a tumor associated antigen, presented by HLA class I molecules, which is recognized by cytotoxic T lymphocytes. The expression of MAGE proteins are confined to malignant tumor tissues, except for the normal testis and placental tissues. Therefore, MAGE may be a potential target for immunotherapy of malignant tumors. However, biological aspects associated with the cell cycle are not yet described. MATERIALS AND METHODS: The material used for this study was a novel human squamous cell carcinoma cell line (PNUH-12) from the hypopharynx, which had one point mutation of 78th base, C to G, in exon 7 of p53 gene. To understand the role of MAGE in relation to cell cycle and its relationship with p53 as the Gl checkpoint regulator, the expressions of MAGE-3 protein and mvtant p53 (Mtp53) were accessed by flow cytometry and immunohistochemistry. Double stains for MAGE-3/Mtp53 was analyzed with bivariate flow cytometry. DNA histograms using MAGE-3/PI (DNA) and Mtp53/PI (DNA) were also analyzed. RESULTS: The expression rate of MAGE-3 and Mtp53 were 83% and 85%, respectively. MAGE-3 was expressed in cytoplasm, while M:p53 were expressed in the nuclei of the tumor cells on the immunohistochemical sections. With bivariate analyses, coexpression rate of MAGE-3/Mtp53 was 0.96, and MAGE-3 and Mtp53 constantly showed high levels throughout the cell cycle except Go. CONCLUSIONS: These results mean that (I) MAGE-3 might have yet unknown relationship with mutant p53, (2) expressions of MAGE-3 and Mtp53 are not dependent on the cell cycle in PNUH-12 hypopharyngeal squamous carcinoma cell line, and suggest that MAGE-3 might have a role as important as p53 during the development of malignant tumors.

Expression of MAGE 3 Gene Product in Squamous Cell Carcinomas of the Head and Neck / 대한이비인후과학회지

BACKGROUND AND OBJECTIVES: MAGE 3 gene may constitute a potential target for cancer immunotherapy since it is expressed in a variety of cancers but not in normal tissues except the testis. In this study, expression and intracellular location of MAGE 3 gene products were investigated using squamous cell carcinomas of the head and neck. MATERIALS AND METHODS: MAGE 3 protein expression was screened in 40 squamous cell carcinomas, 2 tumor lines, 20 benign diseases, and 20 normal tissues of the head and neck. Immunohistochemical staining with anti-MAGE 3 mAb 57B was conducted from fresh frozen specimens. A correlation between MAGE 3 expression and clinicopathological parameters was also evaluated. RESULTS: MAGE 3 gene product was detected in squamous cell carcinomas (18/40, 45%) and in tumor lines (2/2, 100%), but not in benign diseases and normal tissues. MAGE 3 gene product was identified as a cytoplasmic protein of cancer cells without staining in normal epithelia and stromal tissues coexisting adjacent to cancer cells. No significant correlation between MAGE 3 expression and clinicopathological parameters including tumor cell differentiation, age, gender, primary site, tumor stage, and metastasis was drawn. CONCLUSIONS: MAGE 3 antigen could represent a potential target for immunotherapy in head and neck squamous cell carcinomas.

EXPRESSION OF MAGE-3 GENE IN NON-SMALL CELL LUNG CANCER / 解放军医学杂志

To explore the possibility of using tumor antigen encoded by MAGE-3 gene as the target for immunotherapy for non-small cell lung cancer (NSCLC) patients, the expression of MAGE-3 mRNA in 3 human lung cancer cell lines and 56 NSCLC samples together with the adjacent normal lung samples was determined by RT-PCR. The results showed that all 3 human lung cancer cell lines expressed MAGE-3 mRNA; of the 56 NSCLC samples, 30 expressed MAGE-3 mRNA. The expression rate in squamous cell carcinoma was significantly higher than in adenocarcinoma, whereas none of the adjacent normal lung samples expressed MAGE-3 mRNA. This study suggests that MAGE-3 mRNA is expressed in a high percentage in NSCLC specimens, and its encoding tumor antigen may be the target for immunotherapy for NSCLC patients.