| Currently, tumor biotherapy represented by tumor immunotherapy has become the fourth therapeutic pattern for tumor following surgery, chemotherapy and radiotherapy. Among the numerous known tumor-associated antigens, the cancer/testis antigens (CTA) displayed the sepicific expression pattern. They expressed in a variety of tumor tissues but not in normal tissues except the testicular germ cell, and occasionally expressed in the placenta. However, testis is immune privilege organ, which does not cause the specific immune response. Therefore, CTA was a suitable target antigen for specific tumor immunotherapy.As a sub-family of CTA, melanoma antigen gene (MAGE) is a big family which was firstly separated from melanoma cells. MAGE gene encodes tumor-sepicific antigenic peptides which were presented to CD8+T lymphocytes by HLA-I class I molecules, and therefore inducing tumor sepicific killing. Therefore, the study in-depth to MAGE gene will provide experiment data for the molecular diagnosis of tumors and tumor immunotherapy.In the present study, immunohistochemical staining was used to detect the protein expression of MAGE-A9, -A10, -A12 in normal breast tissues and breast cancer tissues, and the correlation between MAGE-A9, -A10, -A12 expressions and the clinical/biological factors of breast cancer was analyzed. MTT assay and colony formation assay were adopted to detect the effect of MAGE-A9 on breast cancer cells. The main research contents and results were shown as follows:Part I Expressions of MAGE-A9, -A10, -A12 in breast cancer tissues and their correlation with the clinical/biological factors of breast cancerObjective: Expressions of MAGE-A9, -A10, -A12 in normal breast tissues and breast cancer tissues and their correlation with the clinical/biological factors of breast cancer were investigated.Methods: Immunohistochemical staining was used to detect the protein expression of MAGE-A9, -A10, -A12 in 16 normal breast tissues and 60 breast cancer tissues, and the correlation between MAGE-A9, -A10, -A12 expressions and the clinical/biological factors of breast caner was analyzed.Results:1 MAGE-A9 was mainly expressed in cytoplasm and cell nuclears of spermatogonia and spermatocytes in normal testis tissues. In breast cancer tissues, MAGE-A9 was mainly expressed in cytoplasm, and part of MAGE-A9 protein was expressed in cell nuclears. MAGE-A9 protein was not expressed in 16 normal breast tissues. 28 of 60 breast cancer tissues showed MAGE-A9 protein expression, and the expression rate was 46.7%, suggesting that MAGE-A9 was tumor specific antigen.With increase expression rates of C-erbB2 (Z=-2.757, P=0.006) and ER-β(Z=-2.187, P=0.029), the MAGE-A9 protein expression rate was also increased. There were no significant differences between MAGE-A9 protein expression and patient's age (χ~2=0.804, P=0.370), pathological types (χ~2=0.000, P=1.000), histological grades (Z=-0.935, P=0.350), clinical stages (χ~2=0.000, P=1.000), tumor size (χ~2=0.741, P=0.690), metastasis of axillary lymph nodes (χ~2=0.268, P=0.605), ER-α(Z=-0.746, P=0.455), PR (Z=-0.031, P=0.975) and AIB-I (Z=-1.159, P=0.247).2 MAGE-A10 was mainly expressed in cytoplasm and cell nuclears of spermatogonia and spermatocytes in normal testis tissues. In breast cancer tissues, MAGE-A10 was mainly expressed in cytoplasm, and part of MAGE-A10 protein was expressed in cell nuclears. MAGE-A10 protein was not expressed in 16 normal breast tissues. 44 of 60 breast cancer tissues showed MAGE-A10 protein expression, and the expression rate was 73.33%, suggesting that MAGE-A10 was tumor specific antigen.There were no significant differences between MAGE-A10 protein expression and patient's age (χ~2=0.114, P=0.736), pathological types (χ~2=0.809, P=0.368), histological grades (Z=-0.772, P=0.440), clinical stages (χ~2=1.378, P=0.241), tumor size (χ~2=0.861, P=0.650), metastasis of axillary lymph nodes (χ~2=0.000, P=1.000), ER-α(Z=-1.362, P=0.173), C-erbB2 (Z=-0.919, P=0.358), ER-β(Z=-0.495, P=0.620), PR (Z=-1.136, P=0.256) and AIB-I (Z=-0.563, P=0.574).3 MAGE-A12 was maily expressed in cytoplasm and cell nuclears of spermatogonia and spermatocytes in normal testis tissues. In breast cancer tissues, MAGE-A12 was mainly expressed in cytoplasm, and part of MAGE-A12 protein was expressed in cell nuclears. MAGE-A12 protein was not expressed in 16 normal breast tissues. 56 of 60 breast cancer tissues showed MAGE-A12 protein expression, and the expression rate was 93.33%, suggesting that MAGE-A12 was tumor specific antigen.There were no significant differences between MAGE-A12 protein expression and patient's age (χ~2=0.357, P=0.550), pathological types (χ~2=1.702, P=0.192), histological grades (Z=-0.058, P=0.954), clinical stages (χ~2=0.000, P=1.000), tumor size (χ~2=0.117, P=0.943), metastasis of axillary lymph nodes (χ~2=0.000, P=1.000), ER-α(Z=-1.366, P=0.172), C-erbB2 (Z=-1.316, P=0.188), ER-β(Z=-1.496, P=0.135), PR (Z=-1.779, P=0.075) and AIB-I (Z=-0.322, P=0.748).4 MAGE-A9, -A10 and -A12 showed hyperdispersion in 60 breast caner tissues. 78.33% (47/60) breast cancer tissues showed at least one kind of MAGE expression. 25 of 60 (41.67%) breast cancer tissues showed MAGE-A9 and MAGE-A10 expression. 21 of 60 (35%) breast cancer tissues showed MAGE-A10 and MAGE-A12 expression. 15 of 60 (25%) breast cancer tissues showed MAGE-A9 and MAGE-A12 expression. 4 of 60 (23.33%) breast cancer tissues showed MAGE-A9, -A10 and -A12 expression. The expression rate of these three proteins was not significantly different in 60 breast cancer tissues (χ~2=0.000, P=1.000>0.05), suggesting that MAGE expression showed a aggregative characteristic.Conclusions: 1 MAGE-A9, -A10, -A12 maybe tumor specific antigens.2 With increase expression rates of C-erbB2 and ER-βin breast cancer tissues, MAGE-A9 expression rate was also increased, suggesting that MAGE-A9 may become an important marker for guiding the treatment and prognosis of breast cancer.3 MAGE expression showed a aggregative characteristic.Part II Effect of MAGE-A9 on proliferation of breast cancer cellsObjective: Effect of MAGE-A9 on proliferation of breast cancer cells was investigated.Methods: Cell culture, gene transfection, RT-PCR and Western-blot were used to detect the expressions of MAGE-A9 and C-erbB2 in human breast cancer line MCF-7 and MDA-MB-231. MTT assay and colony formation assay were used to detect the effect of MAGE-A9 on proliferation of breast cancer cells. Immunoprecipitation was used to detect the interaction between MAGE-A9 and C-erbB2.Results:1 Both MAGE-A9 and C-erbB2 were expressed in MCF-7 and MDA-MB-231 cells at gene transcription level, and C-erbB2 expression in MDA-MB-231 cells was lower than MCF-7 cells.2 MAGE-A9 was not expressed in MCF-7 and MDA-MB-231 cells at protein level. C-erbB2 was expressed in MCF-7 and MDA-MB-231 cells at protein level, and C-erbB2 expression in MDA-MB-231 cells was lower than MCF-7 cells.3 After MAGE-A9 transfection for 48 h, the cell viability of MCF-7 cells was significantly higher than the control group (P<0.05).4 The result of colony formation assay showed that after transfection of MAGE-A9 gene and G418 selection, the number of colonies of MCF-7 cells was 463±53.5, which was significantly higher the control group 117±2.3 (P<0.05).5 There was no direct correlation between MAGE-A9 and C-erbB2. Conclusions:1 Exogenous MAGE-A9 can increases proliferation of MCF-7 cells.2 Effect of MAGE-A9 on proliferative of MCF-7 cells may not be depend on direct interaction between MAGE-A9 and C-erbB2. |
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