Cancer-testis Antigen OY-TES-1 Expression and Immunogenicity in Hepatocellular Carcinoma.

Cancer testis (CT) antigens have received particular attention in cancer immunotherapy. OY-TES-1 is a member of CT antigens. This study was to evaluate OY-TES-1 expression and immunogenicity in hepatocelluar carcinoma (HCC). OY-TES-1 mRNA expression was detected in 56 HCC tissues and 5 normal liver tissues by reverse transcriptase PCR (RT-PCR). Of the 56 cases of HCC tissues tested, 37 cases had tumor and matched adjacent non-cancer tissues and were subjected to both RT-PCR and quantitative real-time PCR. OY-TES-1 protein was subsequently observed on a panel of tissue microarrays. Sera from patients were tested for OY-TES-1 antibody by ELISA. To identify OY-TES-1 capable of inducing cellular immune response, OY-TES-1 protein was used to sensitize dentritic cells and the cytotoxicity effect was measured in vitro. The results showed that OY-TES-1 mRNA was highly expressed in 41 of the 56 (73.21%) HCC tissues, whereas none in 5 normal liver tissues. OY-TES-1 mRNA was frequently expressed not only in HCC tissues (72.97%, 27/37), but also in paired adjacent non-cancer tissues (64.86%, 24/37). But the mean expression level of OY-TES-1 mRNA in HCC tissues was significantly higher than that in adjacent non-cancer tissues (0.76854 vs. 0.09834, P=0.021). Immunohistochemistry showed that OY-TES-1 protein expression was detected in 6 of the 49 cases of HCC tissues, and absent in 9 cases of normal liver and 6 cases of cirrhosis tissues. Seropositivity was detected in 10 of the 45 HCC patients, but not detected in 17 cirrhosis patients and 76 healthy donors. The specific cytotoxic T cells elicited by OY-TES-1 could kill HLA-A2+ HCC cell line which expressed OY-TES-1. The target lysis was mainly HLA class I -dependent and could be blocked by antibodies against monomorphic HLA class I but not HLA class II molecule. In summary, OY-TES-1 expression is up-regulated in HCC tissues and can be recognized by humoral and cellular responses, which suggests that OY-TES-1 is an attractive target for tumor immunotherapy in HCC.

High expression and frequently humoral immune response of melanoma-associated antigen D4 in glioma.

MAGE-D4 is a novel member of MAGE super-family. It has preliminarily been demonstrated that MAGE-D4 mRNA is not expressed in majority of normal tissues except for brain and ovary in which only trace amount of MAGE-D4 mRNA can be detected, but predominantly expressed in glioma. MAGE-D4 protein expression and its immunogenicity in glioma have not been elucidated well. This study was designed to analyze MAGE-D4 expression both at mRNA and protein level, characteristic of humoral immune response, and their relationships with glioma patients' clinicopathological parameters. Recombinant MAGE-D4 protein and antiserum were generated. Quantitative RT-PCR analysis revealed that MAGE-D4 mRNA expression was overall up-regulated in 41 glioma specimens compared with that in 14 normal brain tissues. Immunohistochemistry analysis showed that 78% (21/27) glioma tissues expressed MAGE-D4 protein, which was predominantly located in the cytoplasm of tumor cells, but absent in any neuroglia cell of normal brain tissues. ELISA analysis demonstrated that humoral response against MAGE-D4 was detected in 17% (7/41) of glioma patients' sera but not in 77 healthy donors. No apparent correlation was observed between the expression and immunogenicity of MAGE-D4 with clinicopathological parameters of glioma. In summary, these results indicate that MAGE-D4 is highly expressed in glioma and can develop specifically humoral response in glioma patients, which supports that it may be a promising biomarker for glioma diagnosis and immunotherapy.

MAGED4 expression in glioma and upregulation in glioma cell lines with 5-aza-2'-deoxycytidine treatment.

Melanoma-associated antigen (MAGE) family genes have been considered as potentially promising targets for anticancer immunotherapy. MAGED4 was originally identified as a glioma-specific antigen. Current knowledge about MAGED4 expression in glioma is only based on mRNA analysis and MAGED4 protein expression has not been elucidated. In the present study, we investigated this point and found that MAGED4 mRNA and protein were absent or very lowly expressed in various normal tissues and glioma cell line SHG44, but overexpressed in glioma cell lines A172,U251,U87-MG as well as glioma tissues, with significant heterogeneity. Furthermore, MAGED4 protein expression was positively correlated with the glioma type and grade. We also found that the expression of MAGED4 inversely correlated with the overall methylation status of the MAGED4 promoter CpG island. Furthermore, when SHG44 and A172 with higher methylation were treated with the DNA demethylating agent 5-aza-2'-deoxycytidine (5-AZA-CdR) reactivation of MAGED4 mRNA was mediated by significant demethylation in SHG44 instead of A172. However, 5-AZA-CdR treatment had no effect on MAGED4 protein in both SHG44 and A172 cells. In conclusion, MAGED4 is frequently and highly expressed in glioma and is partly regulated by DNA methylation. The results suggest that MAGED4 might be a promising target for glioma immunotherapy combined with 5-AZA-CdR to enhance its expression and eliminate intratumor heterogeneity.

Cancer testis antigen OY-TES-1 expression and serum immunogenicity in colorectal cancer: its relationship to clinicopathological parameters.

Cancer testis (CT) antigens are attractive targets for cancer immunotherapy because their expression is restricted in normal germ line tissues but frequently detected in variety of tumors. OY-TES-1 is identified as a member of CT antigens. Current knowledge about OY-TES-1 expression in colorectal cancer (CRC) is solely based on mRNA analysis. None of previous researches has studied OY-TES-1 at protein level. In this study, OY-TES-1 polyclonal antibody was generated. The expression of OY-TES-1 mRNA and protein was detected by RT-PCR and immunohistochemistry in 60 CRC and paired adjacent non-tumor tissues, 24 colorectal adenoma and 3 normal colon tissues, respectively. Sera from 73 CRC patients were also tested for OY-TES-1 antibody by ELISA. Our results showed that the frequency of OY-TES-1 mRNA expression was statistically higher in CRC (73.3%, 44/60) than that in adjacent non-tumor tissue (55.0%, 33/60) and colorectal adenoma (45.8%, 11/24). For the first time, OY-TES-1 protein expression was found in (43.3%, 26/60) of CRC tissues, but absent in any of adjacent non-tumor and colorectal adenoma tissues. No OY-TES-1 expression was found in normal colon by either RT-PCR or immunohistochemistry. Furthermore, OY-TES-1 protein expression was correlated with tumor invasion stage (P=0.004) and histological grade (P=0.040). Anti-OY-TES-1 antibody was detected in (9.6%, 7/73) of CRC patients' sera but not in 76 healthy donors. This finding demonstrates that OY-TES-1 is frequently expressed in CRC and is able to induce humoral immune response spontaneously in CRC patients, suggesting that it might be a promising immunotherapy target for CRC.

Knockdown of OY-TES-1 by RNAi causes cell cycle arrest and migration decrease in bone marrow-derived mesenchymal stem cells.

OY-TES-1 is a member of the CTA (cancer-testis antigen) group expressed in a variety of cancer and restrictedly expressed in adult normal tissues, except for testis. To determine whether MSCs (mesenchymal stem cells) express OY-TES-1 and its possible roles on MSCs, OY-TES-1 expression in MSCs isolated from human bone marrow was tested with RT (reverse transcription)-PCR, immunocytochemistry and Western blot. Using RNAi (RNA interference) technology, OY-TES-1 expression was knocked down followed by analysing cell viability, cell cycle, apoptosis and migration ability. MSCs expressed OY-TES-1 at both mRNA and protein levels. The down-regulation of OY-TES-1 expression in these MSCs caused cell growth inhibition, cell cycle arrest, apoptosis induction and migration ability attenuation. Through these primary results it was suggested that OY-TES-1 may influence the biological behaviour of MSCs.

[Construction of eukaryotic expression vector encoding ACRBP and its expression in hepatocarcinoma cells].

AIM: To construct the eukaryotic expression vector pEGFP-N1/ACRBP and stably express ACRBP in human hepatocarcinoma cells, providing functional clues for ACRBP. METHODS: A recombinant plasmid pMAL-C2/ACRBP was used as a template to amplify ACRBP cDNA. The PCR product was ligated into an eukaryotic expression vector pEGFP-N1 to construct a recombinant plasmid pEGFP-N1/ACRBP. Then the pEGFP-N1/ACRBP was transfected by Fugene HD into ACRBP-negative HepG2 cells. The stably transfected clones were selected by G418. RT-PCR and immunohistochemistry were used to detect the expression of ACRBP in HepG2 cells. RESULTS: The eukaryotic expression vector pEGFP-N1/ACRBP was constructed and confirmed by sequencing. The stably transfected HepG2 cells expressed ACRBP. CONCLUSION: The eukaryotic expression vector pEGFP-N1/ACRBP has been successfully constructed and transfected into HepG2 cells, resulting in stable expression of ACRBP.

Cytotoxicity, apoptosis induction, and mitotic arrest by a novel podophyllotoxin glucoside, 4DPG, in tumor cells.

AIM: To define the in vitro cytotoxic activities of 4-demethyl-picropodophyllotoxin 7'-O-beta-D-glucopyranoside (4DPG), a new podophyllotoxin glucoside. METHODS: Antiproliferation activity was measured in several tumor cell lines by using the microculture tetrazolium MTT assays. Cell cycle distribution was analyzed using flow cytometry and mitosis index assays. Furthermore, transmission electron microscopy, TUNEL, DNA agarose electrophoresis, and activated caspase-3 were used to analyze the induction of apoptotic cell death. Moreover, intracellular changes in the cytoskeleton were detected using immunocytochemistry. RESULTS: 4DPG effectively inhibited the proliferation of cancer cells (HeLa, CNE, SH-SY5Y, and K562 cell lines). For the K562 cell line, the antiproliferation effect of 4DPG was much more potent than that of etoposide (IC50 value: 7.79 x 10(-9) mol/L for 4DPG vs 2.23 x 10(-5) mol/L for etoposide). Further, 4DPG blocked the cell cycle in the mitotic phase. The induction of apoptosis and elevated levels of activated caspase-3 were confirmed in cells treated with 4DPG. The microtubule skeleton of HeLa cells was disrupted immediately after treatment with 4DPG. CONCLUSION: The cytotoxicity of 4DPG is due to its inhibition of the microtubule assembly of cancer cells at a low concentration, thus inducing apoptosis. These properties qualify 4DPG to be a potential antitumor drug.