title: Antitumor effect of dendritic cells transfected with prostate-specific membrane antigen recombinant adenovirus on prostate cancer: An in vitro study.
abstract: The aim of the present study was to construct a dendritic cell (DC) vaccine transfected with a prostate-specific membrane antigen (PSMA) recombinant adenovirus and to observe the ability of the recombinant DCs in eliciting a PSMA-directed T‑cell response to prostate cancer (PCa) in vitro. Replication‑defective adenoviral vectors, were constructed using the Adxsi system. The virus titer was measured by TCID50 assay, and the expression of the PSMA gene was identified by polymerase chain reaction (PCR) generation of peripheral blood mononuclear cell (PBMC) derived DCs in vitro. In addition, a PE‑7AAD apoptosis and necrosis kit was applied to detect the survival of DCs at different MOI values to determine the optimal MOI. Morphological changes of DC were observed under a fluorescence microscope, expression of the PSMA protein was detected by western blotting 48 h after transfection, the expression of DC markers prior to and following transfection was detected by direct immunofluorescence, and the interleukin (IL)-12 concentration in the culture supernatant of the three groups was measured by ELISA. The antitumor effect of DC-stimulated cytotoxic T lymphocytes (CTLs) on different PCa cell lines was analyzed using a Cell Counting Kit‑8 assay. The optimal MOI value was determined to be 200. The PSMA protein was expressed in DCs, and the recombinant adenovirus did not impact the maturation and morphological changes of the DCs. The expression levels of the co-stimulatory molecules, CD80, CD83, CD86 and HLA‑DR, were significantly higher in the Ad‑PSMA‑DC group than in the other two groups (P<0.05). The concentration of IL‑12 in the supernatant of the Ad‑PSMA‑DC group (79.51±1.60 pg) was comparable with that of the Ad‑GFP‑DC group (not significantly different), and the two were significantly higher than the non‑transfection group (P<0.05). The optimal effector to target (E:T) ratio was determined to be 40:1. However, at the same E:T ratio, the cytotoxic activity of PSMA‑DC‑T against the LNCap cells was markedly stronger than its activity against the other target cells (DU145 and 22RV; P<0.05); furthermore, the the cytotoxic activity of PSMA-DC-T against the LNCap cells was significantly higher than the anti‑LNCap effect of DC‑T cells in other groups (P<0.05). In vitro experiments indicated that mature DCs transfected with Ad‑PSMA secreted high concentrations of IL‑12 and elicited potent antitumor immune responses targeting PSMA‑expressing cells by stimulating the cytotoxic activity of CTLs.
authors.affiliation: Cancer Research Institute, The First Affiliated Hospital, China Medical University, Shenyang, Liaoning 110001, P.R. China.
authors.forename: Fan-Dong
authors.initials: FD
authors.lastname: Meng
authors.affiliation: Cancer Research Institute, The First Affiliated Hospital, China Medical University, Shenyang, Liaoning 110001, P.R. China.
authors.forename: Shuai
authors.initials: S
authors.lastname: Wang
authors.affiliation: Cancer Research Institute, The First Affiliated Hospital, China Medical University, Shenyang, Liaoning 110001, P.R. China.
authors.forename: You-Hong
authors.initials: YH
authors.lastname: Jiang
authors.affiliation: Cancer Research Institute, The First Affiliated Hospital, China Medical University, Shenyang, Liaoning 110001, P.R. China.
authors.forename: Cheng-Guang
authors.initials: CG
authors.lastname: Sui
mesh_terms: D000256:Adenoviridae / Q000378:metabolism*; D000328:Adult; D000970:Antineoplastic Agents / Q000276:immunology*; D017209:Apoptosis; D015153:Blotting, Western; D048430:Cell Shape; D002478:Cells, Cultured; D003602:Cytotoxicity, Immunologic; D003713:Dendritic Cells / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D005822:Genetic Vectors / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D018664:Interleukin-12 / Q000378:metabolism; D008297:Male; D010957:Plasmids / Q000378:metabolism; D016133:Polymerase Chain Reaction; D017430:Prostate-Specific Antigen / Q000235:genetics / Q000378:metabolism*; D011471:Prostatic Neoplasms / Q000276:immunology / Q000473:pathology / Q000628:therapy*; D011995:Recombination, Genetic / Q000235:genetics*; D014162:Transfection
chemical_list: D000970:Antineoplastic Agents; D049452:Green Fluorescent Proteins; D018664:Interleukin-12; D017430:Prostate-Specific Antigen