In vitro immune response of insulin-like growth factor 2 mRNA-binding protein 3 restricted cytotoxic T lymphocyte epitope from lung cancer immunotherapy target / 解剖学报

Objective Cancer immunotherapy is attractive for antigen-specific T cell-mediated anti-tumor therapy, especially in induction of cytotoxic T lymphocytes(CTL). In this report, we evaluated insulin-like growth factor 2 mRNA-binding protein 3(IGF2P3) restricted epitope-induced cytotoxic T lymphocytes effects in human lung cancer cells. Methods The human leukocyte antigen A2(HLA-A2) restricted epitope peptides of IGF2P3 were selected by NetCTL 1. 2, SYFPEITHI and IEDB software prediction. The binding affinity of the peptides to HLA-A02 molecules was evaluated by T2A2 cells binding assay. enzyme-linked immunosport(ELISPOT) assay was used to investigate the ability of the peptide to induce specific restricted cytotoxic T lymphocytes (CTL) and release of interferon γ(IFN-γ). The ability of the peptides to induce T cell response was investigated by carboxyfluorescein succinimidyl amino ester (CFSE) fluorescent staining. Results The candidate peptide P143, P199, P280, P409 and P515 showed moderate affinity toward HLA-A2 molecule. ELISPOT assay showed P409, P515 were able to induce specific CTL and higher levels of IFN-γ were released. The CTL induced by P409, P515 lysed target cells. Conclusion P409 and P515 have the potential for adoptive immunotherapy and can be used as candidate epitopes for new anti-tumor polypeptide immunotherapy vaccine. P409 and P515 can be used in peptide-based immunotherapy for lung cancer.

Wnt5a modulates vincristine resistance through PI3K/Akt/GSK3β signaling pathway in human ovarian carcinoma SKOV3/VCR cells / 生理学报

The aim of this study was to investigate the effect of Wnt5a on the vincristine (VCR) resistance in human ovarian carcinoma SKOV3 cells and its possible mechanism. The drug-resistant SKOV3/VCR cells were established by stepwise exposure to VCR, and then the SKOV3/VCR cells were stably transfected with specific shRNA interference plasmid vector targeting for Wnt5a. The mRNA expression level of Wnt5a was measured by RT-PCR. CCK-8 assay was used to detect the cell viability of SKOV3/VCR cells. The apoptosis was analyzed by flow cytometry. The protein expression levels of Wnt5a, MDR1, Survivin, β-catenin, Akt, p-Akt(S473), GSK3β and p-GSK3β(Ser9) were detected by Western blot. The result showed that SKOV3/VCR cells had significantly higher protein expression levels of Wnt5a, MDR1, Survivin and β-catenin, phosphorylation levels of Akt and GSK3β, and mRNA expression level of Wnt5a, compared with SKOV3 cells (P < 0.05). WNT5A gene silencing significantly increased the sensitivity of SKOV3/VCR cells to VCR, the IC of VCR being decreased from 38.412 to 9.283 mg/L (P < 0.05), synergistically enhanced VCR-induced apoptosis of SKOV3/VCR cells (P < 0.05), down-regulated the protein expression levels of MDR1, β-catenin and Survivin (P < 0.05), and inhibited phosphorylation of Akt and GSK3β (P < 0.05). Meanwhile, LY294002 (PI3K inhibitor) decreased the protein expression levels of MDR1, β-catenin and Survivin, as well as the phosphorylation levels of Akt and GSK3β in SKOV3/VCR cells (P < 0.05). These results suggest that WNT5A gene silencing reverses VCR resistance in SKOV3/VCR cells possibly through blocking the PI3K/Akt/GSK3β/β-catenin signaling pathway, and thus down-regulating the protein expression levels of MDR1 and Survivin.