Y. Miao

Epithelial mesenchymal transition correlates with CD24+CD44+ and CD133+ cells in pancreatic cancer

The epithelial-mesenchymal transition (EMT) has been linked to induction of a stem-cell like phenotype, characterized by altered cell surface marker expression and increased tumor formation. The aim of this study was to investigate whether EMT correlates with CD24+CD44+ and CD133+ cells in pancreatic cancer. The morphology of untreated and gemcitabine-treated SW1990 gemcitabine-resistant cells and normal SW1990 cells were compared. NF-κB p65 expression was knocked down using siRNA. Vimentin and E-cadherin expression were analyzed using western blotting, and CD24+CD44+, CD133+ cells were quantified by FACS. Additionally, immunohistochemistry of EMT-associated markers and stem cell-associated markers were performed in 41 cases of human pancreatic ductal adenocarcinoma. In SW1990 gemcitabine-resistant cells, gemcitabine induced a mesenchymal cell phenotype, expression of EMT-related molecular markers and increased CD24+CD44+ and CD133+ cells compared to untreated SW1990 gemcitabine-resistant and SW1990 cells. Knockdown of NF-κB p65 inhibited the ability of gemcitabine to increase the proportion of CD24+CD44+ or CD133+ cells and expression of EMT-related molecular markers. In human pancreatic ductal adenocarcinoma, significant correlations were observed between expression of the EMT-associated markers vimentin and E-cadherin, and stem cell-associated markers CD24, CD133 and CD44. This study demonstrated that EMT correlated with CD24+CD44+ and CD133+ cells in pancreatic cancer. This study also suggests that EMT may induce cancer stem-like cells in pancreatic cancer, with different degrees of EMT probability inducing different proportions of CD24+CD44+ and CD133+ cells.

Expression and promoter methylation analysis of ATP-binding cassette genes in pancreatic cancer

We investigated the relationship of ABCB1/MDR1, ABCC1/MRP1 and ABCG2/BCRP expression and promoter methylation with pancreatic cancer tumorigenesis and drug resistance. Gemcitabine-resistant pancreatic cancer cells, SW1990/GZ (33.3-fold increased resistance), were obtained by treating SW1990 cells with gemcitabine. The expression of ABCB1/MDR1, ABCC1/MRP1 and ABCG2/BCRP was determined by quantitative real-time RT-PCR in the cell lines, 3 normal pancreatic tissues, 15 human pancreatic cancer samples and 15 adjacent tissues. Promoter methylation was determined in cell lines by bisulfite genomic sequencing. ABCB1/MDR1, ABCC1/MRP1 and ABCG2/BCRP were upregulated in SW1990 and SW1990/GZ compared with normal pancreatic tissue, and expression in SW1990/GZ was significantly higher than in SW1990 cells. ABCB1/MDR1, ABCC1/MRP1 and ABCG2/BCRP were upregulated in pancreatic cancer tissues, compared to adjacent tissues. The ABCB1/MDR1, ABCC1/MRP1 and ABCG2/BCRP promoter were hypomethylated in all the cell lines. ABCB1/MDR1, ABCC1/MRP1 and ABCG2/BCRP expression correlated with pancreatic cancer tumorigenesis and drug resistance in a mechanism that is independent of promoter methylation.

Dendritic Cells Expressing a Combined PADRE/MUC4-Derived Polyepitope DNA Vaccine Induce Multiple Cytotoxic T-Cell Responses

The tumor-associated antigen, mucin4 (MUC4), is overexpressed on various epithelial malignancies, making it a potentially broadly applicable candidate vaccine therapy. In this paper, we report on the creation of a dendritic cell (DC)-based vaccine, using cells transduced with the universal DR-restricted Th helper epitope (PADRE) combined with human leukocyte antigen (HLA)-A1- and HLA-A2-restricted epitopes from MUC4 (rAd-pE-DCs). We examined this vaccines biologic characteristics and immune activity in vitro, finding that infection with the polyepitope adenovirus did not alter the typical morphology of mature DC and the typical markers of these cells (CD86, CD83, CD80, and HLA-DR) were highly expressed on rAd-pE-DCs. Lymphocytes primed with rAd-pE-DCs generated potent cytotoxic responses. By contrast, lymphocytes primed with a GFP-expressing adenovirus (rAd-GFP-DCs) or mock-transfected DCs were not cytotoxic. Transduction of DCs with an adenovirus encoding PADRE combined with HLA-A1- and HLA-A2-restricted epitopes may be a potential strategy for the immunotherapy of MUC4-associated tumors.

Identification of an HLA-A*0201-restrictive CTL epitope from MUC4 for applicable vaccine therapy

Recent research has indicated that MUC4 plays an important role in the development of many tumors and may prove useful as a novel cancer immunotherapy target. We aimed to identify HLA-A*0201-restrictive cytotoxic T lymphocyte (CTL) epitopes of the cancer-associated antigen MUC4. The MUC4 sequence was scanned for immunogenic peptides using HLA-binding prediction software. Dendritic cells (DCs) from peripheral blood mononuclear cells (PBMCs) were induced by cytokines. Five possible CTL epitopes were selected by software analysis, synthesized, and used to pulse mature DCs. The CD8+ T cells from PBMCs from an HLA-A*0201 healthy donor were stimulated with autologous MUC4-peptide-loaded DCs and expanded in vitro. T cell activation was assessed by ELISPOT, and cytotoxicity was determined by 51chromium (51Cr)-release assays. Our results show that CTLs induced by peptide P01204 could lyse T2 cells pulsed with peptide P01204 and HCT-116 cells (MUC4+, HLA-A2+). Compared with a control peptide, P01204 increased the number of IFN-γ producing T cells. Overall, these results suggest that P01204 is a novel HLA-A*0201-restrictive CTL epitope of the cancer-associated antigen MUC4. This will provide a foundation for the development of tumor-specific peptide vaccines.

Apoptosis and anergy of T cell induced by galectin-1 derived from pancreatic stellate cells in pancreatic cancer

Department of General Surgery, The First Affiliated Hospital of Nanjing Medical University, Nanjing, China Background: Galectin-1 is an immunoregulatory protein which was expressed in the activated pancreatic stellate cells (PSCs). Aim: This study was to investigate the relationships between galectin1 expressed in PSCs and T cells in pancreatic cancer. Methods: Immunohistochemistry was performed in 66 pancreatic cancer and 10 normal pancreas tissues to detect the galectin-1 and CD3 expression. The relationship between galectin-1, CD3 and clinicopathologic variables was assessed. Expression of galectin-1 in freshly isolated PSCs fromhumannormal pancreas (hNPSCs) and human pancreatic cancer tissues (hCaPSCs) were evaluated bywestern blot and quantitative RT-PCR. The apoptosis of Tcells coculturedwith these PSCswere detected byflowcytometry and Tcell IL-2, IL-4, IL-5 and INF-g production levels were quantified by ELISA. Results: Galectin-1 was higher expressed in pancreatic cancer compared to normal pancreas (P<0.05). Galectin-1 expression was gradually increased in well, moderately, poorly differentiated pancreatic cancer (P<0.05, separately), while the CD3 expression is decreased (P<0.05, P<0.01). Galectin-1 expression was associated with tumor size (p1⁄40.007), lymph node metastasis (p1⁄40.019), differentiation (p1⁄40.021) and UICC stage (p1⁄40.01). CD3 expression was associated with tumor differentiation (p1⁄40.009) and UICC stage (p1⁄40.018). Expression of Galectin-1 (p<0.001) and CD3 (p1⁄40.002) were associated with short patient survival. In vitro coculture experiments showed that, compared with hNPSCs, hCaPSCs inducedmore apoptosis of CD3 Tcells, more secretion of Th2 cytokines (IL4 and IL-5) and less secretion of Th1 cytokines (IL-2 and INF-g) (p<0.01). Conclusions: The high expression of galectin-1 in hCaPSCs enhance the apoptosis and anergy of T cells in pancreatic cancer, and thus may play a role in immune evasion of pancreatic cancer.