Sanae Nakajima

To examine the expression of the stromal cell-derived factor 1 (SDF-1)/CXCR4 receptor ligand system in pancreatic cancer cells and endothelial cells, we performed immunohistochemical analysis for 52 pancreatic cancer tissue samples with anti-CXCR4 antibody and reverse transcription-PCR analysis for CXCR4 and SDF-1 in five pancreatic cancer cell lines (AsPC-1, BxPC-3, CFPAC-1, HPAC, and PANC-1), an endothelial cell line (HUVEC), and eight pancreatic cancer tissues. We then performed cell migration assay on AsPC-1 cells, HUVECs, and CFPAC-1 cells in the presence of SDF-1 or MRC-9 fibroblast cells. Immunoreactive CXCR4 was found mainly in pancreatic cancer cells and endothelial cells of relatively large vessels around a tumorous lesion. The immunopositive ratio in the pancreatic cancer was 71.2%. There was no statistically significant correlation with clinicopathological features. SDF-1 mRNA expressions were detected in all pancreatic cancer tissues but not in pancreatic cancer cell lines and HUVECs; meanwhile, CXCR4 mRNA was detected in all pancreatic cancer tissues, cancer cell lines, and HUVECs. The results indicate that the paracrine mechanism is involved in the SDF-1/CXCR4 receptor ligand system in pancreatic cancer. In vitro studies demonstrated that SDF-1 significantly increased the migration ability of AsPC-1 and HUVECs, and these effects were inhibited by CXCR4 antagonist T22, and that the coculture system with MRC-9 also increased the migration ability of CFPAC-1 cells, and this effect was significantly inhibited by T22. Our results suggested that the SDF-1/CXCR4 receptor ligand system may have a possible role in the pancreatic cancer progression through tumor cell migration and angiogenesis.

PURPOSE: Loss of intercellular adhesion and increased cell motility promote tumor cell invasion. In the present study, E- and N-cadherin, members of the classical cadherin family, are investigated as inducers of epithelial-to-mesenchymal transition (EMT) that is thought to play a fundamental role during the early steps of invasion and metastasis of carcinomas. Cell growth factors are known to regulate cell adhesion molecules. The purpose of the study presented here was to investigate whether a gain in N-cadherin in pancreatic cancer is involved in the process of metastasis via EMT and whether its expression is affected by growth factors. EXPERIMENTAL DESIGN: We immunohistochemically examined the expression of N- and E-cadherins and vimentin, a mesenchymal marker, in pancreatic primary and metastatic tumors. Correlations among the expressions of N-cadherin, transforming growth factor (TGF)beta, and fibroblast growth factor 2 was evaluated in both tumors, and the induction of cadherin and vimentin by growth factors was examined in cultured cell lines. RESULTS: N-cadherin expression was observed in 13 of 30 primary tumors and in 8 of 15 metastatic tumors. N-cadherin expression correlated with neural invasion (P = 0.008), histological type (P = 0.043), fibroblast growth factor expression in primary tumors (P = 0.007), and TGF expression (P = 0.004) and vimentin (P = 0.01) in metastatic tumors. Vimentin, a mesenchymal marker, was observed in a few cancer cells of primary tumor but was substantially expressed in liver metastasis. TGF stimulated N-cadherin and vimentin protein expression and decreased E-cadherin expression of Panc-1 cells with morphological change. CONCLUSION: This study provided the morphological evidence of EMT in pancreatic carcinoma and revealed that overexpression of N-cadherin is involved in EMT and is affected by growth factors.

Expression of several members of the Bcl-2 family proteins was investigated by means of both immunohistochemical analysis in 30 invasive ductal adenocarcinomas and 23 intraductal papillary-mucinous tumors (IPMTs) and immunoblot analysis in 6 cancer tissues and 7 pancreatic cancer cell lines. We found that Bcl-2 was expressed in 23%, Bax in 53%, Bcl-X in 90%, and Mcl-1 in 90% of the invasive ductal adenocarcinomas. In intraductal papillary-mucinous adenocarcinomas, the expression rate of Bax was 44% and those of Bcl-XL and Mcl-1 were 88%; these values were higher than those for intraductal papillary-mucinous adenomas. Immunoblot analysis identified Bcl-XL as the predominant form of the Bcl-X protein in both pancreatic cancer tissues and cell lines, and demonstrated that both Bcl-XL and Mcl-1 protein levels were uniformly high in all cell lines. These results suggest that an imbalance between antiapoptosis proteins (such as Bcl-2, Bcl-XL, and Mcl-1) and proapoptotic proteins (such as Bax and Bcl-Xs) is involved in the distinctive biologic features of adenocarcinomas of the pancreas. Furthermore, predominantly high expressions of Bcl-XL and Mcl-1 in intraductal papillary-mucinous adenocarcinomas might be involved in the carcinogenesis in IPMT of the pancreas.

BACKGROUND: The reversion-inducing cysteine-rich protein with Kazal motifs (RECK) gene was initially isolated as a transformation suppressor gene. The RECK gene is expressed widely in normal organs but is undetectable in many tumor-derived cell lines. When artificially expressed in such cell lines, RECK negatively regulates at least matrix metalloprotease (MMP)-9, MMP-2, and MT1-MMP activation and suppresses the invasive and metastatic potentials of these cells. Clinical relevance of these observations, however, is yet to be established. The aim of this study was to examine RECK expression in pancreatic cancer, where intensive invasiveness and metastasis are frequently observed, and investigate its clinical significance. We also analyzed the correlation between RECK expression and MMP activation. METHODS: (a) RECK expression in surgically resected tissue samples of invasive ductal carcinomas of the pancreas (n = 50) was examined immunohistochemically, and its correlation with clinicopathological factors was analyzed; and (b) gelatin zymography was used for the detection of latent and activated forms of MMP-2 and MMP-9 in some of the tissue samples (n = 33). The gelatinase activity was quantified by densitometory, and the ratio of intensity of the active MMP-2 band to the total intensity of the pro- and active MMP-2 bands was evaluated as an indicator of MMP-2 activation. The MMP-9 activation was also studied. RESULTS: Among the 50 ductal carcinoma samples, 26 (52%) were stained positive for RECK. In the normal pancreas, both acinar and beta cells were stained positive, but ductal cells did not. Tumors with positive RECK staining were significantly less invasive as compared with RECK-negative tumors (P = 0.0438). Importantly, patients who had tumors with high RECK expression showed significantly better prognosis than those who had RECK-negative tumors (P = 0.0463, by Log-rank test). Zymographic analysis indicated significant inverse correlation between the level of RECK expression and extent of MMP-2 activation (P = 0.0374). CONCLUSIONS: Our findings support the hypothesis that the RECK protein has negative effects on the invasiveness of pancreatic cancer by inhibiting MMP-2 activation and suggest the potential value of RECK as a prognostic molecular marker for pancreatic cancer.

Retinoids during the embryonic period act as a mesenchymal inducer in many organs, including kidney, lung, central nervous system, and gut. Retinoic acid (RA) demonstrates insulinotropic effects in adult pancreas, but only a limited study has elucidated its role in pancreatic organogenesis. In this study, we have analyzed the existence of RA-signaling machinery in embryonic pancreas and evaluated its role using in vitro tissue culture experiments. Here we show the presence of endogenous retinaldehyde dehydrogenase 2 (RALDH2), the most effective RA-synthesizing enzyme, RA-binding proteins, and RA receptors (RARs) in embryonic pancreatic tissue. RALDH2 is expressed exclusively in the mesenchyme. Exogenously added all-trans-retinoic acid (atRA) in tissue culture experiments stimulated differentiation of endocrine and duct cells and promoted apoptotic cell death of acinar tissue. Furthermore, we demonstrate that atRA upregulates the PDX-1 expression. Taken together, our data suggest that atRA-mediated mesenchymal/epithelial interactions play an important role in determining the cell fate of epithelial cells via regulation of the PDX-1 gene, leading to the proper formation of the endocrine versus exocrine component during pancreatic organogenesis.