Enhanced levels of annexins in pancreatic carcinoma cells of Syrian hamsters and their intrapancreatic allografts.

Annexins are a family of calcium- and phospholipid-binding proteins related by amino acid sequence homology. Annexins I and II are substrates for protein tyrosine kinases. Recent investigations have revealed a possible involvement of annexins I and II in mitogenic signal transduction and cell proliferation. To investigate further the involvement of annexins in cell proliferation, we measured the levels of annexins I and II and the enzyme 3-phosphoglycerate kinase (PGK) (annexin II and PGK are components of the primer recognition protein complex) in normal Syrian hamster pancreas, three hamster pancreatic ductal carcinoma cell lines, and allografts of the three cell lines into hamster pancreas. All three carcinoma cell lines had 5-8-fold higher levels of annexin II compared to normal pancreas. An inverse relationship was seen between level of annexin II and the doubling time of the cell culture. In intrapancreatic allografts, annexin II levels were 3-6-fold higher than in normal pancreas. Annexin I levels were 2-3-fold higher in the allografts. Significant increases (5-6-fold) in specific activity of PGK were seen in all allografts examined. However, the level of PGK, as measured by immunoblotting, was not significantly altered. Immunohistochemical staining revealed heterogeneity in the reactivity of the antiannexin and anti-PGK antibodies with tumor cells. Strikingly, the reactivity and staining intensity were greater in the proliferating regions of the primary tumors and in the metastatic foci. Mitotic cells were either unstained or very weakly stained. We conclude from these findings that annexin II and PGK, as primer recognition proteins, may have a role in cell proliferation.     

Syndecan-1 expression is up-regulated in pancreatic but not in other gastrointestinal cancers

Syndecan-1 belongs to the syndecan family of cell surface transmembrane heparan-sulfate proteoglycans, which participate in cell proliferation, cell migration and cell-matrix interactions. Decreased expression of syndecan-1 has been observed in some gastrointestinal malignancies, and it is thought that high levels of syndecan-1 correlate with the maintenance of epithelial morphology and inhibition of invasiveness. In our study, we characterized the expression of syndecan-1 in normal, chronic pancreatitis and primary and metastatic human pancreatic cancer tissues, in cultured pancreatic cancer cell lines and in esophageal, gastric, colon, and liver cancers. Pancreatic cancer cell lines expressed syndecan-1 mRNA and protein at variable levels. In addition, these cells also released syndecan-1 into the culture medium. Pancreatic cancer tissues markedly over-expressed syndecan-1 mRNA in comparison with both chronic pancreatitis (2.4-fold increase, p < 0.01) and normal pancreatic samples (10.6-fold increase, p < 0.01). There was no difference in syndecan-1 mRNA expression between early and advanced tumors. By in situ hybridization and immunohistochemistry, syndecan-1 expression was evident at relatively low levels in the ductal cells and less frequently in acinar cells of the normal pancreas. In chronic pancreatitis, syndecan-1 was present at low to moderate levels in areas with atrophic acinar cells and ductular complexes. In contrast, in pancreatic cancer tissues, syndecan-1 was present at moderate to high levels in the majority of the cancer cells within the tumor mass and also in metastatic lesions of pancreatic tumors. Syndecan-1 mRNA levels in other gastrointestinal malignancies (esophageal, gastric, colon and liver cancers) were not significantly different from the levels observed in the corresponding normal samples. Together, our findings suggest that syndecan-1 expression by pancreatic cancer cells may be of importance in the pathobiology of this disorder and that its role in pancreatic cancer seems to be different from that in other gastrointestinal malignancies.     

Cell lineage markers in human pancreatic cancer

The normal pancreas consists of three major cell types or lineages that share a common embryologic origin from pluripotent endodermal precursors. The type of cell that undergoes neoplastic transformation to form a pancreatic carcinoma is controversial and may influence the phenotype and biologic behavior of the tumor. In this study, immunohistologic techniques were used to determine the cell lineage differentiation expressed in 29 primary exocrine pancreatic adenocarcinomas, five metastatic exocrine pancreatic adenocarcinomas, and five islet cell neoplasma. Specimens of normal pancreas and chronic pancreatitis were used for comparison. The cell lineage markers consisted of monoclonal and polyclonal antibodies against trypsin and lipase (acinar cells); secretory component, carbonic anhydrase II, and pancreatic cancer mucin SPan-1 (ductal cells); and chromogranin-A and somatostatin (islet cells). The expression of carcinoembryonic antigen (CEA) and lysozyme were also determined. This collection of markers allowed the differentiation between acinar, ductal, and islet cells of normal pancreas and chronic pancreatitis specimens. The expression of cell lineage markers in islet cell tumors was homogeneous and restricted to chromogranin-A. In contrast, the expression of these markers in primary and metastatic exocrine pancreatic adenocarcinomas was variable. Reactivity with monoclonal anti-CEA was absent in normal pancreas, and was present in 83% of chronic pancreatitis specimens as well as 90% of exocrine pancreatic adenocarcinomas. In addition, lysozyme reactivity was absent in normal pancreas; however, lysozyme was expressed in one case of chronic pancreatitis, 17 cases of primary carcinoma, and three cases of metastatic carcinoma. These findings support the concept that the original transformed cell type in many pancreatic exocrine carcinomas resemble endodermal "stem cells" that retain the capability of differentiation along more than one cell lineage pathway.     

Annexin II expression is reduced or lost in prostate cancer cells and its re-expression inhibits prostate cancer cell migration

While studying Bim, a BH3-only proapoptotic protein, we identified an approximately 36 kDa protein, which was abundantly expressed in all five strains of primary normal human prostate (NHP) epithelial cells but significantly reduced or lost in seven prostate cancer cell lines. The approximately 36 kDa protein was subsequently identified as annexin II by proteomic approach and confirmed by Western blotting using an annexin II-specific antibody. Conventional and 2D SDS-PAGE, together with Western blotting, also revealed reduced or lost expression of annexin I in prostate cancer cells. Subcellular localization studies revealed that in NHP cells, annexin II was distributed both in the cytosol and underneath the plasma membrane, but not on the cell surface. Prostate cancer cells showed reduced levels as well as altered expression patterns of annexin II. Since annexins play important roles in maintaining Ca(2+) homeostasis and regulating the cytoskeleton and cell motility, we hypothesized that the reduced or lost expression of annexin I/II might promote certain aggressive phenotypes of prostate cancer cells. In subsequent experiments, we indeed observed that restoration of annexin II expression inhibited the migration of the transfected prostate cancer cells without affecting cell proliferation or apoptosis. Hence, our results suggest that annexin II, and, likely, annexin I, may be endogenous suppressors of prostate cancer cell migration and their reduced or lost expression may contribute to prostate cancer development and progression.     

Angiotensin II activates MAP kinase and NF-kappaB through angiotensin II type I receptor in human pancreatic cancer cells

Pancreatic ductal cancer has higher angiotensin II concentrations compared with normal pancreas or other solid tumors. This study examined angiotensin II type 1 (AT1) receptor expression and the role of angiotensin II in proliferation and survival of human pancreatic cancer cells. All three pancreatic cancer cell lines studied, from well to poorly-differentiated types, HPAF-II, AsPC-1, and Panc-1, showed strong expression of AT1 receptor. In contrast, HT-29 human colon cancer cells showed extremely weak expression. Angiotensin II stimulated the growth of pancreatic cancer cells through MAP kinase activation but had no significant effect on proliferation of HT-29 colon cancer cells. In addition, angiotensin II significantly prevented cisplatin (CDDP)-induced apoptosis through NF-kappaB activation and the subsequent production of anti-apoptotic molecules, including survivin and Bcl-XL, in pancreatic cancer cells. These findings suggest that angiotensin II plays a role in the growth and chemoresistance of AT1-positive pancreatic cancer cells through its action as a potent mitogen and anti-apoptotic molecule.     

Heparanase expression in primary and metastatic pancreatic cancer.

The human endoglycosidase heparanase (hpa) degrades heparan-sulfate proteoglycans, which constitute prominent components of basement membranes and extracellular matrix. Due to the critical function of hpa in cancer cell invasion and metastasis, we have analyzed the expression of hpa in human primary and metastatic pancreatic cancer as well as in the normal pancreas and in chronic pancreatic inflammation. By real-time quantitative PCR, there was a 7.9- and 30.2-fold increase of hpa mRNA in chronic pancreatitis and pancreatic cancer tissue samples, respectively, in comparison with normal pancreatic tissues. There was a significant correlation between enhanced hpa mRNA expression and shorter postoperative patient survival. hpa mRNA and protein localized in the cancer cells of primary and metastatic pancreatic cancer, with a preferentially higher expression at the primary tumor site. Cultured pancreatic cancer cells transfected with a full-length hpa construct displayed enhanced invasiveness in an invasion chamber assay. These results suggest that hpa overexpression in human pancreatic cancers facilitates cancer cell invasion, thereby enhancing the metastatic potential of the tumors.     

Loss of methylthioadenosine phosphorylase and elevated ornithine decarboxylase is common in pancreatic cancer.

PURPOSE: Loss of the methylthioadenosine phosphorylase (MTAP) gene at 9p21 is observed frequently in a variety of human cancers. We have shown previously that MTAP can act as a tumor suppressor gene and that its tumor suppressor function is related to its effect on polyamine homeostasis. Ornithine decarboxylase is a key enzyme in the regulation of polyamine metabolism. The aim of this study is to analyze MTAP and ornithine decarboxylase (ODC) expression in primary pancreatic tumor specimens. EXPERIMENTAL DESIGN: We measured MTAP and ODC activity in protein extracts derived from 30 surgically resected tumor samples and eight normal pancreas samples. In a subset of six samples, we also examined MTAP DNA using interphase fluorescence in situ hybridization. In addition, we examined the effect of the ODC inhibitor difluoromethylornithine on two pancreatic adenocarcinoma-derived cell lines. Result: MTAP activity was 2.8-fold reduced in adenocarcinomas and 6.3-fold reduced in neuroendocrine tumors compared with control pancreas. Conversely, ODC activity was 3.6-fold elevated in adenocarcinomas and 3.9-fold elevated in neuroendocrine tumors compared with control pancreas. Using interphase fluorescence in situ hybridization, we found in tumor samples that 43 to 75% of the nuclei had lost at least one copy of MTAP locus, indicating that loss of MTAP activity was at least partially because of deletion of the MTAP locus. We also show that inhibition of ODC by difluoromethylornithine caused decreased cell growth and increased apoptosis in two MTAP-deleted pancreatic adenocarcinoma-derived cell lines. CONCLUSIONS: MTAP activity is frequently lost, and ODC activity is frequently elevated in both pancreatic adenocarcinoma and neuroendocrine tumors. Inhibition of ODC activity caused decreased cell growth and increased apoptosis in pancreatic tumor-derived cell lines. These findings suggest that MTAP and polyamine metabolism could be potential therapeutic targets in the treatment of pancreatic cancer.     

Loss of BNIP3 expression is a late event in pancreatic cancer contributing to chemoresistance and worsened prognosis

Altered expression of apoptosis-regulating genes plays an important role in the aggressive growth behavior and chemoresistance of pancreatic ductal adenocarcinoma. In the present study, the hypoxia-inducible proapoptotic gene, BNIP3, was analysed in terms of expression, effect on patient survival, and chemo-responsiveness in pancreatic cancer cell lines. cDNA microarray, real-time light cycler quantitative polymerase chain reaction, laser-capture microdissection, and immunohistochemistry analyses were used to evaluate BNIP3 expression in normal and diseased pancreatic specimens. Modulation of BNIP3 expression was achieved using specific siRNA molecules. The effect of chemotherapeutic agents on pancreatic cancer cells was assessed utilizing 3-(4,5-methylthiazol-2-yl)-2,5-diphenyl-tetrazolium-bromide assays. BNIP3 mRNA levels were 3.0- and 6.3-fold lower in chronic pancreatitis and pancreatic cancer compared to the normal pancreas, respectively. Microdissection analysis confirmed the reduction of BNIP3 expression in pancreatic cancer cells compared to normal duct cells. By immunohistochemistry, BNIP3 was predominantly expressed in the acinar cells of the normal and diseased pancreas. Interestingly, while BNIP3 was undetectable in the cancer cells of 59% of the cases, 75-100% of PanIN2/3 lesions displayed BNIP3 immunoreactivity. Loss of BNIP3 expression correlated with poorer survival of patients (8 vs 14 months for BNIP3 negative vs positive tumors). Hypoxia induced BNIP3 expression in four out of eight pancreatic cancer cell lines, while it was absent under normoxic and hypoxic conditions in the remaining four. Downregulation of BNIP3 resulted in increased resistance to 5-fluoro-uracil and gemcitabine. In conclusion, loss of BNIP3 expression occurs late in pancreatic cancer, contributes to resistance to chemotherapy, and correlates with a worsened prognosis.     

NK-1 R在胰腺癌中的表达、组织学定位和临床意义

目的 探讨胰腺癌中P物质（SP）受体NK－1R的表达、组织学定位，并将结果结合临床资料分析，以期找出其中相关性。方法 33个胰腺癌标本取自胰头癌根治术，男民生19例，女性14例，正常胰腺组织取自20个器官捐献者，男性11例，女性9例。应用实时定量逆转录聚合酶链反应（RQ－RT－PCR）技术，检测正常胰腺、胰腺癌组织和7株胰腺癌细胞中NK－1R的mRNA水平，应用Western blot技术检测NK－1R的蛋白水平，应用免疫组织化学方法进行NK－1R的组织学定位。结果 与正常胰腺相比，胰腺癌组织中NK－1R mRNA和蛋白都过度表达，免疫组织化学结果提示：正常胰腺的腺泡和导管中有微弱的NK－1R表达，胰腺癌组织中有较强的NK－1R表达，主要位于胰腺癌细胞、神经纤维和炎性细胞。结论 胰腺癌组织中NK－1R mRNA和蛋白表达水平都明显上调，扰乱了神经激肽的作用环节，促进胰腺癌细胞的发生和发展。     

The kinase Mirk/Dyrk1B mediates cell survival in pancreatic ductal adenocarcinoma

Ductal adenocarcinoma of the pancreas is almost uniformly lethal as this cancer is invariably detected at an advanced stage and is resistant to treatment. The serine/threonine kinase Mirk/Dyrk1B has been shown to be antiapoptotic in rhabdomyosarcomas. We have now investigated whether Mirk might mediate survival in another cancer in which Mirk is widely expressed, pancreatic ductal adenocarcinoma. Mirk was an active kinase in each pancreatic cancer cell line where it was detected. Mirk knockdown by RNA interference (RNAi) reduced the clonogenicity of Panc1 pancreatic cancer cells 4-fold and decreased tumor cell number, showing that Mirk mediates survival in these cells. Mirk knockdown by synthetic duplex RNAis in Panc1, AsPc1, and SU86.86 pancreatic cancer cells induced apoptosis and enhanced the apoptosis induced by gemcitibine. Mirk knockdown did not increase the abundance or activation of Akt. However, four of five pancreatic carcinoma cell lines exhibited either elevated Mirk activity or elevated Akt activity, suggesting that pancreatic cancer cells primarily rely on Mirk or Akt for survival signaling. Mirk protein was detected by immunohistochemistry in 25 of 28 cases (89%) of pancreatic ductal adenocarcinoma, with elevated expression in 11 cases (39%). Increased expression of Mirk was seen in pancreatic carcinomas compared with primary cultures of normal ductal epithelium by serial analysis of gene expression and by immunohistochemistry. Thus, Mirk is a survival factor for pancreatic ductal adenocarcinoma. Because knockout of Mirk does not cause embryonic lethality, Mirk is not essential for normal cell growth and may represent a novel therapeutic target.     

Inhibition of heme oxygenase-1 increases responsiveness of pancreatic cancer cells to anticancer treatment.

Heme oxygenase-1 (HO-1) is believed to represent a key enzyme for the protection of cells against "stress." Its overexpression in different types of human cancers supports the notion that HO-1 provides a growth advantage and contributes to cellular resistance against chemotherapy and radiotherapy. Given the poor survival rates of patients with pancreatic cancer due to its aggressive growth behavior and its exceptional resistance to all known forms of anticancer treatment, we have investigated the expression of HO-1 in human pancreatic cancer cells growth behavior and prognosis. Expression of HO-1 was analyzed in human pancreatic cancer samples in comparison with normal pancreas by quantitative PCR, Western blot, and confocal microscopy. The influence of radiotherapy and chemotherapy on HO-1 expression in pancreatic cancer cell lines was evaluated. Furthermore, HO-1 expression was specifically suppressed by small interfering RNA transfection and subsequently the alterations of growth behavior and resistance to anticancer treatment were tested. Human pancreatic cancer showed a 6-fold and 3.5-fold HO-1 up-regulation in comparison to normal pancreas based on mRNA and protein level, respectively (P < 0.05). Cancer tissues revealed marked HO-1 immunoreactivity in tumor cells and in tumor associated immunocytes. Treatment of the pancreatic cancer cell lines with gemcitabine or radiation strongly induced HO-1 expression. Targeted knockdown of HO-1 expression led to pronounced growth inhibition of the pancreatic cancer cells and made tumor cells significantly more sensitive to radiotherapy and chemotherapy. Therefore, specific inhibition of HO-1 expression may be a new option in pancreatic cancer therapy and may be used as sensitizer to chemotherapy and radiotherapy.     

Nerve growth factor expression correlates with perineural invasion and pain in human pancreatic cancer.

PURPOSE: The reasons for the high frequency of perineural invasion and the presence of pain in pancreatic cancer are still not clear. Nerve growth factor (NGF) and its high-affinity receptor TrkA are involved in stimulating epithelial cancer cell growth and perineural invasion, as well as in pain generation in chronic benign disorders. PATIENTS AND METHODS: NGF and TrkA were examined by Northern blot analysis, in situ hybridization, and immunohistochemistry in 27 normal and 37 pancreatic cancer tissue samples. The molecular findings were correlated with the degree of perineural invasion, pain, and histopathologic tumor characteristics. RESULTS: Northern blot analysis indicated that NGF and TrkA mRNA levels were increased 2.7-fold and 5.6-fold, respectively (P <.05 and P <.05), in pancreatic cancer tissues compared with the normal pancreas tissue. As shown by in situ hybridization and immunohistochemistry, NGF was strongly present in the cytoplasm of pancreatic cancer cells. TrkA was intensely present in the perineurium of pancreatic nerves but not in the cancer cells. There was no difference in NGF and TrkA expression between early (stages I and II) and advanced (stage III) tumor stages and between well-/moderately differentiated (grades 1 and 2) and poorly differentiated (grade 3) tumors. However, tumors with high NGF/TrkA expression levels exhibited more frequent perineural invasion (P <.01). Furthermore, increased NGF/TrkA expression levels were associated with a higher degree of pain (P <. 01). CONCLUSION: Enhanced expression of the NGF/TrkA system may influence perineural invasion and may contribute to the pain syndrome in human pancreatic cancer.     

In vitro modeling of human pancreatic duct epithelial cell transformation defines gene expression changes induced by K-ras oncogenic activation in pancreatic carcinogenesis

Genetic analysis of pancreatic ductal adenocarcinomas and their putative precursor lesions, pancreatic intraepithelial neoplasias (PanIN), has shown a multistep molecular paradigm for duct cell carcinogenesis. Mutational activation or inactivation of the K-ras, p16(INK4A), Smad4, and p53 genes occur at progressive and high frequencies in these lesions. Oncogenic activation of the K-ras gene occurs in >90% of pancreatic ductal carcinoma and is found early in the PanIN-carcinoma sequence, but its functional roles remain poorly understood. We show here that the expression of K-ras(G12V) oncogene in a near diploid HPV16-E6E7 gene immortalized human pancreatic duct epithelial cell line originally derived from normal pancreas induced the formation of carcinoma in 50% of severe combined immunodeficient mice implanted with these cells. A tumor cell line established from one of these tumors formed ductal cancer when implanted orthotopically. These cells also showed increased activation of the mitogen-activated protein kinase, AKT, and nuclear factor-kappaB pathways. Microarray expression profiling studies identified 584 genes whose expression seemed specifically up-regulated by the K-ras oncogene expression. Forty-two of these genes have been reported previously as differentially overexpressed in pancreatic cancer cell lines or primary tumors. Real-time PCR confirmed the overexpression of a large number of these genes. Immunohistochemistry done on tissue microarrays constructed from PanIN and pancreatic cancer samples showed laminin beta3 overexpression starting in high-grade PanINs and occurring in >90% of pancreatic ductal carcinoma. The in vitro modeling of human pancreatic duct epithelial cell transformation may provide mechanistic insights on gene expression changes that occur during multistage pancreatic duct cell carcinogenesis.     

Histone deacetylase 1 is required for exocrine pancreatic epithelial proliferation in development and cancer

Histone deacetylases (HDACs) play important roles in the epigenetic control of development, and aberrant expression of HDACs has been implicated in human diseases including cancer. Among the mammalian HDACs, HDAC1 has been extensively studied, but its role in exocrine pancreatic morphogenesis and cancer is still poorly understood. The goal of this study is to determine the functional role of HDAC1 in normal development of exocrine pancreas using zebrafish as the model organism as well as in human pancreatic adenocarcinoma. The zebrafish germline loss-of-function mutation hdac1(hi1618) caused impaired cell cycle progression in pancreatic epithelia, resulting in growth arrest and dysmorphogenesis of exocrine pancreas. In human pancreatic adenocarcinoma tissues and cell lines, HDAC1 was expressed at variably elevated levels. RNA interference-induced silencing of HDAC1 diminished proliferation of the cancer cells and cell cycle progression. The proliferative arrest in the developing exocrine pancreas and pancreatic cancer cells was associated with up-regulated expression of the cyclin-dependent kinase inhibitors and the sonic hedgehog signaling components. This study indicates that HDAC1 is required for pancreatic epithelial proliferation in development and cancer. We hypothesize that aberrant expression of HDAC1 modulates the developmental and signaling pathways in exocrine pancreatic epithelia and consequently the genes required for cellular proliferation during development and progression of pancreatic neoplasia.     

Expression of neutral endopeptidase (NEP/CD10) on pancreatic tumor cell lines, pancreatitis and pancreatic tumor tissues

Neutral endopeptidase (NEP/CD10) is a cell surface zinc metalloprotease cleaving peptide bounds on the amino terminus of hydrophobic amino acids and inactivating multiple physiologically active peptides. Loss or decrease in NEP/CD10 expression have been reported in many types of malignancies, but the role of NEP/CD10 in pancreatic carcinoma has not yet been identified. Using real-time RT-PCR, flow cytometry as well as immunohistochemistry, NEP/CD10 expression was quantified in both pancreatic carcinoma cell lines and in tumor specimens obtained from patients with primary pancreatic carcinomas. Three out of 8 pancreatic carcinoma cell lines exhibit heterogeneous NEP/CD10 expression levels: PATU-8988T expressed the highest NEP/CD10 levels, whereas HUP-T4 and HUP-T3 cells showed a moderate to low NEP/CD10 expression. NEP/CD10 immunoreactivity was found in 6 of 24 pancreatic ductal adenocarcinomas, but also in 3 of 6 tissues of patients with chronic pancreatitis. NEP/CD10 expression in pancreatic tumor lesions and cell lines was not associated with tumor grading and staging. Treatment of PATU-8988T cells with the histone deacetylase inhibitors sodium butyrate and valproic acid induced an increase of NEP/CD10 expression. This was accompanied by a reduced cell proliferation rate of PATU-8988T cells, which was increased by the addition of the enzyme activity inhibitors phosphoramidon and thiorphan. Thus, NEP/CD10 is differentially expressed in pancreatic tumors and might be involved in the proliferative activity of pancreatic cancer cells. However, further studies are needed to provide more detailed information of the role of NEP/CD10 under physiological and pathophysiological conditions of the pancreas.