Bacterial Quorum Sensing Molecule N-3-Oxo-Dodecanoyl-L-Homoserine Lactone Causes Direct Cytotoxicity and Reduced Cell Motility in Human Pancreatic Carcinoma Cells

In spite of chemotherapeutic and surgical advances, pancreatic cancer continues to have a dismal prognosis. Metastasis due to tumor cell migration remains the most critical challenge in treating pancreatic cancer, and conventional chemotherapy is rarely curative. In the quest for more novel molecules to fight this disease, we tested the hypothesis that the Pseudomonas aeruginosa quorum sensing signal molecule N-3-oxo-dodecanoyl-L-homoserine lactone (O-DDHSL) would be cytotoxic to and reduce mobility of pancreatic carcinoma cells (Panc-1 and Aspc-1). Results showed a decrease in cell viability from apoptosis, diminished colony formation, and inhibition of migration of the evaluated pancreatic carcinoma cell lines. Also, cell viability decreased in the presence of O-DDHSL when cells were grown in matrigel basement membrane matrix. While messenger RNA for IQGAP-1 decreased in Panc-1 and HPDE cells upon exposure to O-DDHSL, no change was observed in Aspc-1 cells. Cofilin mRNA expression was found to be increased in both HPDE and Panc-1 cells with marginal decrease in Aspc-1 cells. RhoC, a Rho-family GTPase involved in cell motility, increased in the presence of O-DDHSL, suggesting a possible compensatory response to alteration in other migration associated genes. Our results indicate that O-DDHSL could be an effective biomolecule in eukaryotic systems with multimodal function for essential molecular targeting in pancreatic cancer.     

Flt3 mutation activates p21WAF1/CIP1 gene expression through the action of STAT5

Metastin, a post-translationally modified variant of KiSS1, was recently identified as an endogenous peptide agonist for a novel G-protein coupled receptor, hOT7T175 (AXOR12, GPR54). In this study, we analyzed the role of KiSS1 and hOT7T175 in both pancreatic cancer tissues and pancreatic cancer cell lines. Furthermore, we synthesized novel short variant forms of metastin and tested the inhibitory effect of those variants on in vitro cell functions that are relevant to metastasis. Pancreatic cancer tissues showed significantly lower expression of KiSS1 mRNA than normal tissues (p=0.018), while cancer tissues showed significantly higher expression of hOT7T175 mRNA than normal pancreatic tissues (p=0.027). In human pancreatic cancer cell lines, KiSS1 mRNA was highly expressed in 2 out of 6 pancreatic cancer cell lines, while hOT7T175 mRNA was expressed in all cell lines at various degrees. PANC-1 cells showed the highest expression of hOT7T175. Exogenous metastin did not suppress cell proliferation but significantly reduced the in vitro migration of PANC-1 cells (p<0.01). Metastin induced activation of ERK1 in PANC-1 and AsPC-1 cells. Finally, we synthesized 3 novel short variant forms of metastin, FM053a2TFA, FM059a2TFA, and FM052a4TFA. These metastin variants significantly suppressed the migration of PANC-1 cells and activated ERK1. These data suggest that the metastin receptor, hOT7T175, is one of the promising targets for suppression of metastasis, and that small metastin variants could be an anti-metastatic agent to pancreatic cancer.     

Pancreatic stellate cells promote epithelial-mesenchymal transition in pancreatic cancer cells

The interaction between pancreatic cancer cells and pancreatic stellate cells (PSCs), a major profibrogenic cell type in the pancreas, is receiving increasing attention. There is accumulating evidence that PSCs promote the progression of pancreatic cancer by increasing cancer cell proliferation and invasion as well as by protecting them from radiation- and gemcitabine-induced apoptosis. Because epithelial-mesenchymal transition (EMT) plays a critical role in the progression of pancreatic cancer, we hypothesized that PSCs promote EMT in pancreatic cancer cells. Panc-1 and SUIT-2 pancreatic cancer cells were indirectly co-cultured with human PSCs isolated from patients undergoing operation for pancreatic cancer. The expression of epithelial and mesenchymal markers was examined by real-time PCR and immunofluorescent staining. The migration of pancreatic cancer cells was examined by scratch and two-chamber assays. Pancreatic cancer cells co-cultured with PSCs showed loose cell contacts and a scattered, fibroblast-like appearance. The expression of E-cadherin, cytokeratin 19, and membrane-associated β-catenin was decreased, whereas vimentin and Snail (Snai-1) expression was increased more in cancer cells co-cultured with PSCs than in mono-cultured cells. The migration of pancreatic cancer cells was increased by co-culture with PSCs. The PSC-induced decrease of E-cadherin expression was not altered by treatment with anti-TGF-β-neutralizing antibody, excluding a central role of TGF-β in this process. In conclusion, PSCs promoted EMT in pancreatic cancer cells suggesting a novel mechanism by which PSCs contribute to the aggressive behavior of pancreatic cancer cells.     

Changes in the distribution of intermediate filament proteins and collagen IV in fetal and adult human pancreas. I. Localization of cytokeratin polypeptides

The expression patterns of individual cytokeratin polypeptides in foetal and adult human pancreatic tissues were examined using monoclonal antibodies. We demonstrated that human pancreatic epithelia in early stages of development (14 weeks of gestation) contain cytokeratins 7, 8, 18 and 19, which are typical of simple epithelia, as well as cytokeratin 4 and 17, which are characteristic of stratified epithelia. In the pancreatic ducts, most of these cytokeratins appeared to be expressed together. Cytokeratins 1, 5, 10, 13, 16 and 20 were not detectable. In contrast, the pancreatic parenchyma was only positive for cytokeratins 8 and 18, except a transient expression of cytokeratins 7 and 19 in pancreatic islets and acinar cells during the foetal development. A focal cytokeratin 7 staining of single acinar cells was seen in newborn and in adult islets. In the stromal tissue, vascular smooth muscle cells were partly reactive with cytokeratin 8 and 18 specific antibodies. The results are discussed in the light of differentiation-dependent changes in the expression of individual cytokeratin polypeptides in developing epithelia.     

Changes in the distribution of intermediate filament proteins and collagen IV in fetal and adult human pancreas

Summary The expression patterns of individual cytokeratin polypeptides in foetal and adult human pancreatic tissues were examined using monoclonal antibodies. We demonstrated that human pancreatic epithelia in early stages of development (14 weeks of gestation) contain cytokeratins 7, 8, 18 and 19, which are typical of simple epithelia, as well as cytokeratin 4 and 17, which are characteristic of stratified epithelia. In the pancreatic ducts, most of these cytokeratins appeared to be expressed together. Cytokeratins 1, 5, 10, 13, 16 and 20 were not detectable. In contrast, the pancreatic parenchyma was only positive for cytokeratins 8 and 18, except a transient expression of cytokeratins 7 and 19 in pancreatic islets and acinar cells during the foetal development. A focal cytokeratin 7 staining of single acinar cells was seen in newborn and in adult islets. In the stromal tissue, vascular smooth muscle cells were partly reactive with cytokeratin 8 and 18 specific antibodies. The results are discussed in the light of differentiation-dependent changes in the expression of individual cytokeratin polypeptides in developing epithelia.     

Reduced expression of the membrane skeleton protein beta1-spectrin (SPTBN1) is associated with worsened prognosis in pancreatic cancer

Spectrins are members of the superfamily of F-actin cross linking proteins that are important as scaffolding proteins for protein sorting, cell adhesion, and migration. In addition, spectrins have been implicated in TGF-beta signaling. The aim of the present study was to analyze the expression and localization of beta1-spectrin (SPTBN1) in pancreatic tissues. mRNA levels of SPTBN1 in cultured pancreatic cancer cell lines, as well as in normal pancreatic tissues (n=18), chronic pancreatitis (n=48) and pancreatic cancer tissues (n=66) were analyzed by real time quantitative RT-PCR. Localization of SPTBN1 in pancreatic tissues was determined by immunohistochemistry. SPTBN1 staining was assessed semi-quantitatively in 55 cancer tissues and survival analysis was carried out using the Kaplan-Meier method. Median SPTBN1 mRNA levels were 6.0-fold higher in pancreatic cancer tissues compared to the normal pancreas (p<0.0001) and 2.2-fold higher compared to chronic pancreatitis tissues (p=0.0002). In the normal pancreas, SPTBN1 was present in the cytoplasm of normal ductal cells and occasionally in pancreatic acinar and centroacinar cells. In pancreatic cancer tissues, SPTBN1 was present in the cytoplasm of pancreatic cancer cells. Low SPTBN1 protein expression indicated a tendency for worsened prognosis with a median survival of 14.0 months, versus 23.8 months for patients whose tumors expressed moderate/high levels of SPTBN1. In conclusion, reduced SPTBN1 expression correlated with shorter survival of pancreatic cancer patients, suggesting a tumor suppressor function of this gene, as has already been shown for other malignancies of the gastrointestinal tract.     

Identification of phosphoenolpyruvate carboxykinase 1 as a potential therapeutic target for pancreatic cancer

Pancreatic cancer is the third leading cause of cancer-related mortalities and is characterized by rapid disease progression. Identification of novel therapeutic targets for this devastating disease is important. Phosphoenolpyruvate carboxykinase 1 (PCK1) is the rate-limiting enzyme of gluconeogenesis. The current study tested the expression and potential functions of PCK1 in pancreatic cancer. We show that PCK1 mRNA and protein levels are significantly elevated in human pancreatic cancer tissues and cells. In established and primary pancreatic cancer cells, PCK1 silencing (by shRNA) or CRISPR/Cas9-induced PCK1 knockout potently inhibited cell growth, proliferation, migration and invasion, and induced robust apoptosis activation. Conversely, ectopic overexpression of PCK1 in pancreatic cancer cells accelerated cell proliferation and migration. RNA-seq analyzing of differentially expressed genes (DEGs) in PCK1-silenced pancreatic cancer cells implied that DEGs were enriched in the PI3K-Akt-mTOR cascade. In pancreatic cancer cells, Akt-mTOR activation was largely inhibited by PCK1 shRNA, but was augmented after ectopic PCK1 overexpression. In vivo, the growth of PCK1 shRNA-bearing PANC-1 xenografts was largely inhibited in nude mice. Akt-mTOR activation was suppressed in PCK1 shRNA-expressing PANC-1 xenograft tissues. Collectively, PCK1 is a potential therapeutic target for pancreatic cancer.Subject terms: Pancreatic cancer, Oncogenes     

Bone morphogenetic protein -4 and -5 in pancreatic cancer--novel bidirectional players

Bone morphogenetic proteins (BMPs) are multifunctional signaling molecules that have gained increasing interest in cancer research. To obtain a systematic view on BMP signaling in pancreatic cancer we first determined the mRNA expression levels of seven BMP ligands (BMP2–BMP8) and six BMP specific receptors in pancreatic cancer cell lines and normal pancreatic tissue. BMP receptor expression was seen in all cancer and normal samples. Low expression levels of BMP5 and BMP8 were detected in cancer cells compared to the normal samples, whereas BMP4 expression was elevated in 25% of the cases. The impact of BMP4 and BMP5 signaling on cell phenotype was then evaluated in five pancreatic cancer cell lines. Both ligands suppressed the growth of three cell lines (up to 79% decrease in BMP4-treated PANC-1 cells), mainly due to cell cycle changes. BMP4 and BMP5 concurrently increased cell migration and invasion (maximally a 10.8-fold increase in invaded BMP4-treated PANC-1 cells). The phenotypic changes were typically associated with the activation of the canonical SMAD pathway, although such activation was not observed in the PANC-1 cells. Taken together, BMP4 and BMP5 simultaneously inhibit the growth and promote migration and invasion of the same pancreatic cells and thus exhibit a biphasic role with both detrimental and beneficial functions in pancreatic cancer progression.     

Cathepsin G-induced malignant progression of MCF-7 cells involves suppression of PAF signaling through induced expression of PAFAH1B2

Breast cancer is primarily classified into ductal and lobular types, as well as into noninvasive and invasive cancer. Invasive cancer involves lymphatic and hematogenous metastasis. In breast cancer patients with distant metastases, a neutrophil-derived serine protease; cathepsin G (Cat G), is highly expressed in breast cancer cells. Cat G induces cell migration and multicellular aggregation of MCF-7 human breast cancer cells; however, the mechanism is not clear. Recently, platelet-activating factor (PAF)-acetylhydrolase (PAF-AH), the enzyme responsible for PAF degradation, was reported to be overexpressed in some tumor types, including pancreatic and breast cancers. In this study, we investigated whether PAF-AH is involved in Cat G-induced aggregation and migration of MCF-7 cells. We first showed that Cat G increased PAF-AH activity and elevated PAFAH1B2 expression in MCF-7 cells. The elevated expression of PAFAH1B2 was also observed in human breast cancer tissue specimens by immunohistochemical analysis. Furthermore, knockdown of PAFAH1B2 in MCF-7 cells suppressed the cell migration and aggregation induced by low concentrations, but not high concentrations, of Cat G. Carbamoyl PAF (cPAF), a nonhydrolyzable PAF analog, completely suppressed Cat G-induced migration of MCF-7 cells. In addition, PAF receptor (PAFR) inhibition induced cell migration of MCF-7 cells even in the absence of Cat G, suggesting that Cat G suppresses the activation of PAFR through enhanced PAF degradation due to elevated expression of PAFAH1B2 and thereby induces malignant phenotypes in MCF-7 cells. Our findings may lead to a novel therapeutic modality for treating breast cancer by modulating the activity of Cat G/PAF signaling.     

Cx31.1 acts as a tumour suppressor in non-small cell lung cancer (NSCLC) cell lines through inhibition of cell proliferation and metastasis

Reduced connexin expression and loss of gap junction function is a characteristic of many cancers, including lung cancer. However, there are little reports about the relation between Cx31.1 and lung cancer. This study was conducted to investigate the effect of Cx31.1 on non-small cell lung cancer (NSCLC). We found that the Cx31.1 was down-regulated in NSCLC cell lines, and the expression levels were reversely related with their metastatic potential. We ectopically expressed Cx31.1 in H1299 NSCLC cell line to examine the influence of Cx31.1 overexpression. The results showed that overexpression of Cx31.1 in H1299 cells reduced cell proliferation, induced a delay in the G(1) phase, inhibited anchorage-independent growth and suppressed cell migration and invasion. The cell cycle delay and cell migration and invasion suppressive effects of Cx31.1 were partially reversed by siRNA targeting mRNA of Cx31.1. Moreover, xenografts of Cx31.1 overexpressing H1299 cells showed reduced tumourigenicity. These results suggested that Cx31.1 has tumour-suppressive properties. Further investigation indicated that cyclin D3 may be responsible for Cx31.1-induced G(1) phase delay. Importantly, Cx31.1 increased the expression of epithelial markers, such as cytokeratin 18, and decreased expression of mesenchymal markers, such as vimentin, indicating a Cx31.1-mediated partial shift from a mesenchymal towards an epithelial phenotype. We concluded that Cx31.1 inhibit the malignant properties of NSCLC cell lines, the mechanisms under this may include regulation of EMT.     

Changes in the expression of proteins associated with aerobic glycolysis and cell migration are involved in tumorigenic ability of two glioma cell lines

ABSTRACT: BACKGROUND: The most frequent and malignant brain cancer is glioblastoma multiforme (GBM). In gliomas, tumor progression and poor prognosis are associated with the tumorigenic ability of the cells. U87MG cells (wild-type p53) are known to be tumorigenic in nude mice, but T98G cells (mutant p53) are not tumorigenic. We investigated the proteomic profiling of these two cell lines in order to gain new insights into the mechanisms that may be involved in tumorigenesis. RESULTS: We found 24 differentially expressed proteins between T98G and U87MG cells. Gene Ontology supports the notion that over-representation of differentially expressed proteins is involved in glycolysis, cell migration and stress oxidative response. Among those associated with the glycolysis pathway, TPIS and LDHB are up-regulated in U87MG cells. Measurement of glucose consumption and lactate production suggests that glycolysis is more effective in U87MG cells. On the other hand, G6PD expression was 3-fold higher in T98G cells and this may indicate a shift to the pentose-phosphate pathway. Moreover, GRP78 expression was also three-fold higher in T98G than in U87MG cells. Under thapsigargin treatment both cell lines showed increased GRP78 expression and the effect of this agent was inversely correlated to cell migration. Quantitative RT-PCR and immunohistochemistry of GRP78 in patient samples indicated a higher level of expression of GRP78 in grade IV tumors compared to grade I and non-neoplastic tissues, respectively. CONCLUSIONS: Taken together, these results suggest an important role of proteins involved in key functions such as glycolysis and cell migration that may explain the difference in tumorigenic ability between these two glioma cell lines and that may be extrapolated to the differential aggressiveness of glioma tumors.     

胰腺癌干细胞中受OCT4调控的表面标志研究

OCT4和Nanog被公认是人ESC的自我更新调控基因,其中OCT4能够转录调控多种表面蛋白的表达,如SEMA6A。该文将人胰腺癌细胞株Panc-1、Bxpc-3、Aspc-1和Cfpac-1培养在无血清条件下,采用EGF、IGF-1和FGF-10诱导球体形成。用免疫荧光法分别检测这4种人胰腺癌细胞株及其球体细胞以及15例胰腺癌组织标本和13例正常胰腺组织标本中OCT4和Nanog的表达,结果显示,4种人胰腺癌细胞株在无血清-DF12培养基中5～10 d即可形成悬浮生长的球体。OCT4和Nanog在4种细胞株均有表达,且球体细胞中表达明显高于亲代细胞。在胰腺癌组织中仅有少量表达自我更新基因,在正常胰腺组织中微量表达。此外,还检测到Panc-1球体细胞表面高表达SEMA6A。由此可见,自我更新基因OCT4和Nanog在胰腺癌细胞中的表达和CSC有关,其表面蛋白SEMA6A作为胰腺癌干细胞表面标志物值得进一步研究。     

Opposite Role of Kindlin-1 and Kindlin-2 in Lung Cancers

Lung cancer is highly heterogenous and is composed of various subtypes that are in diverse differential stages. The newly identified integrin-interacting proteins Kindlin-1 and Kindlin-2 are the activators of transmembrane receptor integrins that play important roles in cancer progression. In this report we present the expression profiles of Kindlin-1 and Kindlin-2 in lung cancers using patient specimens and established their correlation with lung cancer progression. We found that Kindlin-1 was expressed in epithelia-derived non-small-cell lung cancer, especially in squamous cell lung cancer but expressed at low levels in poorly differentiated large cell lung cancer. However, Kindlin-2 was highly expressed in large cell lung cancer. Both Kindlin-1 and Kindlin-2 were found not expressed or expressed at very low levels in neuroendocrine-derived small cell lung cancer. Importantly, the Kindlin-1 expression level was positively correlated with the differentiation of squamous cell lung cancer. Surprisingly, we found that the very homologous Kindlin family proteins, Kindlin-1 and Kindlin-2, displayed counteracting functional roles in lung cancer cells. Ectopic expression of Kindlin-1 in non-small-cell lung cancer cells inhibited in vitro cell migration and in vivo tumor growth, while Kindlin-2 promoted these functions. Mechanistically, Kindlin-1 prohibited epithelail to mesenchymal transition in non-small-cell lung cancer cells, while Kindlin-2 enhanced epithelail to mesenchymal transition in these cells. Taken together, we demonstrated that Kindlin-1 and Kindlin-2 differentially regulate lung cancer cell progression. Further, the expression levels of Kindlin-1 might be potentially used as a marker for lung cancer differentiation and targeting Kindlin-2 might block the invasive growth of large cell lung cancer.     

Biomarker discovery from pancreatic cancer secretome using a differential proteomic approach

Quantitative proteomics can be used as a screening tool for identification of differentially expressed proteins as potential biomarkers for cancers. Candidate biomarkers from such studies can subsequently be tested using other techniques for use in early detection of cancers. Here we demonstrate the use of stable isotope labeling with amino acids in cell culture (SILAC) method to compare the secreted proteins (secretome) from pancreatic cancer-derived cells with that from non-neoplastic pancreatic ductal cells. We identified 145 differentially secreted proteins (>1.5-fold change), several of which were previously reported as either up-regulated (e.g. cathepsin D, macrophage colony stimulation factor, and fibronectin receptor) or down-regulated (e.g. profilin 1 and IGFBP-7) proteins in pancreatic cancer, confirming the validity of our approach. In addition, we identified several proteins that have not been correlated previously with pancreatic cancer including perlecan (HSPG2), CD9 antigen, fibronectin receptor (integrin beta1), and a novel cytokine designated as predicted osteoblast protein (FAM3C). The differential expression of a subset of these novel proteins was validated by Western blot analysis. In addition, overexpression of several proteins not described previously to be elevated in human pancreatic cancer (CD9, perlecan, SDF4, apoE, and fibronectin receptor) was confirmed by immunohistochemical labeling using pancreatic cancer tissue microarrays suggesting that these could be further pursued as potential biomarkers. Lastly the protein expression data from SILAC were compared with mRNA expression data obtained using gene expression microarrays for the two cell lines (Panc1 and human pancreatic duct epithelial), and a correlation coefficient (r) of 0.28 was obtained, confirming previously reported poor associations between RNA and protein expression studies.     

Proteomic analysis of gemcitabine-induced drug resistance in pancreatic cancer cells

Currently, the most effective agent against pancreatic cancer is gemcitabine (GEM), which inhibits tumor growth by interfering with DNA replication and blocking DNA synthesis. However, GEM-induced drug resistance in pancreatic cancer compromises the therapeutic efficacy of GEM. To investigate the molecular mechanisms associated with GEM-induced resistance, 2D-DIGE and MALDI-TOF mass spectrometry were performed to compare the proteomic alterations of a panel of differential GEM-resistant PANC-1 cells with GEM-sensitive pancreatic cells. The proteomic results demonstrated that 33 proteins were differentially expressed between GEM-sensitive and GEM-resistant pancreatic cells. Of these, 22 proteins were shown to be resistance-specific and dose-dependent in the regulation of GEM. Proteomic analysis also revealed that proteins involved in biosynthesis and detoxification are significantly over-expressed in GEM-resistant PANC-1 cells. In contrast, proteins involved in vascular transport, bimolecular decomposition, and calcium-dependent signal regulation are significantly over-expressed in GEM-sensitive PANC-1 cells. Notably, both protein-protein interaction of the identified proteins with bioinformatic analysis and immunoblotting results showed that the GEM-induced pancreatic cell resistance might interplay with tumor suppressor protein p53. Our approach has been shown here to be useful for confidently detecting pancreatic proteins with differential resistance to GEM. Such proteins may be functionally involved in the mechanism of chemotherapy-induced resistance.     

Pancreatic tumor cells influence the composition of the extracellular matrix

The malignant behavior of cancers depends on the microenvironmental context. We investigated compositional alterations of the extracellular matrix (ECM) in pancreatic cancer, with special emphasis on the proteoglycans decorin, lumican, and versican. Compared with normal controls (n=18), marked overexpression of these proteoglycans was observed in pancreatic cancer tissues (n=30) by quantitative RT-PCR (p<0.0001). Immunohistochemistry revealed abundance of proteoglycans in the ECM of pancreatic cancer specimens, whereas tumor cells themselves were devoid of either decorin, lumican or versican. RT-PCR confirmed pancreatic stellate cells (PSCs) as the major source of these proteins. Interestingly, TGFbeta1 and conditioned medium derived from pancreatic cancer cell lines synergistically suppressed the expression of known anti-tumor factors decorin and lumican, but stimulated the expression of pro-metastatic factor versican in cultured PSCs. These findings indicate that malignant cells can actively influence the composition of the ECM through TGFbeta1 and other soluble factors, altering their microenvironment in a tumor-favorable way.     

Claudin-18 Is an Early-Stage Marker of Pancreatic Carcinogenesis

Pancreatic ductal neoplasms exhibit gastric epithelium–like characteristics. In this study, we evaluated the expression of claudin-18 (CLDN18), a gastric epithelium–associated claudin, in pancreatic intraepithelial neoplasias (PanINs), intraductal papillary mucinous neoplasms (IPMNs), mucinous cystic neoplasms (MCNs), and pancreatic ductal adenocarcinomas (PDACs) using immunohistochemistry. We observed a high level of expression of CLDN18 in PanINs (31/32, 97%), IPMNs (61/65, 95%), and MCNs (4/5, 80%) using ordinary tissue section analysis. Furthermore, we observed a high level of CLDN18 expression in PDACs (109/156, 70%) using tissue microarray analysis. However, the normal pancreatic duct or the ductal metaplasia of the acinar cells was not immunoreactive. Comparative analysis of CLDN18 and phenotypic markers in IPMNs revealed that simultaneous expression of CLDN18 and intestinal markers frequently occurred, even in intestinal-type IPMNs. CLDN18 variant 2 mRNA was expressed and was similarly upregulated by phorbol 12–myristate 13–acetate (PMA) treatment in pancreatic cancer cell lines and in a gastric cancer cell line. An inhibitor of pan-PKC (GF109203X) completely suppressed this upregulation in pancreatic cancer cells. These results indicate that CLDN18, a marker for the early carcinogenetic process, is commonly expressed in precursor lesions of PDAC. Activation of the PKC pathway might be involved in CLDN18 expression associated with pancreatic carcinogenesis.     

Expression of secreted frizzled-related protein 2 in a primary canine mammary tumor cell line: a candidate tumor marker for mammary tumor cells

Canine mammary tumors (CMTs) have been proposed to be a good animal model for human breast cancer. To provide a basis for the tumorigenic study of CMTs, cell lines were established using a modified cell culture technique. The epithelial morphology and immunostaining with cytokeratin 18 confirmed the epithelial origin of the cells. In an investigation of possible mammary tumorigenesis-related factors, the expression of Wnt signaling-related proteins was detected in cell lines. Secreted frizzled-related protein 2 (SFRP2) was abundantly expressed in CMT cells but not in normal canine mammary gland (MG) cells. Secreted frizzled-related protein 2 was secreted into the culture medium and was associated with the extracellular matrix. In addition, increased expressions of beta-catenin and cyclin D1 were observed in cells overexpressing SFRP2. The marked differential expression of SFRP2 reveals that this protein may be a potential candidate marker for CMTs. The CMT cell line established in this study provides a useful tool and experimental model for understanding both the tumorigenesis of CMTs and the role of Wnt signaling in cancers.