Notch signaling governs binary cell fate determination in asymmetrically dividing cells. Through a forward genetic screen we identified the fly homologue of Eps15 homology domain containing protein-binding protein 1 (dEHBP1) as a novel regulator of Notch signaling in asymmetrically dividing cells. dEHBP1 is enriched basally and at the actin-rich interface of pII cells of the external mechanosensory organs, where Notch signaling occurs. Loss of function of dEHBP1 leads to up-regulation of Sanpodo, a regulator of Notch signaling, and aberrant trafficking of the Notch ligand, Delta. Furthermore, Sec15 and Rab11, which have been previously shown to regulate the localization of Delta, physically interact with dEHBP1. We propose that dEHBP1 functions as an adaptor molecule for the exocytosis and recycling of Delta, thereby affecting cell fate decisions in asymmetrically dividing cells.

BACKGROUND:X inactive-specific transcript (XIST) RNA is involved in X chromosome silencing in female cells and allows X chromosome equilibration with males. X inactive-specific transcript expression has been found to be dysregulated in a variety of human cancers when compared to normal cells; meanwhile, the inactivated X chromosome has been noted to be conspicuously absent in human cancer specimens, whereas X chromosome duplications are widely noted. The specific pathways whereby changes in X chromosome status and XIST expression occur in cancer remain incompletely described. Nevertheless, a role for XIST in BRCA1-mediated epigenetic activity has been proposed. METHODS:Here we review the data regarding XIST expression and X chromosome status in a variety of female, male, and non-sex-related human cancers. CONCLUSIONS:It is not yet known whether X chromosome duplication, XIST dysregulation, and over-expression of X-linked genes represent important factors in tumorgenesis or are simply a consequence of overall epigenetic instability in these cancers.

MicroRNAs (miRNAs) are involved in cancer pathogenesis, apoptosis, and cell growth, thereby functioning as tumor suppressors or oncogenes. However, expression alterations and roles of these miRNAs in pancreatic cancer are largely unknown. We hypothesized that pancreatic cancer may have a unique miRNA profile, which may play a critical role in pancreatic cancer development, progression, diagnosis, and prognosis.

Objective— The objective of this study was to determine the effects and molecular mechanisms of eotaxin, a newly discovered chemokine (CCL11), on endothelial permeability in the human coronary artery endothelial cells (HCAECs). Methods and Results— Cells were treated with eotaxin, and the monolayer permeability was studied by using a costar transwell system with a Texas Red–labeled dextran tracer. Eotaxin significantly increased monolayer permeability in a concentration-dependent manner. In addition, eotaxin treatment significantly decreased the mRNA and protein levels of endothelial junction molecules including zonula occludens-1 (ZO-1), occludin, and claudin-1 in a concentration-dependent manner as determined by real-time RT-PCR and Western blot analysis, respectively. Increased oxidative stress was observed in eotaxin-treated HCAECs by analysis of cellular glutathione levels. Furthermore, eotaxin treatment substantially activated the phosphorylation of MAPK p38. HCAECs expressed CCR3. Consequently, antioxidants (ginkgolide B and MnTBAP), specific p38 inhibitor SB203580, and anti-CCR3 antibody effectively blocked the eotaxin-induced permeability increase in HCAECs. Eotaxin also increased the phosphorylation of Stat3 and nuclear translocation of NF-κB in HCAECs. Conclusions— Eotaxin increases vascular permeability through CCR3, the downregulation of tight junction proteins, increase of oxidative stress, and activation of MAPK p38, Stat3, and NF-kB pathways in HCAECs.

Introduction: Non-coding XIST RNA gene is located at X chromosome (Xq13.2) and its primary function is to inactivate one of X chromosomes in female cells. Recently, we have first discovered that non-coding XIST RNA may be involved in pancreatic cancer growth. Objective of this study was to determine the expression levels of XIST RNA in human pancreatic cancer and effects of overexpression of XIST RNA on pancreatic cancer growth. Methods: Human pancreatic cancer tissues and normal tissues were collected from surgical specimens. Ten pancreatic cancer cell lines were obtained from ATCC. Expression levels of XIST RNA were determined by microarray and real time PCR. Location of XIST RNA was determined by in situ hybridization. Stable cell lines of overexpression of XIST RNA were established by a retroviral gene delivery system in two pancreatic cancer cell lines Panc-1 and MIA PaCa-2. In vitro cancer proliferation was assayed by MTS and FACS cell cycle analysis. In vivo cancer growth was studied in nude mice with subcutaneous injection. The mRNA levels of two tumor suppressor genes BRCA1 and p21 were determined by real time PCR. Results: in microarray analysis, human pancreatic cancer tissues showed a substantial lower level of XIST RNA than the normal human pancreatic tissues (n=5, p<0.05) and this XIST RNA downregulation was confirmed by real time PCR, and in situ hybridization showed the cancer regions with downreguation of XIST RNA staining. The XIST RNA levels were almost not detectable in ten human pancreatic cancer cell lines by real time PCR analysis, while normal human pancreatic ductal epithelial cells (HPDE) expressed a relatively high level of XIST RNA. Stable cell lines (Panc-1 and MIA-PaCa-2) of overexpression of XIST RNA were confirmed by both real time PCR and in situ hybridization. These cells showed a significant reduced cell proliferation rate as compared to their parental cells and empty vector control cells (n=3, P<0.05). These cells were arrested in G1 phase of the cell cycle with increase of two tumor suppressor genes BRCA1 and p21. More importantly, stable cell line Panc-1 with overexpression of XIST RNA did not grow in the nude mice with subcutaneous injection over 4 weeks, while its parental cells and empty vector control cells had a fast growth in the nude mice (n=10, p<0.05). Conclusions: Non-coding XIST RNA was substantially downregulated in both human pancreatic cancer tissues and multiple cell lines. The functional significance of the loss of XIST RNA expression was demonstrated by overexpression of XIST RNA which resulted in the inhibition of cell proliferation in vitro and tumor growth in vivo. Thus, downregulation of XIST RNA is associated with pancreatic cancer cell growth and malignancy, while increased expression of XIST inhibits pancreatic cancer cell growth in vitro and in vivo. This study also suggests a new strategy of gene therapy to pancreatic cancer.

Functional somatostatin receptors (SSTR) are lost in human pancreatic cancer. Transfection of SSTR‐1 inhibited pancreatic cancer cell proliferation in vitro. We hypothesize that stable transfection of SSTR‐1 may inhibit pancreatic cancer growth in vivo possibly through cell cycle arrest. In this study, we examined the expression of SSTR‐1 mRNA in human pancreatic cancer tissue specimens, and investigated the effect of SSTR‐1 overexpression on cell proliferation, cell cycle, and tumor growth in a subcutaneous nude mouse model. We found that SSTR‐1 mRNA was downregulated in the majority of pancreatic cancer tissue specimens. Transfection of SSTR‐1 caused cell cycle arrest at the G0/G1 growth phase, with a corresponding decline of cells in the S (mitotic) phase. The overexpression of SSTR‐1 significantly inhibited subcutaneous tumor size by 71% and 43% (n = 5, P < 0.05, Student's t‐test), and inhibited tumor weight by 69% and 47% (n = 5, P < 0.05, Student's t‐test), in Panc‐SSTR‐1 and MIA‐SSTR‐1 groups, respectively, indicating the potent inhibitory effect of SSTR‐1 on pancreatic cancer growth. Our data demonstrate that overexpression of SSTR‐1 significantly inhibits pancreatic cancer growth possibly through cell cycle arrest. This study suggests that gene therapy with SSTR‐1 may be a potential adjuvant treatment for pancreatic cancer. (Cancer Sci 2008; 99: 2218–2223)

Introduction: Recent studies indicate a possible correlation of zinc levels with risk of cancers; however, the role of zinc and zinc transporters in cancer progression is unknown. For the first time, we have recently observed that a dietary zinc transporter, ZIP4 (SLC39A4), was overexpressed in human pancreatic cancer. We hypothesize that ZIP4 upregulation may contribute to the pancreatic cancer progression. The objective of this study was to determine the roles and mechanisms of ZIP4 in human pancreatic cancer cell proliferation in vitro and pancreatic cancer progression in vivo. Methods: The expression of ZIP4 in human pancreatic cancer cell lines, normal human pancreatic ductal epithelium (HPDE), and clinical specimens of human pancreatic adenocarcinoma was determined by real-time RT-PCR, western blot, and immunohistochemistry. ZIP4 stable overexpression was established in the pancreatic cancer cell line MIA PaCa-2 using retrovirus vectors. In vitro cell proliferation was performed by MTS assay. In vivo tumor growth was performed in the nude mouse models of subcutaneous and orthotopic xenograft. Zinc uptake was determined by inductively coupled plasma mass spectrometry (ICPMS). Results: ZIP4 was substantially overexpressed in 16 of 17 (94%) clinical pancreatic adenocarcinoma specimens compared with their surrounding normal tissues, and ZIP4 mRNA levels were significantly higher in human pancreatic cancer cells than in HPDE cells (p<0.05). Overexpression of ZIP4 in MIA PaCa-2 cells increased tumor cell proliferation by 2-fold, and increased tumor volume by 13-fold in the nude mouse model of subcutaneous xenograft, compared with the vector control cell line (p<0.05). Overexpression of ZIP4 not only increased the primary tumor weight (7.2-fold) in the orthotopic nude mouse model, but also increased the incidence of peritoneal dissemination and ascites in the mice. Overexpression of ZIP4 also accumulated more zinc in both pancreatic cancer cells, and xenograft tumors. Conclusions: ZIP4 is overexpressed in human pancreatic cancer, and contributes to tumor progression by accumulation of intracellular zinc and stimulation of cell proliferation. Thus, overexpression of ZIP4 is considered a new malignant factor for pancreatic cancer progression. Consequently, targeting ZIP4 may become a new strategy to treat pancreatic cancer.

Interleukin‐8 (IL‐8) is associated with tumorigenesis by promoting angiogenesis and metastasis. Although up‐regulation of IL‐8 is indicated in many cancers, its function in pancreatic cancer has not been well characterized. In this study we examined the expression of IL‐8 on pancreatic cancer cells and clinical tissue specimens, and investigated the effect of exogenous IL‐8 on gene expression, and signaling in human pancreatic cancer cells. We found that pancreatic cancer cells expressed higher amount of IL‐8 mRNA than normal human pancreatic ductal epithelium cells. IL‐8 mRNA was also substantially overexpressed in 11 of 14 (79%) clinical pancreatic‐adenocarcinoma samples compared with that in their surrounding normal tissues. Exogenous IL‐8 up‐regulated the expression of vascular endothelial growth factor 165 , and neuropilin (NRP)‐2 in BxPC‐3 cells, one of human pancreatic cancer cell lines. IL‐8 expression was inducible by hypoxia mimicking reagent cobalt chloride. In addition, IL‐8 activated extracellular signal‐regulated kinase (ERK)1/2 signaling pathway in BxPC‐3 cells. Our studies suggest that IL‐8 might be a malignant factor in human pancreatic cancer by induction of vascular endothelial growth factor and NRP‐2 expression and ERK activation. Targeting IL‐8 along with other antiangiogenesis therapy could be an effective treatment for this malignancy. ( Cancer Sci 2008, 99: 733–737)

The purpose of this study was to determine the effects and mechanisms of sCD40L on endothelial dysfunction in both human coronary artery endothelial cells (HCAECs) and porcine coronary artery rings. HCAECs treated with sCD40L showed significant reductions of endothelial nitric oxide synthase (eNOS) mRNA and protein levels, eNOS mRNA stability, eNOS enzyme activity, and cellular NO levels, whereas superoxide anion (O(2)(-)) production was significantly increased. sCD40L enhanced eNOS mRNA 3'UTR binding to cytoplasmic molecules and induced a unique expression pattern of 95 microRNAs. sCD40L significantly decreased mitochondrial membrane potential, and catalase and SOD activities, whereas it increased NADPH oxidase (NOX) activity. sCD40L increased phosphorylation of MAPKs p38 and ERK1/2 as well as IkappaBalpha and enhanced NF-kappaB nuclear translocation. In porcine coronary arteries, sCD40L significantly decreased endothelium-dependent vasorelaxation and eNOS mRNA levels, whereas it increased O(2)(-) levels. Antioxidant seleno-l-methionine; chemical inhibitors of p38, ERK1/2, and mitochondrial complex II; as well as dominant negative mutant forms of IkappaBalpha and NOX4 effectively blocked sCD40L-induced eNOS down-regulation in HCAECs. Thus, sCD40L reduces eNOS levels, whereas it increases oxidative stress through the unique molecular mechanisms involving eNOS mRNA stability, 3'UTR-binding molecules, microRNAs, mitochondrial function, ROS-related enzymes, p38, ERK1/2, and NF-kappaB signal pathways in endothelial cells.

Zinc is an essential trace element and catalytic/structural component used by many metalloenzymes and transcription factors. Recent studies indicate a possible correlation of zinc levels with the cancer risk; however, the exact role of zinc and zinc transporters in cancer progression is unknown. We have observed that a zinc transporter, ZIP4 (SLC39A4), was substantially overexpressed in 16 of 17 (94%) clinical pancreatic adenocarcinoma specimens compared with the surrounding normal tissues, and ZIP4 mRNA expression was significantly higher in human pancreatic cancer cells than human pancreatic ductal epithelium (HPDE) cells. This indicates that aberrant ZIP4 up-regulation may contribute to the pancreatic cancer pathogenesis and progression. We studied the effects of ZIP4 overexpression in pancreatic cancer cell proliferation in vitro and pancreatic cancer progression in vivo. We found that forced expression of ZIP4 increased intracellular zinc levels, increased cell proliferation by 2-fold in vitro, and significantly increased tumor volume by 13-fold in the nude mice model with s.c. xenograft compared with the control cells. In the orthotopic nude mice model, overexpression of ZIP4 not only increased the primary tumor weight (7.2-fold), it also increased the peritoneal dissemination and ascites incidence. Moreover, increased cell proliferation and higher zinc content were also observed in the tumor tissues that overexpressed ZIP4. These data reveal an important outcome of aberrant ZIP4 expression in contributing to pancreatic cancer pathogenesis and progression. It may suggest a therapeutic strategy whereby ZIP4 is targeted to control pancreatic cancer growth.

Background: Thymosin beta 4 ( T beta 4) has been shown to be associated with tumor metastasis and angiogenesis; however, its role in pancreatic cancer has not been understood. In the current study, we examined the expression of T beta 4 in pancreatic cancer cells, and determined the effect of exogenous T beta 4 on cytokine secretion, and signal transduction in human pancreatic cancer cells.Results: Pancreatic cancer cell lines expressed higher amount of T beta 4 mRNA than normal human pancreatic ductal epithelium ( HPDE) cells. Exogenous T beta 4 increased the secretion of proinflammatory cytokines IL-6, IL-8 and MCP-1 in Panc-1 cells. In addition, T beta 4 activated Jun N-terminal Kinase ( JNK) signaling pathways in pancreatic cancer cells.Methods: The mRNA levels of T beta 4 were determined by realtime RT PCR. Phosphorylation of JNK in pancreatic cancer cells was determined using Bio-Plex phosphoprotein assay. The expression of cytokines in human pancreatic cancer cell lines was determined with Bio-Plex cytokine assay.Conclusions: T beta 4 might be involved in stimulating human pancreatic cancer progression by promoting proinflammatory cytokine environment and activating JNK signaling pathway. Targeting T beta 4 and related molecules may be a novel therapeutic strategy for pancreatic cancer.

BACKGROUND: Somatostatin inhibits cell proliferation through interaction with its cellular receptor, somatostatin receptors (SSTRs). We have previously demonstrated that overexpression of individual SSTR-1 or SSTR-2 genes in receptor-negative pancreatic cancer cells inhibited cell proliferation. We hypothesize that reintroduction of SSTR genes back into pancreatic cancer cells might be an effective gene therapy strategy for pancreatic cancer.STUDY DESIGN: We transfected human pancreatic cancer cell line (Panc-1) with human SSTR-1 and SSTR-2 genes and examined the expression by real-time reverse transcriptase-polymerase chain reaction and immunofluorescence. Panc-1 cell proliferation was determined by [H-3]-thymidine incorporation assay. Activation of phosphorylated c-Jun N-terminal protein kinase (JNK) and cytokine secretion after SSTR-1 and SSTR-2 transfection were detected by Bio-Plex 4-plex phosphoprotein assay and cytokine assay.RESULTS: Panc-1 cells did not express SSTR-1 or SSTR-2, although Panc-1 cells transfected with SSTR-1 and SSTR-2 genes showed a significant amount of SSTR expression. Cell growth rate in Panc-1 cells transfected with SSTR-1 and SSTR-2 was inhibited about 41%, and the cell proliferation of Panc-1 expressing SSTR-1 and SSTR-2 was further reduced about 12% on treatment with somatostatin analogue as compared with the control group. SSTR-1 and SSTR-2 cotransfected Panc-1 cells activated phosphorylation of JNK and increased secretion of interferon-gamma and interleukin-5.CONCLUSIONS: These findings suggest, for the first time, a synergistic inhibitory effect of multiple SSTRs in response to a somatostatin analogue in Panc-1 cells. These studies may improve our understanding of the mechanism by which SSTR inhibits cell growth and lead to novel gene therapies for pancreatic cancer.

Background We previously found that cyclophilin A (CypA) is overexpressed in human pancreatic cancer cells and stimulates cell proliferation through CD147. In this study, we further investigated the effect of CypA on gene expression of several key molecules that are involved in pancreatic cancer cell proliferation. Methods Human pancreatic cancer cell lines (Panc-1, MIA PaCa-2, and BxPC-3) and human pancreatic ductal epithelial (HPDE) cells were used. The messenger RNA (mRNA) levels of CypA, CypB, CD147, neuropilins (NRPs), vascular endothelial growth factor (VEGF), and VEGF receptors upon the treatment of exogenous recombinant human CypA were determined by real-time reverse-transcription polymerase chain reaction. Results Exogenous human recombinant CypA reduced the mRNA levels of NRP-1 and VEGF, but not endogenous CypA, CypB, and CD147, in Panc-1, MIA PaCa-2, and BxPC-3 cells. In contrast, HPDE cells showed a decrease of endogenous CypA and CD147 mRNA, but not detectable changes of CypB, NRPs, and VEGF mRNA levels upon exogenous CypA treatment. Conclusions These data show that exogenous CypA downregulates NRP-1 and VEGF expression in pancreatic cancer cells. This effect is different in normal HPDE cells. Thus, soluble CypA may affect cell growth of pancreatic cancer.