Human Pancreatic Ductal Cells Research Articles

Abstract 1337: Novel model of pancreatic cancer induced by smoking compound NNK

Abstract Background & Aims: Cigarette smoking is a major risk factor for pancreatic cancer. However, the molecular signaling mechanisms through which smoking promotes pancreatic cancer remain unknown. Experimental models of smoking-induced pancreatic cancer have not been developed. Here we studied the effect of cigarette smoking compound: nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) on cancer progression in Elastase-Kras (EL-Kras) mice, which have mutated/activated Kras. We further determined the effect of NNK and cigarette smoking extract (CSE) on cell death pathways both in vivo and in vitro in human pancreatic ductal cells. Methods: Wild type and EL-Kras transgenic mice were injected with NNK (100mg/Kg) once a week for 4 weeks. Mice were sacrificed one week later. Analysis of pancreatic lesions, adenocarcinoma, as well as measurement of fibrosis, inflammation, and proliferation were performed by immunohistochemistry. Apoptosis in tissue was assessed by TUNEL assay. Immortalized human ductal cells HPDE6 were cultured for up to 1 week in the presence or absence of NNK (1nM-1µM) or CSE (4ng/ml-4µg/ml). Apoptosis was assessed by measuring DNA fragmentation. Autophagy in cells and in mice pancreatic tissue was assessed by measuring autophagic marker LC3-II with western blot and with immunofluorescence. Results: NNK injections significantly increased (8 times) the number of pancreatic neoplastic lesions in EL-Kras mice. Importantly, NNK induced progression of the pancreatic lesions to the adenocarcinoma stage. Staining of pancreatic tumors with amylase and cytokeratines 7 and 9 showed that cancer cells present the phenotype of both acinar and ductal cells indicating that cells are at the stage of progression to the ductal adenocarcinoma phenotype. Stimulation of pancreatic adenocarcinoma was associated with a decrease in autophagy and apoptosis of exocrine pancreatic cells. NNK inhibited autophagy and apoptosis in pancreas from wild type mice but the effect was much less pronounced. Importantly, NNK injection stimulated fibrosis and stellate cells recruitment and activation. Furthermore, NNK stimulated inflammation. Similarly, exposure to NNK or CSE inhibited apoptosis and autophagy of human ductal cells. NNK and CSE increased Akt phosphorylation both in vivo and in vitro. Pharmacological and molecular inhibition of Akt kinase reversed the smoking inhibitory effect on apoptosis and autophagy, indicating a central role of Akt in the progression of pancreatic cancer by smoking compounds. Further, Akt inhibited autophagy through regulation of AMPK phosphorylation. Conclusion: NNK-induced cancer progression in EL-Kras mice represents the first mouse model of pancreatic cancer induced by smoking and can be utilized to understand the mechanism of progression of the disease. Akt kinase is a key mediator of smoking-induced pancreatic cancer progression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1337. doi:1538-7445.AM2012-1337

Chronic GLP-1 Receptor Activation by Exendin-4 Induces Expansion of Pancreatic Duct Glands in Rats and Accelerates Formation of Dysplastic Lesions and Chronic Pancreatitis in the KrasG12D Mouse Model

Pancreatic duct glands (PDGs) have been hypothesized to give rise to pancreatic intraepithelial neoplasia (PanIN). Treatment with the glucagon-like peptide (GLP)-1 analog, exendin-4, for 12 weeks induced the expansion of PDGs with mucinous metaplasia and columnar cell atypia resembling low-grade PanIN in rats. In the pancreata of Pdx1-Cre; LSL-KrasG12D mice, exendin-4 led to acceleration of the disruption of exocrine architecture and chronic pancreatitis with mucinous metaplasia and increased formation of murine PanIN lesions. PDGs and PanIN lesions in rodent and human pancreata express the GLP-1 receptor. Exendin-4 induced proproliferative signaling pathways in human pancreatic duct cells, cAMP–protein kinase A and mitogen-activated protein kinase phosphorylation of cAMP-responsive element-binding protein, and increased cyclin D1 expression. These GLP-1 effects were more pronounced in the presence of an activating mutation of Kras and were inhibited by metformin. These data reveal that GLP-1 mimetic therapy may induce focal proliferation in the exocrine pancreas and, in the context of exocrine dysplasia, may accelerate formation of neoplastic PanIN lesions and exacerbate chronic pancreatitis.

Purinergic regulation of ion transport in Capan‐1 cell monolayers

The aim of the present study was to develop a human pancreatic duct model and elucidate the purinergic signaling regulation of ion transport in pancreatic ducts. Human pancreatic duct cells, capan‐1 were grown on permeable supports. Transepithelial potential (Vte) was measured in open‐circuit Ussing chamber in physiological buffers. Capan‐1 cells formed monolayers with resting transepithelial resistance 404±25 Ω·cm2 (n=35). Serosal ATP (100 μM) transiently increased Vte from −1.4±0.2 to −4.7±0.4 mV and short‐circuit current (Isc) by 14± 0.8 μA/cm2, while mucosal ATP increased Vte by −3.6±0.3 mV and Isc by 13±1 μA/cm2 (n=28). The Ca2+‐activated Cl− channel (CaCC) blocker niflumic acid (NA, mucosa, 100 μM, n=5) inhibited 44% of mucosal but not serosal ATP effect. The CFTR Cl− channel blocker CFTRinh172 blocked both mucosal and serosal ATP effects (mucosa,10 μM, n=5). The intermediate conductance K+ (IK) channel opener DCEBIO (mucosa, 100 μM, n=5) increased mucosal ATP effect, and DCEBIO stimulation was completely inhibited by NA and CFTRinh172 (n=8). The IK channel blocker, clotrimazole, abolished mucosal and serosal ATP effects (30 μM, n=6). In conclusion, mucosal ATP opens both CFTR and CaCC and may stimulate pancrea secretion, while serosal ATP regulates secretion partly through CFTR. In addition, ATP stimulation also regulates IK channels that provide the driving force for Cl− channel dependent secretions.

Purified Human Pancreatic Duct Cell Culture Conditions Defined by Serum-Free High-Content Growth Factor Screening

The proliferation of pancreatic duct-like CK19+ cells has implications for multiple disease states including pancreatic cancer and diabetes mellitus. The in vitro study of this important cell type has been hampered by their limited expansion compared to fibroblast-like vimentin+ cells that overgrow primary cultures. We aimed to develop a screening platform for duct cell mitogens after depletion of the vimentin+ population. The CD90 cell surface marker was used to remove the vimentin+ cells from islet-depleted human pancreas cell cultures by magnetic-activated cell sorting. Cell sorting decreased CD90+ cell contamination of the cultures from 34±20% to 1.3±0.6%, yielding purified CK19+ cultures with epithelial morphology. A full-factorial experimental design was then applied to test the mitogenic effects of bFGF, EGF, HGF, KGF and VEGF. After 6 days in test conditions, the cells were labelled with BrdU, stained and analyzed by high-throughput imaging. This screening assay confirmed the expected mitogenic effects of bFGF, EGF, HGF and KGF on CK19+ cells and additionally revealed interactions between these factors and VEGF. A serum-free medium containing bFGF, EGF, HGF and KGF led to CK19+ cell expansion comparable to the addition of 10% serum. The methods developed in this work should advance pancreatic cancer and diabetes research by providing effective cell culture and high-throughput screening platforms to study purified primary pancreatic CK19+ cells.

Concomitant inhibition of HSP90, its mitochondrial localized homologue TRAP1 and HSP27 by green tea in pancreatic cancer HPAF‐II cells

Pancreatic cancer is a deadly disease characterized by poor prognosis and patient survival. Green tea polyphenols have been shown to exhibit multiple antitumor activities in various cancers, but studies on the pancreatic cancer are very limited. To identify the cellular targets of green tea action, we exposed a green tea extract (GTE) to human pancreatic ductal adenocarcinoma HPAF-II cells and performed two-dimensional gel electrophoresis of the cell lysates. We identified 32 proteins with significantly altered expression levels. These proteins are involved in drug resistance, gene regulation, motility, detoxification and metabolism of cancer cells. In particular, we found GTE inhibited molecular chaperones heat-shock protein 90 (Hsp90), its mitochondrial localized homologue Hsp75 (tumor necrosis factor receptor-associated protein 1, or Trap1) and heat-shock protein 27 (Hsp27) concomitantly. Western blot analysis confirmed the inhibition of Hsp90, Hsp75 and Hsp27 by GTE, but increased phosphorylation of Ser78 of Hsp27. Furthermore, we showed that GTE inhibited Akt activation and the levels of mutant p53 protein, and induced apoptosis and growth suppression of the cells. Our study has identified multiple new molecular targets of GTE and provided further evidence on the anticancer activity of green tea in pancreatic cancer.

Synthesis of steroidal quinones and hydroquinones from bile acids by Barton radical decarboxylation and benzoquinone addition. Studies on their cytotoxic and antifungal activities

Twelve new hydroquinones and quinones ( 4a–c to 7a–c) derived from free or peracetylated bile acids were prepared by a Barton decarboxylation reaction, with subsequent trapping of the resulting free radical by benzoquinone. All new compounds were completely characterized by 2D NMR techniques and screened for antifungal and cytotoxic activity. One of the new hydroquinones ( 7b) showed promising results against the human pancreatic ductal carcinoma cell line PANC1, with similar cytotoxic activity as the commercial chemotherapy drug doxorubicin.

Combination Therapy with a Dipeptidyl Peptidase-4 Inhibitor and a Proton Pump Inhibitor Induces β-Cell Neogenesis from Adult Human Pancreatic Duct Cells Implanted in Immunodeficient Mice

Combination therapy with a dipeptidyl peptidase-4 inhibitor (DPP-4i) and a proton pump inhibitor (PPI) raises endogenous levels of GLP-1 and gastrin, respectively, and restores pancreatic β-cell mass and normoglycemia in nonobese diabetic (NOD) mice with autoimmune diabetes. The aim of this study was to determine whether a DPP-4i and PPI combination could increase β-cell mass in the adult human pancreas. Pancreatic cells from adult human pancreas donors were implanted in NOD-severe combined immunodeficient (NOD-scid) mice and the mice were treated with a DPP-4i and a PPI for 16 weeks. Human grafts were examined for insulin content and insulin-stained cells. Graft β-cell function was assessed by intravenous glucose tolerance tests (IVGTT) and by glucose control in human cell-engrafted mice treated with streptozotocin (STZ) to delete mouse pancreatic β-cells. Plasma GLP-1 and gastrin levels were raised to two- to threefold in DPP-4i- and PPI-treated mice. Insulin content and insulin-stained cells in human pancreatic cell grafts were increased 9- to 13-fold in DPP-4i and PPI-treated mice and insulin-stained cells were colocalized with pancreatic exocrine duct cells. Plasma human C-peptide responses to IVGTT were significantly higher and STZ-induced hyperglycemia was more completely prevented in DPP-4i- and PPI-treated mice with grafts than in vehicle-treated mice with grafts. In conclusion, DPP-4i and PPI combination therapy raises endogenous levels of GLP-1 and gastrin and greatly expands the functional β-cell mass in adult human pancreatic cells implanted in immunodeficient mice, largely from pancreatic duct cells. This suggests that a DPP-4i and PPI combination treatment may provide a pharmacologic therapy to correct the β-cell deficit in type 1 diabetes.

Hypoxia-inducible factor-1α expression and gemcitabine chemotherapy for pancreatic cancer

The normal pancreas has an abundant blood flow, in contrast to pancreatic cancer, which is a hypovascular tumor. During hypoxia under a hypovascular environment, the transcription factor hypoxia-inducible factor-1α (HIF-1α) is activated. High HIF-1α expression reduces sensitivity to gemcitabine (GEM) which is used as a treatment for pancreatic cancer. The objective of this study was to clarify HIF-1α expression in pancreatic cancer and the association of its effects to GEM treatment. We used the human pancreatic ductal carcinoma cell lines AsPC-1 and BxPC-3 to evaluate cell proliferation, HIF-1α protein expression and sensitivity to GEM in a hypoxic environment of 1% O2 in 48 pancreatic cancer patients who received adjuvant GEM treatment after pancreatectomy. We divided the patients according to HIF-1α expression and the presence of single nucleotide polymorphisms, and we based our evaluation on the adverse events associated with GEM chemotherapy and patient outcome. The hypoxic environment promoted cell proliferation, induced HIF-1α expression and increased GEM resistance, especially in AsPC-1 cells, which included a mutant homozygote for HIF-1α(C1772T). There were no significant differences between the HIF-1α(-) and HIF-1α(+) groups in either adverse events or patient outcomes. HIF-1α enhanced neo-microvascularity in a hypoxic environment and increased drug resistance. The period until recurrence was shorter in the patients with a strong HIF-1α expression, than that in those with a weak HIF-1α expression.

The Id3/E47 Axis Mediates Cell-Cycle Control in Human Pancreatic Ducts and Adenocarcinoma

Pancreatic ductal adenocarcinoma (PDA) has a 5-year survival rate of less than 5%, and therapeutic advances have been hampered by gaps in our understanding of cell-cycle control in the adult pancreas. Previously, we reported that basic Helix-Loop-Helix (bHLH) transcription factors regulate cell fate specification in the pancreas. In the present study, we found that a repressor of bHLH activity, Id3, was profoundly upregulated in ductal cells in murine models of pancreatitis and pancreatic intraepithelial neoplasia (PanIN). Id3 was also pervasively expressed in neoplastic lesions in human PDA in situ. We hypothesized that an imbalance in bHLH versus Id activity controlled cell growth in PDA. Consistent with this model, cell-cycle progression in PDA cells was impeded by siRNA-mediated depletion of Id3 or overexpression of the bHLH protein E47. The precursors of human PDA are normally quiescent duct cells which do not proliferate in response to high serum or growth factors. The finding that Id3 was expressed in pancreatitis, as well as PDA, suggested that Id3 might induce cell-cycle entry in ducts. To test this hypothesis, primary human pancreatic duct cells were transduced with an adenovirus-expressing Id3. Remarkably, Id3 expression alone was sufficient to trigger efficient cell-cycle entry, as manifested by expression of the proliferation markers Ki67, phospho-cyclin E, and phospho-histone H3. Collectively, the data establish dysregulation of the Id/bHLH axis as an early and sustained feature of ductal pathogenesis and mark this axis as a potential therapeutic target for intervention in pancreatitis and PDA.

Abstract 2817: An RNA aptamer is a potential biomarker for pancreatic cancer

Abstract Introduction: The symptoms of pancreatic cancer are usually non-specific and late. Existing tumor markers (such as CA19-9 and CEA) are neither sensitive nor specific enough to serve as useful screening tests. Tools for earlier detection could increase the proportion of patients who are cured with surgical resection. Aptamers are oligonucleotide ligands that are identified from large libraries of RNA or DNA molecules using an iterative selection process known as SELEX (Systematic Evolution of Ligands by Exponential Enrichment). Aptamers bind their specific targets with high affinity and specificity and can be used for the dual purpose of identifying novel biomarkers and detecting them in biological specimens. Methods: In order to select for aptamers against molecules that are selectively secreted by cancerous cells but not by normal cells, “positive/negative” SELEX was performed using the conditioned media (the “secretome”) of the MiaPaCa2 human pancreatic ductal adenocarcinoma cell line (positive selection) and the immortalized, non-cancerous human pancreatic ductal cell line HPDE (negative selection). Standard radioactive filter-based binding assays were used to measure binding. Results/Conclusion: We have successfully selected several nuclease resistant (2′-fluoro-modified) RNA aptamers that differentially bind the secretome of pancreatic cancer cells compared to non-cancerous cells. One of the aptamers (M9-5) binds pancreatic cancer patient sera with high affinity compared to control sera collected from healthy donors. So far, the aptamer has demonstrated specific binding in 20 of 22 patients with histologically-proven pancreatic cancer and none of 20 healthy donors. In addition, paired serum samples obtained from two patients pre- and post-chemoradiation therapy and one patient pre- and two months post-resection have demonstrated a decrease in M9-5 binding with decreased tumor burden. Notably, the serum CA19-9 and CEA levels in several of these patients were normal, indicating that our biomarker might be more sensitive than these existing serum biomarkers for pancreatic cancer. M9-5 binding to the MiaPaCa2 “secretome” is sensitive to Proteinase K treatment indicating that the aptamer target is a protein. Currently, we are using a biotinylated version of the aptamer to affinity purify the target and identify it by mass-spectrometric techniques. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2817. doi:10.1158/1538-7445.AM2011-2817

Prosurvival role of heat shock factor 1 in the pathogenesis of pancreatobiliary tumors.

Several mechanisms have evolved to ensure the survival of cells under adverse conditions. The heat shock response is one such evolutionarily conserved survival mechanism. Heat shock factor-1 (HSF1) is a transcriptional regulator of the heat shock response. By the very nature of its prosurvival function, HSF1 may contribute to the pathogenesis of cancer. The current study investigates the role of HSF1 in the pathogenesis of pancreatobiliary tumors. HSF1 was downregulated in pancreatic cancer (MIA PaCa-2 and S2-013) and cholangiocarcinoma (KMBC and KMCH) cell lines by HSF1-specific small interfering RNA (siRNA). Nonsilencing siRNA was used as control. The effect of HSF1 downregulation on viability and apoptosis parameters, i.e., annexin V, terminal deoxynucleotidyl transferase dUTP-mediated nick end labeling (TUNEL), and caspase-3, was measured. To evaluate the cancer-specific effects of HSF1, the effect of HSF1 downregulation on normal human pancreatic ductal cells was also evaluated. HSF1 is abundantly expressed in human pancreatobiliary cancer cell lines, as well as in pancreatic cancer tissue, as demonstrated by Western blot and immunohistochemistry, respectively. Inhibition of HSF1 expression by the HSF1 siRNA sequences leads to time-dependent death in pancreatic and cholangiocarcinoma cell lines. Downregulation of HSF1 expression induces annexin V and TUNEL positivity and caspase-3 activation, suggesting activation of a caspase-dependent apoptotic pathway. Although caspase-3 inhibition protects against cell death induced by HSF1 expression, it does not completely prevent it, suggesting a role for caspase-independent cell death. HSF1 plays a prosurvival role in the pathogenesis of pancreatobiliary tumors. Modulation of HSF1 activity could therefore emerge as a novel therapeutic strategy for cancer treatment.

Pancreatic cancer bears overexpression of neurotensin and neurotensin receptor subtype-1 and SR 48692 counteracts neurotensin induced cell proliferation in human pancreatic ductal carcinoma cell line PANC-1

The presence of neurotensin and neurotensin receptors has been demonstrated in human pancreatic carcinomas using autoradiography and Northern blot analysis. In vitro studies have reported that the neurotensin antagonist SR 48692 could inhibit the growth of MIA PaCa-2 cells in a neurotensin mediated fashion, and neurotensin could overcome this inhibition or stimulate proliferation. However, it is currently unknown whether such actions are exerted on PANC-1 cells. In addition, the immunolocation of neurotensin and neurotensin receptors is still unclear in human pancreatic ductal carcinoma tissues. Immunohistochemistry was applied to detect the distribution of neurotensin and neurotensin receptor subtype-1 in human pancreatic ductal carcinoma and normal pancreatic tissues. Furthermore, an in vitro study was carried out to test the pharmacological profile of neurotensin and SR 48692 in human pancreatic ductal carcinoma cell line PANC-1. Compared with normal pancreatic tissues, pancreatic ductal carcinoma tissues have higher neurotensin and neurotensin receptor subtype-1 expression rates. Pancreatic ductal carcinomas (32/40) bear the expression of both neurotensin and neurotensin receptor subtype-1. We observed that neurotensin (10⁻¹¹-10⁻⁷ M) significantly stimulated the proliferation of PANC-1 and SR 48692 (10⁻¹¹-10⁻⁷ M) alone had no effect on the growth of PANC-1 cells; however, SR 48692 (10⁻¹⁰-10⁻⁶ M) inhibited the stimulatory effect of neurotensin (10⁻⁹ M). Considering the overexpression of both neurotensin and neurotensin receptor subtype-1 in pancreatic ductal carcinomas, it could enable us to develop markers for pancreatic cancer diagnosis. As SR 48692 could inhibit neurotensin induced cell growth, neurotensin receptor subtype-1 may serve as a therapeutic target for the therapy of pancreatic carcinomas. Furthermore, our study indicates that the counteraction of neurotensin and neurotensin receptor subtype-1 regulates the genesis and development of pancreatic carcinomas.

Transforming growth factor‐β induces epithelial to mesenchymal transition and suppresses the proliferation and transdifferentiation of cultured human pancreatic duct cells

Pancreatic duct cells are considered a potential source of β-cell regeneration, and transforming growth factor-β (TGF-β) has been suggested to perform an important role in these processes, but the underlying mechanism of the signal pathways, especially in humans, remains poorly understood. To evaluate the role of TGF-β1, pancreatic duct cells were isolated from three brain-dead organ donors. Pancreatic cell clusters harvested after islet isolation were dispersed to single cells and cultured in monolayers, then treated with TGF-β1. We analyzed the characteristics of the cultured cells, the TGF-β1 intracellular signaling pathway, the proliferation, and transdifferentiation rates of the duct cells. We also evaluated the genes and protein expression patterns after TGF-β1 treatment. After TGF-β1 treatment, typical morphologic changes representative of EMT were observed and Erk1/2, JNK, and AKT phosphorylation, Ras downstream effectors, were increased. β cell-specific transcription factors including PDX-1, Beta2/NeuroD, Ist-1, and NGN3 were markedly suppressed and the rate of transdifferentiation into β cells was also suppressed. Genomic and proteomic analyses suggested that TGF-β1 induces marked changes in a variety of structural genes and proteins associated with EMT. In conclusion, TGF-β1 induces EMT in cultured human pancreatic duct cells, but suppresses its proliferation and transdifferentiation into β cells. Our results are the first report of TGF-β1 effects for EMT and ductal cell transdifferentiation and proliferation at the protein level in human pancreatic duct cells.

MicroRNA miR-17-5p is overexpressed in pancreatic cancer, associated with a poor prognosis, and involved in cancer cell proliferation and invasion

The microRNA-17-92 cluster is an oncogene in human B cell lymphomas and lung cancers. Previous microRNA microarray data revealed that miR-17-5p, a member of the miR-17-92 cluster, is upregulated in pancreatic cancer. However, the involvement of miR-17-5p expression in pancreatic carcinogenesis has not well been studied. In the present study, we measured the miR-17-5p expression levels in pancreatic cancer cell lines, primary cultures of normal human pancreatic ductal cells, formalin-fixed paraffin-embedded (FFPE) tissue samples derived from 80 patients who underwent pancreatectomy for pancreatic cancer and microdissected cells (including normal ductal epithelial, pancreatic intraepithelial neoplasia-1B and invasive ductal carcinoma cells) by qRT-PCR. Furthermore, we investigated the effects of upregulation of miR-17-5p expression on the proliferation and invasion of pancreatic cancer cells. We found that pancreatic cancer cells expressed higher levels of miR-17-5p than primary cultured normal ductal cells. miR-17-5p was also overexpressed in pancreatic cancer in FFPE and microdissected samples. Furthermore, analysis of macrodissected FFPE samples revealed that high miR-17-5p expression was associated with a poor prognosis (p = 0.03). In addition, in vitro experiments revealed that SUIT-2 and KP-2 pancreatic cancer cells transfected with the miR-17-5p precursor showed significantly higher cell growth ratios than the corresponding control cells (p < 0.001 and p = 0.012, respectively), as well as significantly higher numbers of invading cells (p < 0.0001 for both). The present findings suggest that miR-17-5p plays important roles in pancreatic carcinogenesis and cancer progression, and is associated with a poor prognosis in pancreatic cancer.

Molecular mechanisms underlying Ca2+-mediated motility of human pancreatic duct cells

We recently reported that transforming growth factor-β (TGF-β) induces an increase in cytosolic Ca(2+) ([Ca(2+)](cyt)) in pancreatic cancer cells, but the mechanisms by which TGF-β mediates [Ca(2+)](cyt) homeostasis in these cells are currently unknown. Transient receptor potential (TRP) channels and Na(+)/Ca(2+) exchangers (NCX) are plasma membrane proteins that play prominent roles in controlling [Ca(2+)](cyt) homeostasis in normal mammalian cells, but little is known regarding their roles in the regulation of [Ca(2+)](cyt) in pancreatic cancer cells and pancreatic cancer development. Expression and function of NCX1 and TRPC1 proteins were characterized in BxPc3 pancreatic cancer cells. TGF-β induced both intracellular Ca(2+) release and extracellular Ca(2+) entry in these cells; however, 2-aminoethoxydiphenyl borate [2-APB; a blocker for both inositol 1,4,5-trisphosphate (IP(3)) receptor and TRPC], LaCl(3) (a selective TRPC blocker), or KB-R7943 (a selective inhibitor for the Ca(2+) entry mode of NCX) markedly inhibited the TGF-β-induced increase in [Ca(2+)](cyt). 2-APB or KB-R7943 treatment was able to dose-dependently reverse membrane translocation of PKCα induced by TGF-β. Transfection with small interfering RNA (siRNA) against NCX1 almost completely abolished NCX1 expression in BxPc3 cells and also inhibited PKCα serine phosphorylation induced by TGF-β. Knockdown of NCX1 or TRPC1 by specific siRNA transfection reversed TGF-β-induced pancreatic cancer cell motility. Therefore, TGF-β induces Ca(2+) entry likely via TRPC1 and NCX1 and raises [Ca(2+)](cyt) in pancreatic cancer cells, which is essential for PKCα activation and subsequent tumor cell invasion. Our data suggest that TRPC1 and NCX1 may be among the potential therapeutic targets for pancreatic cancer.

C-Kit and stem cell factor regulate PANC-1 cell differentiation into insulin- and glucagon-producing cells

Recent evidence has shown that stem cell factor (SCF) and its receptor, c-Kit, have an important role in pancreatic islet development by promoting islet cell differentiation and proliferation. In this study, we examined the role of c-Kit and SCF in the differentiation and proliferation of insulin- and glucagon-producing cells using a human pancreatic duct cell line (PANC-1). Our study showed that increased expression of endocrine cell markers (such as insulin and glucagon) and transcription factors (such as PDX-1 and PAX-6) coincided with a decrease in CK19+ and c-Kit+ cells (P<0.001) during PANC-1 cell differentiation, determined by immunofluorescence and qRT-PCR. Cells cultured with exogenous SCF showed an increase in insulin+ (26%) and glucagon+ (35%) cell differentiation (P<0.01), an increase in cell proliferation (P<0.05) and a decrease in cell apoptosis (P<0.01). siRNA knockdown of c-Kit resulted in a decrease in endocrine cell differentiation with a reduction in PDX-1 and insulin mRNA, as well as the number of cells immunostaining for PDX-1 and insulin. Taken together, these results show that c-Kit/SCF interactions are involved in mediating islet-like cluster formation and islet-like cell differentiation in a human pancreatic duct cell line.

DCB-3503, a Tylophorine Analog, Inhibits Protein Synthesis through a Novel Mechanism

BackgroundDCB-3503, a tylophorine analog, inhibits the growth of PANC-1 (human pancreatic ductal cancer cell line) and HepG2 (human hepatocellular cancer cell line) tumor xenografts in nude mice. The inhibition of growth leads to cancer cell differentiation instead of cell death. However, the mechanisms of action of tylophorine analogs is unknown.Methodology/Principal FindingsIn this study, we show that DCB-3503 suppresses the expression of pro-oncogenic or pro-survival proteins with short half-lives, including cyclin D1, survivin, β-catenin, p53, and p21, without decreasing their mRNA levels. Proteasome inhibitor reversed the inhibitory effect of DCB-3503 on expression of these proteins. DCB-3503 inhibited the incorporation of radiolabeled amino acid and thymidine, and to a much lesser degree of uridine, in a panel of cell lines. The mechanism of inhibition of protein synthesis is different from that of cycloheximide (CHX) as assayed in cell culture and HeLa in vitro translation system. Furthermore, in contrast to rapamycin, DCB-3503 does not affect protein synthesis through the mTOR pathway. DCB-3503 treatment shifts the sedimentation profiles of ribosomes and mRNAs towards the polysomal fractions while diminishing monosome abundance, indicative of the inhibition of the elongation step of protein synthesis. Preferential down regulation of several studied proteins under these conditions is likely due to the relative short half-lives of these proteins.Conclusion/SignificanceThe inhibitory effect of DCB-3503 on translation is apparently distinct from any of the current anticancer compounds targeting protein synthesis. Translation inhibitors with novel mechanism could complement current chemotherapeutic agents for the treatment of human cancers and suppress the occurrence of drug resistance.

Abstract 2456: Mechanism of pancreatic cancer induction by smoking

Abstract Background &amp; Aims: Cigarette smoking is a major risk factor for pancreatic cancer. However, the molecular signaling mechanisms of cigarette smoking remain poorly understood. We studied the effects of the cigarette smoking extract (CSE) and the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) on apoptosis, proliferation and autophagy and underlying signaling mechanisms both in vitro in human pancreatic ductal cells and in vivo in mouse pancreas. We also studied the effect of K-ras mutation on the smoking-induced cell survival and proliferation. Methods: Immortalized human ductal cells HPDE6 were cultured for up to 1 week in the presence or absence of CSE (0.04-4µg/ml) or NNK (1nM-1µM). Pancreas-specific K-ras transgenic mice EL-K-ras were used as a cancer model. Wild type and EL-K-ras mice were injected with NNK (100mg/Kg) once weekly for 4 weeks and animals sacrificed 72h after the last injection. Apoptosis was assessed by measuring DNA fragmentation with Cell death ELISA or TUNEL and proliferation by 3H-thymidine incorporation into DNA or BrdUrd labeling. Autophagy was measured with its marker LC3 using immunofluorescence staining. Molecular signaling mechanisms were evaluated with Western. Results: Exposure of human ductal cells to CSE and NNK for 24h to 1 week time-dependently decreased apoptosis and autophagy and increased proliferation. Exposure to CSE and NNK decreased AMPK phosphorylation and increased Akt phosphorylation. Akt activation occurred at the times of AMPK inhibition. Pharmacological Akt inactivation reversed the inhibition of AMPK phosphorylation by CSE and NNK and prevented the effects of CSE and NNK on apoptosis and autophagy, indicating a central role of Akt in the prosurvival effects of tobacco compounds. In K-ras mice model of pancreatic cancer NNK injections induced increase in Akt kinase and decrease in AMPK phosphorylation. NNK decreased autophagy and apoptosis and stimulated proliferation in the pancreas. The NNK inhibitory effect on apoptosis and autophagy and the stimulation of proliferation were more pronounced in K-ras mice compared to wild type mice. Conclusion: Our in vitro and in vivo data indicate that smoking compounds create conditions favorable for cancer promotion through activating survival pathways including activation of Akt kinase and inhibition of AMPK activities; this leads to a decrease in apoptosis and autophagy and stimulation of proliferation. Our data suggest that smoking works in concert with K-ras mutation to induce adenocarcinoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2456.

Dynamic Regulation of CFTR Bicarbonate Permeability by [Cl−]i and Its Role in Pancreatic Bicarbonate Secretion

Pancreatic bicarbonate (HCO3-) secretion is important for a healthy pancreas as well as digestive physiology. However, how human pancreatic duct cells secrete copious amounts of HCO3- has long been a puzzle. Here, we report that a dynamic increase in the cystic fibrosis transmembrane conductance regulator (CFTR) HCO3- permeability by intracellular Cl- concentration ([Cl-]i)-sensitive mechanisms plays a pivotal role in pancreatic HCO3- secretion. The role of [Cl-]i-sensitive kinases in CFTR-mediated HCO3- transport was examined in heterologous expression systems, PANC1 human pancreatic duct cells, and human and guinea pig pancreatic tissues using an integrated molecular and physiologic approach. In human pancreatic tissues, CFTR-positive duct cells abundantly expressed with-no-lysine (WNK1) kinase, oxidative stress-responsive kinase 1 (OSR1), and sterile 20/SPS1-related proline/alanine-rich kinase (SPAK), which are known to be activated by low [Cl-]i. Interestingly, CFTR activation rapidly decreased [Cl-]i in response to luminal Cl- depletion in polarized PANC1 human pancreatic duct cells. Notably, the WNK1-mediated OSR1 and SPAK activation by low [Cl-]i strongly increased CFTR HCO3- permeability in CFTR-transfected HEK 293T, PANC1, and guinea pig pancreatic duct cells, making CFTR primarily an HCO3- channel, which is essential for the secretion of pancreatic juice containing HCO3- at a concentration greater than 140 mmol/L. In contrast, OSR1 and SPAK activation inhibited CFTR-dependent Cl-/HCO3- exchange activity that may reabsorb HCO3- from the high HCO3--containing pancreatic juice. These results indicate that the [Cl-]i-sensitive activation of the WNK1-OSR1/SPAK pathway is the molecular switch to generate HCO3--rich fluid in the human pancreatic duct.

CFTR Expression But Not Cl− Transport Is Involved in the Stimulatory Effect of Bile Acids on Apical Cl−/HCO3 − Exchange Activity in Human Pancreatic Duct Cells

Low doses of chenodeoxycholate (CDC) stimulate apical anion exchange and HCO3(-) secretion in guinea pig pancreatic duct cells (Gut. 2008;57:1102-1112). We examined the effects of CDC on intracellular pH (pHi), intracellular Ca(2+) concentration ([Ca(2+)]i), and apical Cl(-)/HCO3(-) exchange activity in human pancreatic duct cells and determined whether any effects were dependent on cystic fibrosis transmembrane conductance regulator (CFTR) expression and Cl(-) channel activity. Polarized CFPAC-1 cells (expressing F508del CFTR) were transduced with Sendai virus constructs containing complementary DNAs for either wild-type CFTR or beta-galactosidase. Microfluorimetry was used to record pHi and [Ca(2+)]i and apical Cl(-)/HCO3(-) exchange activity. Patch clamp experiments were performed on isolated guinea pig duct cells. Chenodeoxycholate induced a dose-dependent intracellular acidification and a marked increase in [Ca(2+)]i in CFPAC-1 cells. CFTR expression slightly reduced the rate of acidification but did not affect the [Ca(2+)]i changes. Luminal administration of 0.1 mmol/L of CDC significantly elevated apical Cl(-)/HCO3(-) exchange activity but only in cells that expressed CFTR. However, CDC did not activate CFTR Cl(-) conductance. Bile salts modulate pHi, [Ca(2+)]i, and apical anion exchange activity in human pancreatic duct cells. The stimulatory effect of CDC on anion exchangers requires CFTR expression but not CFTR channel activity.