Pantoprazole and riluzole target H+/K+-ATPases and pH-sensitive K+ channels in pancreatic cancer cells.

Pancreatic ductal adenocarcinoma (PDAC) remains the most lethal cancer type. PDAC is characterized by fibrotic, hypoxic, and presumably acidic tumor microenvironment (TME). Acidic TME is an important player in tumor development, progression, aggressiveness, and chemoresistance. The dysregulation of ductal ion transporters/channels might contribute to extracellular pH (pHe) acidification and PDAC progression. Our aim was to test whether H+/K+-ATPases and pH-sensitive K+ channels contribute to these processes and could be targeted by clinically approved drugs. We used human pancreatic cancer cells adapted to various pHe conditions and grown in monolayers and spheroids. First, we created cells expressing pHoran4 at the outer plasma membrane and showed that pantoprazole, the H+/K+-ATPase inhibitor, alkalinized pHe. Second, we used FluoVolt to monitor the membrane voltage (Vm) and showed that riluzole hyperpolarized Vm, most likely by opening of pH-sensitive K+ channels such as TREK-1. Third, we show that pantoprazole and riluzole inhibited cell proliferation and viability of monolayers and spheroids of cancer cells adapted to various pHe conditions. Most importantly, combination of the two drugs had significantly larger inhibitory effects on PDAC cell survival. We propose that co-targeting H+/K+-ATPases and pH-sensitive K+ channels by re-purposing of pantoprazole and riluzole could provide novel acidosis-targeted therapies of PDAC.

Human P2X7 receptor variants Gly150Arg and Arg276His polymorphisms have differential effects on risk association and cellular functions in pancreatic cancer.

BACKGROUND: The purinergic P2X7 receptor (P2X7R) plays an important role in the crosstalk between pancreatic stellate cells (PSCs) and cancer cells, thus promoting progression of pancreatic ductal adenocarcinoma (PDAC). Single nucleotide polymorphisms (SNPs) in the P2X7R have been reported for several cancers, but have not been explored in PDAC. MATERIALS AND METHODS: Blood samples from PDAC patients and controls were genotyped for 11 non-synonymous SNPs in P2X7R and a risk analysis was performed. Relevant P2X7R-SNP GFP variants were expressed in PSCs and cancer cells and their function was assayed in the following tests. Responses in Ca2+ were studied with Fura-2 and dye uptake with YO-PRO-1. Cell migration was monitored by fluorescence microscopy. Released cytokines were measured with MSD assay. RESULTS: Risk analysis showed that two SNPs 474G>A and 853G>A (rs28360447, rs7958316), that lead to the Gly150Arg and Arg276His variants, had a significant but opposite risk association with PDAC development, protecting against and predisposing to the disease, respectively. In vitro experiments performed on cancer cells and PSCs expressing the Gly150Arg variant showed reduced intracellular Ca2+ response, fluorescent dye uptake, and cell migration, while the Arg276His variant reduced dye uptake but displayed WT-like Ca2+ responses. As predicted, P2X7R was involved in cytokine release (IL-6, IL-1ß, IL-8, TNF-α), but the P2X7R inhibitors displayed varied effects. CONCLUSION: In conclusion, we provide evidence for the P2X7R SNPs association with PDAC and propose that they could be considered as potential biomarkers.

Synergistic effects of agonists and two-pore-domain potassium channels on secretory responses of human pancreatic duct cells Capan-1.

Mechanisms of synergistic agonist stimulation and modulation of the electrochemical driving force for anion secretion are still not fully explored in human pancreatic duct epithelial cells. The first objective of this study was therefore to test whether combined agonist stimulation augments anion transport responses in the Capan-1 monolayer model of human pancreatic duct epithelium. The second objective was to test the influence of H+,K+-ATPase inhibition on anion transport in Capan-1 monolayers. The third objective was to analyze the expression and function of K+ channels in Capan-1, which could support anion secretion and cooperate with H+,K+-ATPases in pH and potassium homeostasis. The human pancreatic adenocarcinoma cell line Capan-1 was cultured conventionally or as polarized monolayers that were analyzed by Ussing chamber electrophysiological recordings. Single-cell intracellular calcium was assayed with Fura-2. mRNA isolated from Capan-1 was analyzed by use of the nCounter assay or RT-PCR. Protein expression was assessed by immunofluorescence and western blot analyses. Combined stimulation with different physiological agonists enhanced anion transport responses compared to single agonist stimulation. The responsiveness of Capan-1 cells to histamine was also revealed in these experiments. The H+,K+-ATPase inhibitor omeprazole reduced carbachol- and riluzole-induced anion transport responses. Transcript analyses revealed abundant TASK-2, TWIK-1, TWIK-2, TASK-5, KCa3.1, and KCNQ1 mRNA expression. KCNE1 mRNA and TREK-1, TREK-2, TASK-2, and KCNQ1 protein expression were also shown. This study shows that the Capan-1 model recapitulates key physiological aspects of a bicarbonate-secreting epithelium and constitutes a valuable model for functional studies on human pancreatic duct epithelium.

Bile acid effects are mediated by ATP release and purinergic signalling in exocrine pancreatic cells.

BACKGROUND: In many cells, bile acids (BAs) have a multitude of effects, some of which may be mediated by specific receptors such the TGR5 or FXR receptors. In pancreas systemic BAs, as well as intra-ductal BAs from bile reflux, can affect pancreatic secretion. Extracellular ATP and purinergic signalling are other important regulators of similar secretory mechanisms in pancreas. The aim of our study was to elucidate whether there is interplay between ATP and BA signalling. RESULTS: Here we show that CDCA (chenodeoxycholic acid) caused fast and concentration-dependent ATP release from acini (AR42J) and duct cells (Capan-1). Taurine and glycine conjugated forms of CDCA had smaller effects on ATP release in Capan-1 cells. In duct monolayers, CDCA stimulated ATP release mainly from the luminal membrane; the releasing mechanisms involved both vesicular and non-vesicular secretion pathways. Duct cells were not depleted of intracellular ATP with CDCA, but acinar cells lost some ATP, as detected by several methods including ATP sensor AT1.03(YEMK). In duct cells, CDCA caused reversible increase in the intracellular Ca(2+) concentration [Ca(2 +)]i, which could be significantly inhibited by antagonists of purinergic receptors. The TGR5 receptor, expressed on the luminal side of pancreatic ducts, was not involved in ATP release and Ca(2+) signals, but could stimulate Na(+)/Ca(2+) exchange in some conditions. CONCLUSIONS: CDCA evokes significant ATP release that can stimulate purinergic receptors, which in turn increase [Ca(2+)]i. The TGR5 receptor is not involved in these processes but can play a protective role at high intracellular Ca(2+) conditions. We propose that purinergic signalling could be taken into consideration in other cells/organs, and thereby potentially explain some of the multifaceted effects of BAs.

Imaging of extracellular and intracellular ATP in pancreatic beta cells reveals correlation between glucose metabolism and purinergic signalling.

Adenosine triphosphate (ATP) is a universal energy molecule and yet cells release it and extracellular ATP is an important signalling molecule between cells. Monitoring of ATP levels outside of cells is important for our understanding of physiological and pathophysiological processes in cells/tissues. Here, we focus on pancreatic beta cells (INS-1E) and test the hypothesis that there is an association between intra- and extracellular ATP levels which depends on glucose provision. We imaged real-time changes in extracellular ATP in pancreatic beta cells using two sensors tethered to extracellular aspects of the plasma membrane (eATeam3.10, iATPSnFR1.0). Increase in glucose induced fast micromolar ATP release to the cell surface, depending on glucose concentrations. Chronic pre-treatment with glucose increased the basal ATP signal. In addition, we co-expressed intracellular ATP sensors (ATeam1.30, PercevalHR) in the same cultures and showed that glucose induced fast increases in extracellular and intracellular ATP. Glucose and extracellular ATP stimulated glucose transport monitored by the glucose sensor (FLII12Pglu-700uDelta6). In conclusion, we propose that in beta cells there is a dynamic relation between intra- and extracellular ATP that depends on glucose transport and metabolism and these processes may be tuned by purinergic signalling. Future development of ATP sensors for imaging may aid development of novel approaches to target extracellular ATP in, for example, type 2 diabetes mellitus therapy.

Live-cell imaging of viral RNA genomes using a Pumilio-based reporter.

We describe a method for localizing plant viral RNAs in vivo using Pumilio, an RNA-binding protein, coupled to bimolecular fluorescence complementation (BiFC). Two Pumilio homology domain (PUMHD) polypeptides, fused to either the N- or C-terminal halves of split mCitrine, were engineered to recognize two closely adjacent eight-nucleotide sequences in the genomic RNA of tobacco mosaic virus (TMV). Binding of the PUMHDs to their target sites brought the split mCitrine halves into close proximity, allowing BiFC to occur and revealing the localization of viral RNA within infected cells. The bulk of the RNA was sequestered in characteristic inclusion bodies known as viral replication complexes (VRCs), with a second population of RNA localized in discrete particles distributed throughout the peripheral cytoplasm. Transfer of the TMV Pumilio recognition sequences into the genome of potato virus X (PVX) allowed the PVX RNA to be localized. Unlike TMV, the PVX RNA was concentrated in distinctive 'whorls' within the VRC. Optical sectioning of the PVX VRCs revealed that one of the viral movement proteins was localized to the centres of the RNA whorls, demonstrating significant partitioning of viral RNA and proteins within the VRC. The utility of Pumilio as a fluorescence-based reporter for viral RNA is discussed.

Proton Pump Inhibitors Reduce Pancreatic Adenocarcinoma Progression by Selectively Targeting H+, K+-ATPases in Pancreatic Cancer and Stellate Cells.

Pancreatic duct cells are equipped with acid/base transporters important for exocrine secretion. Pancreatic ductal adenocarcinoma (PDAC) cells may utilize such transporters to acidify extracellular tumor microenvironment, creating a niche favoring cell proliferation, fibrosis and resistance to chemotherapy-all contributing to the notoriously bad prognosis of this disease. Here, we report that gastric and non-gastric H+, K+-ATPases (coded by ATP4A and ATP12A) are overexpressed in human and murine pancreatic cancer and that we can target them specifically with proton pump inhibitors (PPIs) and potassium-competitive acid blockers (P-CABs) in in vitro models of PDAC. Focusing on pantoprazole, we show that it significantly reduced human cancer cell proliferation by inhibiting cellular H+ extrusion, increasing K+ conductance and promoting cyclin D1-dependent cell cycle arrest and preventing STAT3 activation. Pantoprazole also decreased collagen secretion from pancreatic stellate cells. Importantly, in vivo studies show that pantoprazole treatment of tumor-bearing mice reduced tumor size, fibrosis and expression of angiogenic markers. This work provides the first evidence that H+, K+-ATPases contribute to PDAC progression and that these can be targeted by inhibitors of these pumps, thus proving a promising therapeutic strategy.

Phytoplasmas and their interactions with hosts.

Phytoplasmas are bacteria without cell walls and are responsible for plant diseases that have large economic impacts. Knowledge of their biology is limited because they are uncultivable and experimentally inaccessible in their hosts. It is a mystery how these bacteria use the sugar-rich phloem sap in which they live and how they interact with the host. This makes it difficult to develop means to control them. Recently, the full genomes of two phytoplasmas have been sequenced, allowing new insights into their requirements. Phytoplasmas contain a minimal genome and lack genes coding for ATP synthases and sugar uptake and use, making them dependent on their host. This dependency can be exploited to elucidate the particular physiology of the phloem.

Evaluating nanoparticle sensor design for intracellular pH measurements.

Particle-based nanosensors have over the past decade been designed for optical fluorescent-based ratiometric measurements of pH in living cells. However, quantitative and time-resolved intracellular measurements of pH in endosomes and lysosomes using particle nanosensors are challenging, and there is a need to improve measurement methodology. In the present paper, we have successfully carried out time-resolved pH measurements in endosomes and lyosomes in living cells using nanoparticle sensors and show the importance of sensor choice for successful quantification. We have studied two nanoparticle-based sensor systems that are internalized by endocytosis and elucidated important factors in nanosensor design that should be considered in future development of new sensors. From our experiments it is clear that it is highly important to use sensors that have a broad measurement range, as erroneous quantification of pH is an unfortunate result when measuring pH too close to the limit of the sensitive range of the sensors. Triple-labeled nanosensors with a pH measurement range of 3.2-7.0, which was synthesized by adding two pH-sensitive fluorophores with different pK(a) to each sensor, seem to be a solution to some of the earlier problems found when measuring pH in the endosome-lysosome pathway.

Advances in imaging RNA in plants.

Increasing evidence shows that many RNAs are targeted to specific locations within cells, and that RNA-processing pathways occur in association with specific subcellular structures. Compartmentation of mRNA translation and RNA processing helps to assemble large RNA-protein complexes, while RNA targeting allows local protein synthesis and the asymmetric distribution of transcripts during cell polarisation. In plants, intercellular RNA trafficking also plays an additional role in plant development and pathogen defence. Methods that allow the visualisation of RNA sequences within a cellular context, and preferably at subcellular resolution, can help to answer important questions in plant cell and developmental biology. Here, we summarise the approaches currently available for localising RNA in vivo and address the specific limitations inherent with plant systems.

Development of Cuscuta species on a partially incompatible host: induction of xylem transfer cells.

The growth of dodders, Cuscuta reflexa and Cuscuta japonica, on the partially incompatible host poinsettia ( Euphorbia pulcherrima) is studied. Poinsettia responds by bark growths to the formation of the dodder haustoria and prevents dodder from obtaining normal growth. The growth instead becomes extremely branched, coral-like, and dodder lacks the ability to form haustoria. After a period of coral-like growth, long shoots sprout, resembling the normal growth. These long shoots mark an ending phase for dodder, which dies shortly after without having flowered. During the coral-like growth phase, dodder develops transfer cells in the parenchyma cells bordering the vessels of the xylem in the shoot. The transfer cells have not been observed when dodder is grown on the compatible host Pelargonium zonale. A coral-like growth phase has also been observed at the establishing phase when dodder is grown in vitro on agar; later a more normal growth form takes over. In this coral phase, xylem transfer cells are also developed. The fluorochromes carboxyfluorescein and Texas Red were loaded into the host in the phloem and xylem, respectively, and detection of these fluorochromes in the dodder stem indicated that a functional haustorial contact developed for both vascular systems. The results show that Cuscutaspp. have the genetic ability to develop xylem transfer cells and use this in response to developmental stress.