Investigating the influence of taurochenodeoxycholic acid (TCDCA) on pancreatic cancer cell behavior: An RNA Sequencing Approach.

Pancreatic cancer (PC) poses a substantial global health challenge, ranking as the fourth leading cause of cancer-related deaths due to its high mortality rate. Late-stage diagnoses are common due to the absence of specific symptoms. Pancreatic ductal adenocarcinoma (PDAC) accounts for the majority of PC cases. Recent research has suggested a potential link between elevated serum levels of bile acids (BAs) and tumorigenesis of PDAC. This study aims to understand how taurochenodeoxycholic acid (TCDCA), a secondary BA, influences PDAC using RNA sequencing techniques on the Capan-1 cell line. We identified 2,950 differentially expressed genes (DEGs) following TCDCA treatment, with 1,597 upregulated and 1,353 downregulated genes. These DEGs were associated with critical PDAC pathways, including coagulation, angiogenesis, cell migration, and signaling regulation. Furthermore, we reviewed relevant literature highlighting genes like DKK-1, KRT80, UPLA, and SerpinB2, known for their roles in PDAC tumorigenesis and metastasis. Our study sheds light on the complex relationship between BAs and PDAC, offering insights into potential diagnostic markers and therapeutic targets. Further research is needed to unravel these findings' precise mechanisms and clinical implications, potentially improving PDAC diagnosis and treatment.

Role of CFTR in diabetes-induced pancreatic ductal fluid and HCO3 - secretion.

Type 1 diabetes is a disease of the endocrine pancreas; however, it also affects exocrine function. Although most studies have examined the effects of diabetes on acinar cells, much less is known regarding ductal cells, despite their important protective function in the pancreas. Therefore, we investigated the effect of diabetes on ductal function. Diabetes was induced in wild-type and cystic fibrosis transmembrane conductance regulator (CFTR) knockout mice following an i.p. administration of streptozotocin. Pancreatic ductal fluid and HCO3 - secretion were determined using fluid secretion measurements and fluorescence microscopy, respectively. The expression of ion transporters was measured by real-time PCR and immunohistochemistry. Transmission electron microscopy was used for the morphological characterization of the pancreas. Serum secretin and cholecystokinin levels were measured by an enzyme-linked immunosorbent assay. Ductal fluid and HCO3 - secretion, CFTR activity, and the expression of CFTR, Na+ /H+ exchanger-1, anoctamine-1 and aquaporin-1 were significantly elevated in diabetic mice. Acute or chronic glucose treatment did not affect HCO3 - secretion, but increased alkalizing transporter activity. Inhibition of CFTR significantly reduced HCO3 - secretion in both normal and diabetic mice. Serum levels of secretin and cholecystokinin were unchanged, but the expression of secretin receptors significantly increased in diabetic mice. Diabetes increases fluid and HCO3 - secretion in pancreatic ductal cells, which is associated with the increased function of ion and water transporters, particularly CFTR. KEY POINTS: There is a lively interaction between the exocrine and endocrine pancreas not only under physiological conditions, but also under pathophysiological conditions The most common disease affecting the endocrine part is type-1 diabetes mellitus (T1DM), which is often associated with pancreatic exocrine insufficiency Compared with acinar cells, there is considerably less information regarding the effect of diabetes on pancreatic ductal epithelial cells, despite the fact that the large amount of fluid and HCO3 - produced by ductal cells is essential for maintaining normal pancreatic functions Ductal fluid and HCO3 - secretion increase in T1DM, in which increased cystic fibrosis transmembrane conductance regulator activation plays a central role. We have identified a novel interaction between T1DM and ductal cells. Presumably, the increased ductal secretion represents a defence mechanism in the prevention of diabetes, but further studies are needed to clarify this issue.

Calcium imaging in intact mouse acinar cells in acute pancreas tissue slices.

The physiology and pathophysiology of the exocrine pancreas are in close connection to changes in intra-cellular Ca2+ concentration. Most of our knowledge is based on in vitro experiments on acinar cells or acini enzymatically isolated from their surroundings, which can alter their structure, physiology, and limit our understanding. Due to these limitations, the acute pancreas tissue slice technique was introduced almost two decades ago as a complementary approach to assess the morphology and physiology of both the endocrine and exocrine pancreas in a more conserved in situ setting. In this study, we extend previous work to functional multicellular calcium imaging on acinar cells in tissue slices. The viability and morphological characteristics of acinar cells within the tissue slice were assessed using the LIVE/DEAD assay, transmission electron microscopy, and immunofluorescence imaging. The main aim of our study was to characterize the responses of acinar cells to stimulation with acetylcholine and compare them with responses to cerulein in pancreatic tissue slices, with special emphasis on inter-cellular and inter-acinar heterogeneity and coupling. To this end, calcium imaging was performed employing confocal microscopy during stimulation with a wide range of acetylcholine concentrations and selected concentrations of cerulein. We show that various calcium oscillation parameters depend monotonically on the stimulus concentration and that the activity is rather well synchronized within acini, but not between acini. The acute pancreas tissue slice represents a viable and reliable experimental approach for the evaluation of both intra- and inter-cellular signaling characteristics of acinar cell calcium dynamics. It can be utilized to assess many cells simultaneously with a high spatiotemporal resolution, thus providing an efficient and high-yield platform for future studies of normal acinar cell biology, pathophysiology, and screening pharmacological substances.

Inhibition of NHE-1 Increases Smoke-Induced Proliferative Activity of Barrett's Esophageal Cell Line.

Several clinical studies indicate that smoking predisposes its consumers to esophageal inflammatory and malignant diseases, but the cellular mechanism is not clear. Ion transporters protect esophageal epithelial cells by maintaining intracellular pH at normal levels. In this study, we hypothesized that smoking affects the function of ion transporters, thus playing a role in the development of smoking-induced esophageal diseases. Esophageal cell lines were treated with cigarettesmoke extract (CSE), and the viability and proliferation of the cells, as well as the activity, mRNA and protein expression of the Na+/H+ exchanger-1 (NHE-1), were studied. NHE-1 expression was also investigated in human samples. For chronic treatment, guinea pigs were exposed to tobacco smoke, and NHE-1 activity was measured. Silencing of NHE-1 was performed by using specific siRNA. CSE treatment increased the activity and protein expression of NHE-1 in the metaplastic cells and decreased the rate of proliferation in a NHE-1-dependent manner. In contrast, CSE increased the proliferation of dysplastic cells independently of NHE-1. In the normal cells, the expression and activity of NHE-1 decreased due to in vitro and in vivo smoke exposure. Smoking enhances the function of NHE-1 in Barrett's esophagus, and this is presumably a compensatory mechanism against this toxic agent.

Mechanisms of Post-Pancreatitis Diabetes Mellitus and Cystic Fibrosis-Related Diabetes: A Review of Preclinical Studies.

Anatomical proximity and functional correlations between the exocrine and endocrine pancreas warrant reciprocal effects between the two parts. Inflammatory diseases of the exocrine pancreas, such as acute or chronic pancreatitis, or the presence of cystic fibrosis disrupt endocrine function, resulting in diabetes of the exocrine pancreas. Although novel mechanisms are being increasingly identified, the intra- and intercellular pathways regulating exocrine-endocrine interactions are still not fully understood, making the development of new and more effective therapies difficult. Therefore, this review sought to accumulate current knowledge regarding the pathogenesis of diabetes in acute and chronic pancreatitis, as well as cystic fibrosis.

Mouse organoid culture is a suitable model to study esophageal ion transport mechanisms.

Altered esophageal ion transport mechanisms play a key role in inflammatory and cancerous diseases of the esophagus, but epithelial ion processes have been less studied in the esophagus because of the lack of a suitable experimental model. In this study, we generated three-dimensional (3D) esophageal organoids (EOs) from two different mouse strains and characterized the ion transport processes of the EOs. EOs form a cell-filled structure with a diameter of 250-300 µm and were generated from epithelial stem cells as shown by FACS analysis. Using conventional PCR and immunostaining, the presence of Slc26a6 Cl-/HCO3- anion exchanger (AE), Na+/H+ exchanger (NHE), Na+/HCO3- cotransporter (NBC), cystic fibrosis transmembrane conductance regulator (CFTR), and anoctamin 1 Cl- channels was detected in EOs. Microfluorimetric techniques revealed high NHE, AE, and NBC activities, whereas that of CFTR was relatively low. In addition, inhibition of CFTR led to functional interactions between the major acid-base transporters and CFTR. We conclude that EOs provide a relevant and suitable model system for studying the ion transport mechanisms of esophageal epithelial cells, and they can be also used as preclinical tools to assess the effectiveness of novel therapeutic compounds in esophageal diseases associated with altered ion transport processes.

Bile accelerates carcinogenic processes in pancreatic ductal adenocarcinoma cells through the overexpression of MUC4.

Pancreatic cancer (PC) is one of the leading causes of mortality rate globally and is usually associated with obstructive jaundice (OJ). Up to date, there is no clear consensus on whether biliary decompression should be performed prior to surgery and how high levels of serum bile affects the outcome of PC. Therefore, our study aims were to characterise the effect of bile acids (BAs) on carcinogenic processes using pancreatic ductal adenocarcinoma (PDAC) cell lines and to investigate the underlying mechanisms. Liquid chromatography-mass spectrometry was used to determine the serum concentrations of BAs. The effects of BAs on tumour progression were investigated using different assays. Mucin expressions were studied in normal and PDAC cell lines and in human samples at gene and protein levels and results were validated with gene silencing. The levels of BAs were significantly higher in the PDAC + OJ group compared to the healthy control. Treating PDAC cells with different BAs or with human serum obtained from PDAC + OJ patients enhanced the rate of proliferation, migration, adhesion, colony forming, and the expression of MUC4. In PDAC + OJ patients, MUC4 expression was higher and the 4-year survival rate was lower compare to PDAC patients. Silencing of MUC4 decreased BAs-induced carcinogenic processes in PDAC cells. Our results show that BAs promote carcinogenic process in PDAC cells, in which the increased expression of MUC4 plays an important role. Based on these results, we assume that in PC patients, where the disease is associated with OJ, the early treatment of biliary obstruction improves life expectancy.

A Novel in situ Approach to Studying Pancreatic Ducts in Mice.

Introduction: The tissue slice technique offers several benefits compared to isolated cells and cell clusters that help us understand the (patho)physiology of several organs in situ. The most prominent features are preserved architecture and function, with intact homotypic and heterotypic interactions between cells in slices. In the pancreas, this technique has been utilized successfully to study acinar and endocrine islet cells. However, it has never been used to investigate ductal function. Since pancreatic ductal epithelial cells (PDECs) play an essential role in the physiology of the pancreas, our aim was to use this technique to study PDEC structure and function in situ. Materials and methods: Eight- to sixteen weeks old C57BL/6 mice were used for preparation of pancreas tissue slices. Low melting point agarose was injected into the common bile duct and the whole organ was extracted. For morphological studies, pieces of tissue were embedded in agarose and cryosectioned to obtain 15 µm thick slices. In order to visualize pancreatic ducts, (i) the Giemsa dye was added to the agarose and visualized using light microscopy or (ii) immunostaining for the cystic fibrosis transmembrane conductance regulator (CFTR) was performed. For functional characterization, agarose-embedded tissue was immediately cut to 140 µm thick tissue slices that were loaded with the cell permeant form of the Oregon Green 488 BAPTA-1 dye and used for confocal calcium imaging. Results: Giemsa staining has shown that the injected agarose reaches the head and body of the pancreas to a greater extent than the tail, without disrupting the tissue architecture. Strong CFTR expression was detected at the apical membranes of PDECs and acinar cells, whereas islet cells were completely negative for CFTR. Stimulation with chenodeoxycholic acid (CDCA, 1 mM) resulted in a robust transient increase in intracellular calcium concentration that was readily visible in >40 ductal cells per slice. Conclusion: Our results confirm that the acutely-isolated pancreas tissue slice technique is suitable for structural and functional investigation of PDECs and their relationship with other cell types, such as acini and endocrine cells in situ. In combination with different genetic, pharmacological or dietary approaches it could become a method of choice in the foreseeable future.

The Importance of Aquaporin 1 in Pancreatitis and Its Relation to the CFTR Cl- Channel.

Aquaporins (AQPs) facilitate the transepithelial water flow involved in epithelial fluid secretion in numerous tissues; however, their function in the pancreas is less characterized. Acute pancreatitis (AP) is a serious disorder in which specific treatment is still not possible. Accumulating evidence indicate that decreased pancreatic ductal fluid secretion plays an essential role in AP; therefore, the aim of this study was to investigate the physiological and pathophysiological role of AQPs in the pancreas. Expression and localization of AQPs were investigated by real-time PCR and immunocytochemistry, whereas osmotic transmembrane water permeability was estimated by the dye dilution technique, in Capan-1 cells. The presence of AQP1 and CFTR in the mice and human pancreas were investigated by immunohistochemistry. Pancreatic ductal HCO3- and fluid secretion were studied on pancreatic ducts isolated from wild-type (WT) and AQP1 knock out (KO) mice using microfluorometry and videomicroscopy, respectively. In vivo pancreatic fluid secretion was estimated by magnetic resonance imaging. AP was induced by intraperitoneal injection of cerulein and disease severity was assessed by measuring biochemical and histological parameters. In the mice, the presence of AQP1 was detected throughout the whole plasma membrane of the ductal cells and its expression highly depends on the presence of CFTR Cl- channel. In contrast, the expression of AQP1 is mainly localized to the apical membrane of ductal cells in the human pancreas. Bile acid treatment dose- and time-dependently decreased mRNA and protein expression of AQP1 and reduced expression of this channel was also demonstrated in patients suffering from acute and chronic pancreatitis. HCO3- and fluid secretion significantly decreased in AQP1 KO versus WT mice and the absence of AQP1 also worsened the severity of pancreatitis. Our results suggest that AQP1 plays an essential role in pancreatic ductal fluid and HCO3- secretion and decreased expression of the channel alters fluid secretion which probably contribute to increased susceptibility of the pancreas to inflammation.

Na+/Ca2+ exchangers regulate the migration and proliferation of human gastric myofibroblasts.

Gastrointestinal myofibroblasts are contractile, electrically nonexcitable, transitional cells that play a role in extracellular matrix production, in ulcer healing, and in pathophysiological conditions they contribute to chronic inflammation and tumor development. Na+/Ca2+ exchangers (NCX) are known to have a crucial role in Ca2+ homeostasis of contractile cells, however, no information is available concerning the role of NCX in the proliferation and migration of gastrointestinal myofibroblasts. In this study, our aim was to investigate the role of NCX in the Ca2+ homeostasis, migration, and proliferation of human gastrointestinal myofibroblasts, focusing on human gastric myofibroblasts (HGMs). We used microfluorometric measurements to investigate the intracellular Ca2+ and Na+ concentrations, PCR analysis and immunostaining to show the presence of the NCX, patch clamp for measuring NCX activity, and proliferation and migration assays to investigate the functional role of the exchanger. We showed that 53.0±8.1% of the HGMs present Ca2+ oscillations, which depend on extracellular Ca2+ and Na+, and can be inhibited by NCX inhibitors. NCX1, NCX2, and NCX3 were expressed at both mRNA and protein levels in HGMs, and they contribute to the intracellular Ca2+ and Na+ homeostasis as well, regardless of the oscillatory activity. NCX inhibitors significantly blocked the basal and insulin-like growth factor II-stimulated migration and proliferation rates of HGMs. In conclusion, we showed that NCX plays a pivotal role in regulating the Ca2+ homeostasis, migration, and proliferation of HGMs. The inhibition of NCX activity may be a potential therapeutic target in hyperproliferative gastric diseases.