Human Cholangiocyte Cell Line Research Articles

Preparation and application of silver nanoparticle capped carbon dots composite as fluorescent probe for cobalt determination in vitamin B12 supplement samples

A simple and economical material of fluorescence nanocomposite was developed to quantify vitamin B12 through cobalt(II) ion measurement. The material was derived from carbon dots capped with silver nanoparticles, forming spherical AgNPs@CDs composite with the average diameter of 8.7 nm, expressing unique optical properties, and was used as an optical probe for Co(II) ion determination. The synthesized AgNPs@CDs showed a maximum absorption peak at the wavelength of 406 nm, while their fluorescence peak appeared at the wavelength of 526 nm under the excitation wavelength of 406 nm. The nanocomposites exhibited stable fluorescence with IC50 value against human cholangiocyte cell line of 137.7 ppm after 24 h of the treatment. The interaction between Co(II) ions and AgNPs@CDs effervesced molecular oxygen and caused fluorescence quenching of the composite material. The optimum conditions for Co(II) ion detection were pH 10, volume ratio between AgNPs@CDs and Co(II) ion of 0.10:0.90, and reaction time of 30 min. The linearity range based on the Stern–Volmer plot was 0.05–10.00 ppm, and the limit of detection and limit of quantification were 0.032 ppm and 0.095 ppm, respectively. The developed approach was used to determine the extracted Co(II) ions in commercial vitamin B12 supplement samples and their spiked extracts. The results showed good agreement with those from a conventional method of atomic emission spectrometry, and acceptable range of recovery from 90.0 to 105.0 % was obtained. The fluorescence quenching mechanism of the AgNPs@CDs was elucidated based on the characterization information from several analytical techniques, and electrocatalytic reaction between the nanocomposite and Co(II) species was proposed.

Curcumin-loaded albumin submicron particles with potential as a cancer therapy: an in vitro study.

Curcumin (CUR), a polyphenolic compound, shows promising biological properties, particularly antioxidant activity. However, its medical applications are limited due to its low water solubility, bioavailability, and pH-instability. CUR-loaded albumin microparticles (CUR-HSA-MPs) of submicron size in the range of 800 to 900 nm and a zeta potential of -15 mV were prepared. The CUR loading efficiency was up to 65%. A maximum release of 37% of the encapsulated CUR was observed within 6 h when the CUR-HSA-MPs were dispersed in 50% ethanol in PBS at pH 7, while in RPMI 1640 medium the release was 7%. This demonstrates a sustainable release. The in vitro cytotoxicity of CUR-HSA-MPs showed promising anticancer potential against human hepatocellular carcinoma (Huh-7) and human breast adenocarcinoma (MCF-7) cell lines, although this effect was less pronounced in human dermal fibroblasts (HDFB) and human cholangiocyte (MMN) cell lines. Confocal microscopy was used to confirm the uptake of CUR-HSA-MPs by cancer cells. Our studies revealed that HSA-MPs are potentially promising vehicles for increasing the solubility and bioavailability of CUR.

Green synthesized apigenin conjugated gold nanoparticles inhibit cholangiocarcinoma cell activity and endothelial cell angiogenesis in vitro

Cholangiocarcinoma (CCA) is a rare malignancy of the biliary tract with extremely poor clinical outcomes due to a lack of effective therapies to improve disease management. The emerging green synthesis of gold nanoparticles (AuNPs) has extensively provided their use in biomedical applications. In this study, we developed AuNPs via reducing gold salts with apigenin (4′,5,7-trihydroxyflavone). The synthesized apigenin-conjugated AuNPs (api-AuNPs) were physicochemically characterized by various techniques before evaluation their biological and functional inhibition in a CCA cell line, KKU-M055. The mean size of api-AuNPs was 90.34 ± 22.82 nm with zeta potential of -36 ± 0.55. The half-maximal inhibitory concentration (IC50, 0.8 mg/mL) of api-AuNPs on cell proliferation of KKU-M055 was 1.9-fold less than that of an immortalized human cholangiocyte cell line, MMNK1 (IC50, 1.5 mg/mL). Moreover, api-AuNPs induced cell apoptosis via the up-regulation of Bax, Bid, and Caspase 3, and down-regulation of Bcl2, leading to elevated caspase 3/7, 8, 9 activities and reactive oxygen species (ROS) production. The api-AuNPs significantly inhibited the migration of KKU-M055 cells and suppressed the proliferation, migration, and in vitro tube formation of vascular endothelial cells. Collectively, our findings indicate the dual abilities of api-AuNPs that potentially inhibit cancer cell growth and motility as well as endothelial cell-mediated angiogenesis, which may offer a novel therapeutic avenue to treat CCA patients effectively.

FGF1 Signaling Modulates Biliary Injury and Liver Fibrosis in the Mdr2-/- Mouse Model of Primary Sclerosing Cholangitis.

Fibroblast growth factor 1 (FGF1) belongs to a family of growth factors involved in cellular growth and division. MicroRNA 16 (miR‐16) is a regulator of gene expression, which is dysregulated during liver injury and insult. However, the role of FGF1 in the progression of biliary proliferation, senescence, fibrosis, inflammation, angiogenesis, and its potential interaction with miR‐16, are unknown. In vivo studies were performed in male bile duct–ligated (BDL, 12‐week‐old) mice, multidrug resistance 2 knockout (Mdr2 −/−) mice (10‐week‐old), and their corresponding controls, treated with recombinant human FGF1 (rhFGF1), fibroblast growth factor receptor (FGFR) antagonist (AZD4547), or anti‐FGF1 monoclonal antibody (mAb). In vitro, the human cholangiocyte cell line (H69) and human hepatic stellate cells (HSCs) were used to determine the expression of proliferation, fibrosis, angiogenesis, and inflammatory genes following rhFGF1 treatment. PSC patient and control livers were used to evaluate FGF1 and miR‐16 expression. Intrahepatic bile duct mass (IBDM), along with hepatic fibrosis and inflammation, increased in BDL mice treated with rhFGF1, with a corresponding decrease in miR‐16, while treatment with AZD4547 or anti‐FGF1 mAb decreased hepatic fibrosis, IBDM, and inflammation in BDL and Mdr2 −/− mice. In vitro, H69 and HSCs treated with rhFGF1 had increased expression of proliferation, fibrosis, and inflammatory markers. PSC samples also showed increased FGF1 and FGFRs with corresponding decreases in miR‐16 compared with healthy controls. Conclusion: Our study demonstrates that suppression of FGF1 and miR‐16 signaling decreases the presence of hepatic fibrosis, biliary proliferation, inflammation, senescence, and angiogenesis. Targeting the FGF1 and miR‐16 axis may provide therapeutic options in treating cholangiopathies such as PSC.

Therapeutic effects of dexamethasone-loaded hyaluronan nanogels in the experimental cholestasis

A major function of the intrahepatic biliary epithelium is bicarbonate excretion in bile. Recent reports indicate that budesonide, a corticosteroid with high receptor affinity and hepatic first pass clearance, increases the efficacy of ursodeoxycholic acid, a choleretic agent, in primary biliary cholangitis patients. We have previously reported that bile ducts isolated from rats treated with dexamethasone or budesonide showed an enhanced activity of the Na+/H+ exchanger isoform 1 (NHE1) and Cl−/HCO3− exchanger protein 2 (AE2) . Increasing the delivery of steroids to the liver may result in three beneficial effects: increase in the choleresis, treatment of the autoimmune or inflammatory liver injury and reduction of steroids’ systemic harmful effects. In this study, the steroid dexamethasone was loaded into nanohydrogels (or nanogels, NHs), in order to investigate corticosteroid-induced increased activities of transport processes driving bicarbonate excretion in the biliary epithelium (NHE-1 isoform) and to evaluate the effects of dexamethasone-loaded NHs (NHs/dex) on liver injury induced by experimental cholestatis. Our results showed that NHs and NHs/dex do not reduce cell viability in vitro in human cholangiocyte cell lines. Primary and immortalized human cholangiocytes treated with NHs/dex show an increase in the functional marker expression of NHE1 cholangiocytes compared to control groups. A mouse model of cholangiopathy treated with NHs/dex shows a reduction in markers of hepatocellular injury compared to control groups (NHs, dex, or sham group). In conclusion, we believe that the NHs/dex formulation is a suitable candidate to be investigated in preclinical models of cholangiopathies.Graphical abstract

Impaired Regulatory Volume Decrease and Characterization of Underlying Volume-Activated Currents in Cystic Fibrosis Human Cholangiocyte Cell Line.

The volume-activated chloride channel (VACC) serves vital cellular functions in secretion and cell volume regulation via regulatory volume decrease (RVD) in various epithelia. Previously, we have shown that RVD in primary CF mouse cholangiocytes is impaired. Thus, the effect of CFTR defect on VACC and RVD in CF human immortalized cholangiocyte cell (HBDC) was examined in comparison with those in normal HBDC by using cell volume measurement and whole-cell patch clamp techniques, respectively. The CF HBDC had an impaired RVD, which was not further inhibited by removing the extracellular calcium or administering BAPTA-AM, NPPB, or DIDS. When exposed to a hypotonic solution, CF HBDC exhibited large, outwardly rectified currents with time-dependent inactivation at a positive potential. The amplitude of the outward currents was about three times that of the inward currents. The amplitude and reversal potential of VACC was dependent on chloride concentration. The VACC was significantly inhibited by replacing chloride with gluconate, glutamate, sucrose, or acetate in the hypotonic solution as well as by an administration of NPPB or tamoxifen, classical VACC inhibitors. Surprisingly, the VACC amplitude is greater in CF HBDC than in normal HBDC, suggesting that the channel density or open probability of VACC is increased, thus CFTR may have inhibitory effects on VACC. On the contrary, the amplitude of the volume-activated potassium current is lower in CF HBDC, suggesting the potassium channel density or open probability is decreased in CF cholangiocytes and/or CFTR may have regulatory effects on volume-activated potassium current. In conclusion, RVD is impaired in CF human cholangiocytes. The VACC of CF human cholangiocytes has similar electrophysiological characteristics as that of normal cholangiocytes but its activity is augmented in CF cholangiocytes, while volume-activated potassium current is decreased in CF human cholangiocytes, providing a fundamental underlying pathophysiologic mechanism for the impaired RVD in CF cholangiocytes.

Critical alterations in cellular bioenergetics and epithelial‐mesenchymal transition mediated by crosstalk between tumor cells and lymphatic vasculature augments tumor progression in cholangiocarcinoma

Cholangiocarcinoma is a rare but highly aggressive cancer of bile duct cells with a dismal 5-year survival rate following surgical tumor resection of 2%. Despite the presence of a high degree of lymphangiogenesis in the prominently inflamed CCA tumor microenvironment (TME), the role of lymphatic-CCA crosstalk in CCA metastasis has been unexplored. Increased glucose metabolism and oxidative phosphorylation (OXPHOS) in CCA cells increase CCA metastasis and therapeutic resistance. Further mitochondrial dysfunction is a driver of epithelial-mesenchymal transition (EMT), critical for tumor progression. We hypothesized that CCA-LEC interactions in the tumor microenvironment alters CCA metabolism and drives EMT and lymphangiogenesis. We used CCA (HuCCT1, Mz-ChA1 and CCLP1), LEC (human dermal and lymph node LECs) and normal human cholangiocyte (H69 and HiBEC) cell lines as well as human CCA tissue arrays. Conditioned medium (CM) was collected from CCA, control and inflamed LECs (LECs stimulated with lipopolysaccharide (LPS, 100ng/ml for 24hr). To define the CCA-LEC crosstalk, both CCA and LECs were treated with LEC/inflamed LEC CM and CCA-CM respectively, and alterations of mitochondrial gene expression, alterations in cellular metabolism, and migration and proliferation of CCA and LECs was determined. CCA cells showed enhanced migration and proliferation in response to LPS-LEC-CM. Real time PCR revealed that this was associated with altered expression of EMT genes such as Gli1, SNAI1, SLUG and TWIST1 or changes in matrix metalloproteinases (MMP) as MMP2, MMP9 and MMP21. The seahorse metabolic data showed that stimulation of CCA cells by LPS-LEC-CM, caused a distinct shift towards higher glycolysis and oxidative phosphorylation (OXPHOS), along with increased ROS and ATP production. This was associated with dysregulation of some critical metabolic genes such as PFKP and ATP6 involved in increasing glycolysis and OXPHOS rate critical for aggressive tumor growth and metastasis. Further, analysis of mitochondrial genes in CCA-CM treated LECs showed increased expression of PFKP, GLUT3, FASN, CO I, HK2 and ATP6, critical genes involved in glycolysis, OXPHOS and fatty acid synthesis, key pathways known to drive tumor-lymphangiogenesis. Dysregulation of these mitochondrial genes was also observed in clinical samples of CCA. siRNA mediated silencing of a number of these genes including SLUG1 and ATP-6 show decreased CCA migration, EMT and reversal of the increased glycolysis as well as significantly reduced LEC tube formation. Thus, alterations in cellular metabolism due to tumor-lymphatic crosstalk alter oncogenic pathways and regulates lymphangiogenesis in progression of CCA.

Activated cholangiocytes release macrophage-polarizing extracellular vesicles bearing the DAMP S100A11.

In mouse models of biliary tract diseases, macrophages are recruited to the periductal milieu and promote injury and cholestasis. Although cell necrosis with release of biomolecules termed damage-associated molecular patterns (DAMPs) promotes recruitment and activation of macrophages, necrosis was not observed in these studies. Because extracellular vesicles (EVs) are important in cell-to-cell communication, we postulated that activated cholangiocytes may release EVs containing DAMPs as cargo. Both the human (NHC) and mouse cholangiocyte (603B) cell lines display constitutive activation with mRNA expression of chemokines. Proteomic analysis revealed that EVs from both cell lines contained the DAMP S100A11, a ligand for the receptor for advanced glycation end products (RAGE). Bone marrow-derived macrophages (BMDM) incubated with EVs derived from the mouse 603B cell line increased mRNA expression of proinflammatory cytokines. Genetic or pharmacologic inhibition of RAGE reduced BMDM expression of proinflammatory cytokines treated with EVs. RAGE signaling resulted in activation of the canonical NF-κB pathway, and consistently, proinflammatory cytokine expression was blunted by the IKKα/β inhibitor TPCA-1 in BMDM incubated with EVs. We also demonstrated that primary mouse cholangiocyte-derived organoids express chemokines indicating cholangiocyte activation, release EVs containing S100A11, and stimulate proinflammatory cytokine expression in BMDM by a RAGE-dependent pathway. In conclusion, these observations identify a non-cell death mechanism for cellular release of DAMPs by activated cholangiocytes, namely by releasing DAMPs as EV cargo. These data also suggest RAGE inhibitors may be salutary in macrophage-associated inflammatory diseases of the bile ducts.

Pinealectomy or light exposure exacerbates biliary damage and liver fibrosis in cholestatic rats through decreased melatonin synthesis

Melatonin, a neuroendocrine hormone synthesized by the pineal gland and cholangiocytes, decreases biliary hyperplasia and liver fibrosis during cholestasis-induced biliary injury via melatonin-dependent autocrine signaling through increased biliary arylalkylamine N-acetyltransferase (AANAT) expression and melatonin secretion, downregulation of miR-200b and specific circadian clock genes. Melatonin synthesis is decreased by pinealectomy (PINX) or chronic exposure to light. We evaluated the effect of PINX or prolonged light exposure on melatonin-dependent modulation of biliary damage/ductular reaction/liver fibrosis. Studies were performed in male rats with/without BDL for 1 week with 12:12 h dark/light cycles, continuous light or after 1 week of PINX. The expression of AANAT and melatonin levels in serum and cholangiocyte supernatant were increased in BDL rats, while decreased in BDL rats following PINX or continuous light exposure. BDL-induced increase in serum chemistry, ductular reaction, liver fibrosis, inflammation, angiogenesis and ROS generation were significantly enhanced by PINX or light exposure. Concomitant with enhanced liver fibrosis, we observed increased biliary senescence and enhanced clock genes and miR-200b expression in total liver and cholangiocytes. In vitro, the expression of AANAT, clock genes and miR-200b was increased in PSC human cholangiocyte cell lines (hPSCL). The proliferation and activation of HHStecs (human hepatic stellate cell lines) were increased after stimulating with BDL cholangiocyte supernatant and further enhanced when stimulated with BDL rats following PINX or continuous light exposure cholangiocyte supernatant via intracellular ROS generation. Conclusion: Melatonin plays an important role in the protection of liver against cholestasis-induced damage and ductular reaction.

Neoplastic Transformation of the Peribiliary Stem Cell Niche in Cholangiocarcinoma Arisen in Primary Sclerosing Cholangitis.

Primary sclerosing cholangitis (PSC) is a chronic inflammatory cholangiopathy frequently complicated by cholangiocarcinoma (CCA). Massive proliferation of biliary tree stem/progenitor cells (BTSCs), expansion of peribiliary glands (PBGs), and dysplasia were observed in PSC. The aims of the present study were to evaluate the involvement of PBGs and BTSCs in CCA which emerged in PSC patients. Specimens from normal liver (n = 5), PSC (n = 20), and PSC-associated CCA (n = 20) were included. Samples were processed for histology, immunohistochemistry, and immunofluorescence. In vitro experiments were performed on human BTSCs, human mucinous primary CCA cell cultures, and human cholangiocyte cell lines (H69). Our results indicated that all CCAs emerging in PSC patients were mucin-producing tumors characterized by PBG involvement and a high expression of stem/progenitor cell markers. Ducts with neoplastic lesions showed higher inflammation, wall thickness, and PBG activation compared to nonneoplastic PSC-affected ducts. CCA showed higher microvascular density and higher expression of nuclear factor kappa B, interleukin-6, interleukin-8, transforming growth factor β, and vascular endothelial growth factor-1 compared to nonneoplastic ducts. CCA cells were characterized by a higher expression of epithelial-to-mesenchymal transition (EMT) traits and by the absence of primary cilia compared to bile ducts and PBG cells in controls and patients with PSC. Our in vitro study demonstrated that lipopolysaccharide and oxysterols (PSC-related stressors) induced the expression of EMT traits, the nuclear factor kappa B pathway, autophagy, and the loss of primary cilia in human BTSCs. Conclusion: CCA arising in patients with PSC is characterized by extensive PBG involvement and by activation of the BTSC niche in these patients, the presence of duct lesions at different stages suggests a progressive tumorigenesis.

Defined Small Molecules Produced by Himalayan Medicinal Plants Display Immunomodulatory Properties.

Plant-derived compounds that modulate the immune responses are emerging as frontline treatment agents for cancer, infectious diseases and autoimmunity. Herein we have isolated 40 phytochemicals from five Bhutanese Sowa Rigpa medicinal plants—Aconitum laciniatum, Ajania nubegina, Corydalis crispa, Corydalis dubia and Pleurospermum amabile—and tested 14 purified compounds for their immunomodulatory properties using a murine dendritic cell (DC) line, and cytotoxicity against a human cholangiocyte cell line using xCELLigence real time cell monitoring. These compounds were: pseudaconitine, 14-veratryolpseudaconitine, 14-O-acetylneoline, linalool oxide acetate, (E)-spiroether, luteolin, luteolin-7-O-β-d-glucopyranoside, protopine, ochrobirine, scoulerine, capnoidine, isomyristicin, bergapten, and isoimperatorin. Of the 14 compounds tested here, scoulerine had adjuvant-like properties and strongly upregulated MHC-I gene and protein expression whereas bergapten displayed immunosuppressive properties and strongly down-regulated gene and protein expression of MHC-I and other co-stimulatory molecules. Both scoulerine and bergapten showed low cytotoxicity against normal healthy cells that were consistent with their immunoregulatory properties. These findings highlight the breadth of immunomodulatory properties of defined compounds from Bhutanese medicinal plants and show that some of these compounds exert their mechanisms of action by modulating DC activity.

Role of lactoferrin and its receptors on biliary epithelium.

Human lactoferrin is an iron-binding glycoprotein present at high concentrations in breast milk and colostrum. It is produced by many exocrine glands and widely distributed in a variety of body fluids. This protein has antimicrobial, immunomodulatory, antioxidant, and anticancer properties. Two important hLf receptors have been identified: LDL receptor related protein (LRP1), a low specificity receptor, and intelectin-1 (ITLN1), a high specificity receptor. No data are present on the role of hLf on the biliary epithelium. Our aims have been to evaluate the expression of Lf and its receptors in human and murine cholangiocytes and its effect on proliferation. Immunohistochemistry and immunofluorescence (IF) were conducted on human healthy and primary biliary cholangitis (PBC) liver samples as well as on liver samples obtained from normal and bile duct ligated (BDL) mice to evaluate the expression of Lf, LRP1 and ITLN1. Cell proliferation in vitro studies were performed on human cholangiocyte cell lines via 3-(4,5-dimetiltiazol-2-il)-2,5-diphenyltetrazolium assay as well as IF to evaluate proliferating cell nuclear antigen (PCNA) expression. Our results show that mouse and human cholangiocytes express Lf, LRP1 and ITLN1, at higher extent in cholangiocytes from BDL and PBC samples. Furthermore, the in vitro addition of bovine Lf (bLf) has a proliferative effect on human cholangiocyte cell line. The results support a proliferative role of hLf on the biliary epithelium; this pro-proliferative effect of hLf and bLf on cholangiocytes could be particularly relevant in human cholangiopathies such as PBC, characterized by cholangiocyte death and ductopenia.

Abstract 361: Loss of cholangiocyte primary cilia induces LKB1 downregulation and defective AMPK signaling

Abstract Cholangiocarcinoma (CCA) is a malignancy arising from cholangiocytes, the epithelial cells lining the biliary tree. CCA is an uncommon, but devastating cancer that is increasing in incidence. Over the past 3 decades, 5-years survival rates have remained at 10%. Although surgical resection and liver transplantation are potentially curative therapies, most patients are diagnosed at late stages and are not eligible for these options. Therefore, it is imperative to identify novel targets leading to new therapeutic strategies for this devastating disease. Cholangiocytes express primary cilia that function as chemo, mechano, and osmosensors controlling several molecular pathways. We showed cilia are absent in CCA cells, and experimental deciliation of normal cholangiocytes induced a malignant like phenotype, with significant invasion and proliferation, suggesting the loss of cilia could be associated with CCA development. LKB1 is a tumor suppressor described to be expressed in primary cilia in MDCK cells, and is involved in AMPK activation through a ciliary dependent mechanism. AMPK functions as metabolic and stress check points. Interestingly, patients with intrahepatic CCA and low expression of LKB1 have poor prognosis. Therefore, we hypothesized that primary cilia function as tumor suppressor organelles through a LKB1-AMPK-p53 pathway. To test this hypothesis, first LKB1 subcellular localization was evaluated by confocal microscopy. We show for the first time that LKB1 is enriched in cholangiocyte primary cilia in a normal human cholangiocyte cell line (NHC). However, in experimental deciliated NHC and the CCA cell line KMCH, LKB1 is found dispersed in the cell cytoplasm. Next, we analyzed cellular expression levels of LKB1, AMPK (T172), p53 (s15), p53 and p21 by western blot. We found experimentally deciliated cholangiocytes and CCA cells exhibit defective AMPK signaling characterized by lower levels of LKB1 (-70% and -85%), AMPK (T172) (-72% and -71%), p53 (-37% and -83%), p53 (S15) (-38% and -100%) and p21 (-59% and -87%) relative to NHC. These molecular characteristics correlated with increased cell proliferation in deciliated cells (28%) and CCA cells (27%). Finally, we attempted to rescue the phenotype by using the AMPK activator, ampkinone. This treatment induced 20% inhibition of proliferation on experimental deciliated cholangiocytes and 18% in CCA cells, while not affecting NHC cells and the inhibition was associated with phosphorylation of AMPK and p21 up-regulation. In summary, our data suggest cholangiocyte primary cilia may normally function as tumor suppressors via a mechanism involving LKB1 and AMPK. The loss of cilia in CCA impairs LKB1-AMPK-p53-p21 signaling inducing a proliferative phenotype that may be rescued by specific activation of AMPK, warranting further studies to assess its use as a potential therapeutic approach. Citation Format: Adrian P. Mansini, Kristen M. Thelen, Sergio A. Gradilone. Loss of cholangiocyte primary cilia induces LKB1 downregulation and defective AMPK signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 361. doi:10.1158/1538-7445.AM2017-361

Development of a Potent Wound Healing Agent Based on the Liver Fluke Granulin Structural Fold.

Granulins are a family of protein growth factors that are involved in cell proliferation. An orthologue of granulin from the human parasitic liver fluke Opisthorchis viverrini, known as Ov-GRN-1, induces angiogenesis and accelerates wound repair. Recombinant Ov-GRN-1 production is complex and poses an obstacle for clinical development. To identify the bioactive region(s) of Ov-GRN-1, four truncated N-terminal analogues were synthesized and characterized structurally using NMR spectroscopy. Peptides that contained only two native disulfide bonds lack the characteristic granulin β-hairpin structure. Remarkably, the introduction of a non-native disulfide bond was critical for formation of β-hairpin structure. Despite this structural difference, both two and three disulfide-bonded peptides drove proliferation of a human cholangiocyte cell line and demonstrated potent wound healing in mice. Peptides derived from Ov-GRN-1 are leads for novel wound healing therapeutics, as they are likely less immunogenic than the full-length protein and more convenient to produce.

The biliary epithelium presents antigens to and activates natural killer T cells.

Cholangiocytes express antigen-presenting molecules, but it has been unclear whether they can present antigens. Natural killer T (NKT) cells respond to lipid antigens presented by the major histocompatibility complex class I-like molecule CD1d and are abundant in the liver. We investigated whether cholangiocytes express CD1d and present lipid antigens to NKT cells and how CD1d expression varies in healthy and diseased bile ducts. Murine and human cholangiocyte cell lines as well as human primary cholangiocytes expressed CD1d as determined by flow cytometry and western blotting. Murine cholangiocyte cell lines were able to present both exogenous and endogenous lipid antigens to invariant and noninvariant NKT cell hybridomas and primary NKT cells in a CD1d-dependent manner. A human cholangiocyte cell line, cholangiocarcinoma cell lines, and human primary cholangiocytes also presented exogenous CD1d-restricted antigens to invariant NKT cell clones. CD1d expression was down-regulated in the biliary epithelium of patients with late primary sclerosing cholangitis, primary biliary cirrhosis, and alcoholic cirrhosis compared to healthy controls. Cholangiocytes express CD1d and present antigens to NKT cells and CD1d expression is down-regulated in diseased biliary epithelium, findings which show that the biliary epithelium can activate an important lymphocyte subset of the liver. This is a potentially important immune pathway in the biliary system, which may be capable of regulating inflammation in the context of biliary disease.

Breast tumor kinase/protein tyrosine kinase 6 (Brk/PTK6) activity in normal and neoplastic biliary epithelia

Breast tumor kinase (BRK) augments proliferation and promotes cell survival in breast cancers via interactions with SH2 and SH3 ligand-containing proteins, such as receptor tyrosine kinases (RTK; e.g. EGFR, ErbB2/neu). Since RTK contribute to cholangiocarcinoma (CC) evolution we probed BRK protein expression and function in normal and CC livers. Immunohistochemical staining of normal livers and CC (n=93) in a tissue microarray and three CC and an immortalized human cholangiocyte cell lines (real-time PCR, Western blotting, siRNA) were used to study the functional relationships between BRK, EGFR, ErbB2, SAM68, and SPRR2a. BRK protein was expressed in normal human intrahepatic bile ducts; all CC cell lines and a majority of CC showed strong BRK protein expression. Multiplex immunostaining/tissue cytometry and immunoprecipitation studies showed: 1) BRK co-localized with EGFR and ErbB2/neu; 2) BRK(high)/EGFR(high)-co-expressing CC cells had significantly higher Ki67 labeling and; 3) stronger BRK protein expression was seen in perihilar and distal CC than intrahepatic CC and directly correlated with CC differentiation. In cell lines, BRK expression augmented proliferation in response to exogenous EGF, whereas BRK siRNA significantly reduced growth. The SH3 ligand-containing, SPRR2A activated pTyr342 BRK, which in turn, phosphorylated SAM68, causing nuclear localization and increased cell proliferation similar to observations in breast cancers. BRK expression in a majority of CC can interact with RTK, augmenting growth and interfering with proliferation inhibitors (SAM68). Therapeutically targeting BRK function (in addition to RTK) should be of benefit for CC treatment.

Suppression of Ov-grn-1 encoding granulin of Opisthorchis viverrini inhibits proliferation of biliary epithelial cells

Multistep processes likely underlie cholangiocarcinogenesis induced by chronic infection with the fish-borne liver fluke, Opisthorchis viverrini. One process appears to be cellular proliferation of the host bile duct epithelia driven by excretory–secretory (ES) products of this pathogen. Specifically, the secreted growth factor Ov-GRN-1, a liver fluke granulin, is a prominent component of ES and a known driver of hyper-proliferation of cultured human and mouse cells in vitro. We show potent hyper-proliferation of human cholangiocytes induced by low nanomolar levels of recombinant Ov-GRN-1 and similar growth produced by low microgram concentrations of ES products and soluble lysates of the adult worm. To further explore the influence of Ov-GRN-1 on the flukes and the host cells, expression of Ov-grn-1 was repressed using RNA interference. Expression of Ov-grn-1 was suppressed by 95% by day 3 and by ~100% by day 7. Co-culture of Ov-grn-1 suppressed flukes with human cholangiocyte (H-69) or human cholangiocarcinoma (KKU-M214) cell lines retarded cell hyper-proliferation by 25% and 92%, respectively. Intriguingly, flukes in which expression of Ov-grn-1 was repressed were less viable in culture, suggesting that Ov-GRN-1 is an essential growth factor for survival of the adult stage of O. viverrini, at least in vitro. To summarize, specific knock down of Ov-grn-1 reduced in vitro survival and capacity of ES products to drive host cell proliferation. These findings may help to contribute to a deeper understanding of liver fluke induced cholangiocarcinogenesis.

Secreted cyclophilin A mediates G1/S phase transition of cholangiocarcinoma cells via CD147/ERK1/2 pathway.

Cyclophilin A (CypA) was shown to be upregulated in human cholangiocarcinoma (CCA) tissues. Suppression of intracellular CypA (inCypA) significantly reduces cell proliferation in vitro and tumor growth in nude mice. In the present study, the effect and potential mechanism of secreted CypA (sCypA) on cell proliferation of CCA cell lines were further investigated. CCA cells were treated with sCypA-containing conditioned media (CM) or with purified recombinant human CypA (rhCypA). Cell proliferation, cell cycle, ERK1/2, p38 MAPK, NF-κB,and STAT3 activities were examined by MTS assay, flow cytometry, and Western blot. sCypA was detected in CM from MMNK1 (an immortalized human cholangiocyte cell line) and six CCA cell lines. The sCypA levels corresponded to the inCypA levels indicating the intracellular origin of sCypA. Both sCypA-containing CM and rhCypA significantly increased proliferation of CCA cells. CD147 depletion by shRNA-knockdown or neutralizing with a CD147-monoclonal antibody significantly reduced sCypA-, and rhCypA-mediated cell proliferation. Upon rhCypA treatment, ERK1/2 was rapidly phosphorylated; whereas neutralizing CD147 inhibited ERK1/2 phosphorylation. Cell cycle analysis showed a significant increase in S phase and decrease in G1 population in rhCypA-treated cells. The expression levels of cyclin D1 and phosphorylated-retinoblastoma protein in the rhCypA-treated cells were increased compared with those in the non-treated control cells. p38 MAPK pathway was shown to be suppressed in siCypA-treated cells. In summary, CypA is secreted from CCA cells and enhances cell proliferation in an autocrine/paracrine manner, at least via direct binding with CD147, which may activate the ERK1/2 and p38 MAPK signaling pathways.

Characterization of cultured cholangiocytes isolated from livers of patients with primary sclerosing cholangitis

Primary sclerosing cholangitis (PSC) is a chronic, idiopathic cholangiopathy. The role of cholangiocytes (biliary epithelial cells) in PSC pathogenesis is unknown and remains an active area of research. Here, through cellular, molecular and next-generation sequencing (NGS) methods, we characterize and identify phenotypic and signaling features of isolated PSC patient-derived cholangiocytes. We isolated cholangiocytes from stage 4 PSC patient liver explants by dissection, differential filtration and immune-magnetic bead separation. We maintained cholangiocytes in culture and assessed for: (i) cholangiocyte, cell adhesion and inflammatory markers; (ii) proliferation rate; (iii) transepithelial electrical resistance (TEER); (iv) cellular senescence; and (v) transcriptomic profiles by NGS. We used two well-established normal human cholangiocyte cell lines (H69 and NHC) as controls. Isolated PSC cells expressed cholangiocyte (eg, cytokeratin 7 and 19) and epithelial cell adhesion markers (EPCAM, ICAM) and were negative for hepatocyte and myofibroblast markers (albumin, α-actin). Proliferation rate was lower for PSC compared with normal cholangiocytes (4 vs 2 days, respectively, P<0.01). Maximum TEER was also lower in PSC compared with normal cholangiocytes (100 vs 145 Ωcm2, P<0.05). Interleukin-6 (IL-6) and IL-8 (protein and mRNA) were both increased compared with NHCs and H69s (all P<0.01). The proportion of cholangiocytes staining positive for senescence-associated β-galactosidase was higher in PSC cholangiocytes compared with NHCs (48% vs 5%, P<0.01). Finally, NGS confirmed cholangiocyte marker expression in isolated PSC cholangiocytes and extended our findings regarding pro-inflammatory and senescence-associated signaling. In conclusion, we have demonstrated that high-purity cholangiocytes can be isolated from human PSC liver and grown in primary culture. Isolated PSC cholangiocytes exhibit a phenotype that may reflect their in vivo contribution to disease and serve as a vital tool for in vitro investigation of biliary pathobiology and identification of new therapeutic targets in PSC.

Annexin A1: A new immunohistological marker of cholangiocarcinoma

To evaluate a new immunohistological marker, annexin A1 (ANXA1), in cholangiocarcinoma (CCA) and hepatocellular carcinoma (HCC). Expression of ANXA1 protein was investigated in liver tissues from patients with CCA and HCC by immunohistochemistry. Its expression on differences stages of tumor development was investigated in hamster CCA tissues induced by Opisthorchis viverrini and N-nitrosodimethylamine. Moreover, mRNA expression of ANXA1 was assessed in CCA cell lines by quantitative real-time polymerase chain reaction and silencing of ANXA1 gene expression using small interfering RNA. In human CCA tissue arrays, immunohistochemical analysis revealed that the positive expression of ANXA1 was 94.1% (64/68 cases) consisting of a high expression (66.2%, 45/68 cases) and a low expression (33.8%, 23/68 cases). However, expression of ANXA1 protein was negative in all histologic patterns for HCC (46/46 cases) and healthy individuals (6/6 cases). In hamster with opisthorchiasis-associated CCA, the expression of ANXA1 was observed in the cytoplasm of inflammatory cells, bile duct epithelia and tumor cells. Grading scores of ANXA1 expression were significantly increased with tumor progression. In addition, mRNA expression of ANXA1 significantly increased in all of the various CCA cell lines tested compared to an immortalized human cholangiocyte cell line (MMNK1). Suppressing the ANXA1 gene significantly reduced the matrix metalloproteinase (MMP) 2 and MMP9, and transforming growth factor-β genes, but increased nuclear factor-κB gene expression. ANXA1 is highly expressed in CCA, but low in HCC, suggesting it may serve as a new immunohistochemical marker of CCA. ANXA1 may play a role in opisthorchiasis-associated cholangiocarcinogenesis.