Autocrine and Paracrine Mechanisms Promoting Chemoresistance in Cholangiocarcinoma.

Resistance to conventional chemotherapeutic agents, a typical feature of cholangiocarcinoma, prevents the efficacy of the therapeutic arsenal usually used to combat malignancy in humans. Mechanisms of chemoresistance by neoplastic cholangiocytes include evasion of drug-induced apoptosis mediated by autocrine and paracrine cues released in the tumor microenvironment. Here, recent evidence regarding molecular mechanisms of chemoresistance is reviewed, as well as associations between well-developed chemoresistance and activation of the cancer stem cell compartment. It is concluded that improved understanding of the complex interplay between apoptosis signaling and the promotion of cell survival represent potentially productive areas for active investigation, with the ultimate aim of encouraging future studies to unveil new, effective strategies able to overcome current limitations on treatment.

Leukemia inhibitory factor protects cholangiocarcinoma cells from drug-induced apoptosis via a PI3K/AKT-dependent Mcl-1 activation.

Cholangiocarcinoma is an aggressive, strongly chemoresistant liver malignancy. Leukemia inhibitory factor (LIF), an IL-6 family cytokine, promotes progression of various carcinomas. To investigate the role of LIF in cholangiocarcinoma, we evaluated the expression of LIF and its receptor (LIFR) in human samples. LIF secretion and LIFR expression were assessed in established and primary human cholangiocarcinoma cell lines. In cholangiocarcinoma cells, we tested LIF effects on proliferation, invasion, stem cell-like phenotype, chemotherapy-induced apoptosis (gemcitabine+cisplatin), expression levels of pro-apoptotic (Bax) and anti-apoptotic (Mcl-1) proteins, with/without PI3K inhibition, and of pSTAT3, pERK1/2, pAKT. LIF effect on chemotherapy-induced apoptosis was evaluated after LIFR silencing and Mcl-1 inactivation.Results show that LIF and LIFR expression were higher in neoplastic than in control cholangiocytes; LIF was also expressed by tumor stromal cells. LIF had no effects on cholangiocarcinoma cell proliferation, invasion, and stemness signatures, whilst it counteracted drug-induced apoptosis. Upon LIF stimulation, decreased apoptosis was associated with Mcl-1 and pAKT up-regulation and abolished by PI3K inhibition. LIFR silencing and Mcl-1 blockade restored drug-induced apoptosis.In conclusion, autocrine and paracrine LIF signaling promote chemoresistance in cholangiocarcinoma by up-regulating Mcl-1 via a novel STAT3- and MAPK-independent, PI3K/AKT-dependent pathway. Targeting LIF signaling may increase CCA responsiveness to chemotherapy.

A three-year prospective longitudinal cohort study of medical students' attitudes toward anatomy teaching and their career aspirations.

Today's medical students are faced with numerous learning needs. Continuously developing curricula have reduced time for basic science subjects such as anatomy. This study aimed to determine the students' views on the relevance of anatomy teaching, anatomical knowledge, and the effect these have on their career choices. A Likert scale questionnaire was distributed to second year medical students [response rate 91% (n = 292/320)]. The same questionnaire was subsequently distributed to the cohort three years later when they were final year students [response rate 37% (n = 146/392)]. Students in both the cohorts of study agreed strongly that clinically correlated anatomical teaching was relevant to clinical practice (92% and 86% of second and final year respondents, respectively) and helped them during their clinical placements (73% and 92%, respectively). Only 28% of the second year and 31% of the final year students agreed that their anatomy teaching prepared them to interpret clinical images (P = 0.269). Only 14% of the final year students felt confident in their knowledge of anatomy. Of the final year students, 30% felt that they had enough opportunity to scrub in the operating room. Nearly half of those students who would consider surgery as a career (19%) think that they will eventually become surgeons (11%). This data suggests that modern anatomy curriculum should focus on clinical correlations and clinical image interpretation. Students would value more opportunities to participate in surgeries. Vertical integration of anatomy teaching throughout the full medical course may be useful.

Human intrahepatic biliary epithelial cell lineages: studies in vitro.

The human intrahepatic biliary epithelium is composed of a morphologically heterogeneous population of epithelial cells. During liver cirrhosis, new biliary ductular structures develop at the portal margins that express markers of immaturity such as CD56 and Bcl-2. These markers are also expressed transiently on immature biliary duct precursors during embryological development; thus their reappearance during cirrhosis suggests a recapitulation of ontogenesis during some liver conditions. Here we describe methods, based on the differential expression of membrane markers, for separating immature biliary epithelial cells from those associated with mature ducts. We also describe two- and three-dimensional culture models for the maintenance of mature and immature populations in vitro. Both populations readily establish colonies in monolayer culture but only cells from mature ducts can be maintained in medium-term culture as serially proliferating, passageable cultures; immature cells deteriorate and detach within 2-3 weeks of isolation. In three-dimensional collagen gel culture, both mature and immature populations form duct-like structures with clearly definable lumena that persist for up to 6 weeks.

Combination of ursodeoxycholic acid and glucocorticoids upregulates the AE2 alternate promoter in human liver cells.

Primary biliary cirrhosis (PBC) is a cholestatic disease associated with autoimmune phenomena and alterations in both biliary bicarbonate excretion and expression of the bicarbonate carrier AE2. The bile acid ursodeoxycholic acid (UCDA) is currently used in treatment of cholestatic liver diseases and is the treatment of choice in PBC; however, a subset of PBC patients respond poorly to UDCA monotherapy. In these patients, a combination of UDCA and glucocorticoid therapy appears to be beneficial. To address the mechanism of this benefit, we analyzed the effects of UDCA and dexamethasone on AE2 gene expression in human liver cells from hepatocyte and cholangiocyte lineages. The combination of UDCA and dexamethasone, but not UDCA or dexamethasone alone, increased the expression of liver-enriched alternative mRNA isoforms AE2b1 and AE2b2 and enhanced AE2 activity. Similar effects were obtained after replacing UDCA with UDCA conjugates. In in vitro and in vivo reporter assays, we found that a UDCA/dexamethasone combination upregulated human AE2 alternate overlapping promoter sequences from which AE2b1 and AE2b2 are expressed. In chromatin immunoprecipitation assays, we demonstrated that combination UCDA/dexamethasone treatment induced p300-related interactions between HNF1 and glucocorticoid receptor on the AE2 alternate promoter. Our data provide a potential molecular explanation for the beneficial effects of the combination of UDCA and glucocorticoids in PBC patients with inadequate response to UDCA monotherapy.

Effects of angiogenic factor overexpression by human and rodent cholangiocytes in polycystic liver diseases.

Liver involvement in autosomal dominant polycystic kidney disease (ADPKD) is characterized by altered remodeling of the embryonic ductal plate (DP) with presence of biliary cysts and aberrant portal vasculature. The genetic defect causing ADPKD has been identified, but mechanisms of liver cyst growth remain uncertain. To investigate the possible role of angiogenic mechanisms, we have studied the immunohistochemical expression of vascular endothelial growth factor (VEGF), angiopoietin-1 (Ang-1), angiopoietin-2 (Ang-2) and their receptors (VEGFR-1, VEGFR-2, Tie-2) in ADPKD, Caroli's disease, normal and fetal livers. In ADPKD and control livers Ang-1 and Ang-2 gene expression was studied by real-time-PCR. Effects of VEGF on cholangiocyte proliferation were studied by PCNA Western Blot in isolated rat cholangiocytes and by MTS assay in cultured cholangiocytes isolated from ADPKD patients and from an ADPKD mouse model (Pkd2(WS25/-)). Cholangiocytes were strongly positive for VEGF, VEGFR-1, VEGFR-2 and Ang-2 in ADPKD and Caroli, and also for Ang-1 and Tie-2 in ADPKD, similar to fetal ductal plate cells. VEGF stimulated proliferation in both normal and ADPKD cholangiocytes, but the effect was particularly evident in the latter. Ang-1 alone had no effect, but was synergic to VEGF. VEGF expression on cholangiocytes positively correlated with microvascular density. In conclusion, consistent with the immature phenotype of the cystic epithelium, expression of VEGF, VEGFRs, Ang-1 and Tie-2 is strongly upregulated in cholangiocytes from polycystic liver diseases. VEGF and Ang-1 have autocrine proliferative effect on cholangiocyte growth and paracrine effect on portal vasculature, thus promoting the growth of the cysts and their vascular supply. Supplementary material for this article can be found on the HEPATOLOGY website (http://interscience.wiley.com/jpages/0270-9139/suppmat/index.html).

Preservation of the synthetic and metabolic capacity of isolated human hepatocytes by coculture with human biliary epithelial cells.

Bioartificial liver support systems have demonstrated limited efficacy in compensation of liver detoxification and substitution of liver-derived factors. However, in these devices, the biological substitution of the complex liver function has been restricted to xenogeneic or transformed hepatocytes. Therefore, we have examined the long-term effect of coculturing normal human hepatocytes (HCs) with allogeneic biliary epithelial cells (BECs). We applied functional in vitro assays to examine their metabolic potential by ammonia detoxification to urea, cytochrome P450-dependent lignocaine conversion to mono-ethyl-glycine-xylidide (MEGX), and protein expression and secretion. As the liver has a pivotal role in the synthesis of coagulation factors, we measured antithrombin III (AT III), factor VII, and albumin, comparing HCs plated on collagen or inside 3-dimensional collagen gels. Over 30 days, expression and secretion of albumin and clotting factors by human HCs were augmented by culture inside collagen gel, but were also enhanced and better maintained by coculture with BECs. Higher proportions of BECs cocultured with HCs substantially increased the protein synthesis and urea production. Remarkably, the almost absent cytochrome P450 activity of HC alone after 1 week could be reversed and maintained over 3 weeks by coculture with BECs. The pattern of these effects differed from the extent of interleukin-6 (IL-6) production and HC viability under the compared conditions. In conclusion, coculture of human HCs with BECs impressively restores the synthetic and metabolic liver function in vitro. These results suggest mechanisms of improved liver epithelial differentiation supported by coculture conditions. This technique offers new perspectives in bioartificial liver support, hepatocyte transplantation, and stem cell differentiation.

Does a betaretrovirus infection trigger primary biliary cirrhosis?

Patients with primary biliary cirrhosis develop progressive ductopenia associated with the production of antimitochondrial antibodies that react with a protein aberrantly expressed on biliary epithelial cells and peri-hepatic lymph nodes. Although no specific microbe has been identified, it is thought that an infectious agent triggers this autoimmune liver disease in genetically predisposed individuals. Previous serologic studies have provided evidence to suggest a viral association with primary biliary cirrhosis. Here we describe the identification of viral particles in biliary epithelium by electron microscopy and the cloning of exogenous retroviral nucleotide sequences from patients with primary biliary cirrhosis. The putative agent is referred to as the human betaretrovirus because it shares close homology with the murine mammary tumor virus and a human retrovirus cloned from breast cancer tissue. In vivo, we have found that the majority of patients with primary biliary cirrhosis have both RT-PCR and immunohistochemistry evidence of human betaretrovirus infection in lymph nodes. Moreover, the viral proteins colocalize to cells demonstrating aberrant autoantigen expression. In vitro, we have found that lymph node homogenates from patients with primary biliary cirrhosis can induce autoantigen expression in normal biliary epithelial cells in coculture. Normal biliary epithelial cells also develop the phenotypic manifestation of primary biliary cirrhosis when cocultivated in serial passage with supernatants containing the human betaretrovirus or the murine mammary tumor virus, providing a model to test Koch's postulates in vitro.

Defective regulation of cholangiocyte Cl-/HCO3(-) and Na+/H+ exchanger activities in primary biliary cirrhosis.

Primary biliary cirrhosis (PBC) is a disorder of unknown origin with autoimmune features. Recently, impaired biliary secretion of bicarbonate has been shown in patients with PBC. Here we have investigated whether bile duct epithelial cells isolated from PBC patients exhibit defects in transepithelial bicarbonate transport by analyzing the activities of 2 ion exchangers, Cl(-)/HCO3(-) anion exchanger 2 (AE2) and Na(+)/H(+) exchanger (NHE) in isolated cholangiocytes. AE2 and NHE activities were studied in basal conditions and after stimulation with cyclic adenosine monophosphate (cAMP) and extracellular adenosine triphosphate (ATP), respectively. Cholangiocytes were grown from needle liver biopsies from 12 PBC patients, 8 normal controls, and 9 patients with other liver diseases. Also, intrahepatic cholangiocytes were cultured after immunomagnetic isolation from normal liver tissue (n = 6), and from recipients undergoing liver transplantation for end-stage PBC (n = 9) and other forms of liver disease (n = 8). In needle-biopsy cholangiocytes, basal AE2 activity was significantly decreased in PBC as compared with normal livers and disease controls. In addition, we observed that though cAMP increased AE2 activity in cholangiocytes from both normal and non-PBC livers, this effect was absent in PBC cholangiocytes. Similarly, though in cholangiocytes from normal and disease control livers extracellular ATP induced a marked enhancement of NHE activity, cholangiocytes from PBC patients failed to respond to purinergic stimulation. In conclusion, our findings provide functional evidence that PBC cholangiocytes exhibit a widespread failure in the regulation of carriers involved in transepithelial H(+)/HCO3(-) transport, thus, providing a molecular basis for the impaired bicarbonate secretion in this cholestatic syndrome.