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Background and Aims: Endoscopic resection is accepted as standard treatment for intramucosal esophageal adenocarcinoma (EAC) that is well or moderately differentiated. Poor differentiation (PD) is judged as a risk factor for lymph node metastasis (LNM), and surgery is recommended. However, the evidence for this recommendation is weak. The aim of this study was to analyze the clinical course of patients after endoscopic resection of EAC with PD., Methods: Patients undergoing endoscopic submucosal dissection for EAC were included from 16 German centers. Inclusion criteria were PD in the resection specimen, R0 resection, and endoscopic follow-up. Primary outcome was the metastasis rate during follow-up. Analysis was performed retrospectively in a prospectively collected database., Results: Twenty-five patients with PD as single risk factor (group A) and 15 patients with PD and additional risk factors (submucosal invasion and/or lymphovascular invasion) (group B) were included. The metastasis rate was was 1 of 25 (4.0%; 95% CI, .4%-17.2%) in group A and 3 of 15 (20.0%; 95% CI, 6.0%-44.4%) in group B, respectively (P = .293). The rate of EAC-associated deaths was 1 of 25 (4%; 95% CI, .4%-17.2%) versus 3 of 15 (20%; 95% CI, 6.0%-44.4%) in group B (P = .293). The overall death rate was 7 of 25 (28.0%; 95% CI, 13.5%-47.3%) versus 3 of 15 (20%; 95% CI, 6.0%-44.4%) (P = .715). Median follow-up was 30 months (interquartile range, 15-53 months)., Conclusions: During long-term follow-up, the risk of metastasis is low after endoscopic resection of mucosal EAC with PD as a single risk factor. A conservative approach seems justified in this small patient group. However, the treatment strategy must be determined on an individualized basis until further prospective data are available., Competing Interests: Disclosure All authors disclosed no financial relationships., (Copyright © 2024 American Society for Gastrointestinal Endoscopy. Published by Elsevier Inc. All rights reserved.)

Background: In most clinical studies older people are underrepresented compared to the demographic reality. However, risk for some severe diseases like cancer typically increase with age. Most insight into cancer treatment comes from mixed-age patient cohorts, leading to a lack of detailed understanding of cancer drug effects in the elderly population. There is growing evidence that cancer drug effects can be influenced by dehydration conditions often found in older people. Colon cancer remains the second leading cause of death by cancer in Europe. Inter- and intra-heterogeneity of tumors contribute to why some individuals do not respond to specific cancer therapies or may often suffer a relapse., Objective: Our study applies an in vitro drug test system for simulating treatment with cytostatics of colorectal cancer in elderly patients with dehydration condition., Methods: Two well-known colon cancer cell lines, Caco-2 and RKO, harboring defined cancer-related mutations, were step-wisely adapted from routine culture medium to a severe hyperosmotic condition (397 mOmol/kg) by adding sodium chloride to the medium. We investigated the effects of these cell culture conditions, which should mimic cellular dehydration in elderly people, on the growth characteristics of the cells. Therefore, cell proliferation was investigated by measuring population doubling times. Furthermore, we investigated how the metabolic activity of the cells was influenced by treatment with different concentrations of cyclophosphamide (CPA) under normal and hyperosmotic conditions., Results: We found that Caco-2 and RKO cell lines have an identical cell doubling time of 23 hours in normosmotic medium. However, hyperosmotic medium lifted the doubling time of Caco-2 cells to 31 hours while that of RKO cells did not change. Despite reduced cell proliferation rates, hyperosmotic medium sensitized Caco-2 cells to treatment with 10 mM CPA for 48 hours as measured by metabolic activity assays on ATP levels., Conclusions: The two investigated colon cancer cells lines reacted differently to hyperosmotic conditions. Only the growth of Caco-2 cells was reduced by increased osmolality. Despite this reduced growth their sensitivity to an alkylating cytostatic agent was even slightly increased. We are now in line to examine these effects in more detail and with more tumor cell lines.

History and Admission Findings: We report on the case of an elderly patient with persisting diarrhea. Few weeks previous of admission the patient had received antibiotic therapy because of respiratory infection. On admission he seemed exsiccated and feeble., Examinations: Macroscopic findings in colonoscopy showed proctosigmoiditis and membranous exsudations. Stool culture provided the evidence for an antibiotic-associated infection with pseudomonas aeruginosa., Treatment and Course: The recommended oral therapy with ciprofloxacin proved to be effective., Conclusion: Complications with elderly patients are multimorbidity and diarrhea-induced prerenal failure. Frail patients can react strongly to antibiotic therapy with enteritis and dysbacteriosis., Competing Interests: Disclosure The authors report no conflicts of interest in this work., (© Georg Thieme Verlag KG Stuttgart · New York.)

The calcium-activated potassium channel KCa3.1 controls different cellular processes such as proliferation and volume homeostasis. We investigated the role of KCa3.1 in experimental and human liver fibrosis. KCa3.1 gene expression was investigated in healthy and injured human and rodent liver. Effect of genetic depletion and pharmacological inhibition of KCa3.1 was evaluated in mice during carbon tetrachloride induced hepatic fibrogenesis. Transcription, protein expression and localisation of KCa3.1 was analysed by reverse transcription polymerase chain reaction, Western blot and immunohistochemistry. Hemodynamic effects of KCa3.1 inhibition were investigated in bile duct-ligated and carbon tetrachloride intoxicated rats. In vitro experiments were performed in rat hepatic stellate cells and hepatocytes. KCa3.1 expression was increased in rodent and human liver fibrosis and was predominantly observed in the hepatocytes. Inhibition of KCa3.1 aggravated liver fibrosis during carbon tetrachloride challenge but did not change hemodynamic parameters in portal hypertensive rats. In vitro, KCa3.1 inhibition leads to increased hepatocyte apoptosis and DNA damage, whereas proliferation of hepatic stellate cells was stimulated by KCa3.1 inhibition. Our data identifies KCa3.1 channels as important modulators in hepatocellular homeostasis. In contrast to previous studies in vitro and other tissues this channel appears to be anti-fibrotic and protective during liver injury.

Submucosal tumors of stomach and esophagus are often detected incidentally during endoscopy and further characterized by endoscopic ultrasonography. After risk estimation such submucosal tumors are either controlled by watchful waiting or surgically resected. Nevertheless, symptomatic submucosal tumors should be treated. Endoscopic submucosal excavation (ESE) and submucosal tunneling endoscopic resection (STER) may represent an alternative non-surgical therapeutic option. Two cases of complete endoscopic resection of symptomatic submucosal tumors are reported: a small gastrointestinal stroma tumor (GIST) of the antrum and a 12  cm long esophageal lipoma. For selected cases, ESE of symptomatic submucosal tumors of stomach and esophagus represents a useful alternative compared to surgical removal particularly if mass is located in antrum or corpus, sized < 20  mm and clearly defined by endoscopic ultrasonography., (© Georg Thieme Verlag KG Stuttgart · New York.)

Insulin is known to induce hepatocyte swelling, which triggers via integrins and c-Src kinase an activation of the epidermal growth factor receptor (EGFR) and subsequent cell proliferation (1). Free fatty acids (FFAs) are known to induce lipoapoptosis in liver cells in a c-Jun-NH2-terminal kinase (JNK)-dependent, but death receptor-independent way (2). As non-alcoholic steatohepatitis (NASH) is associated with hyperinsulinemia and increased FFA-blood levels, the interplay between insulin and FFA was studied with regard to hepatocyte proliferation and apoptosis in isolated rat and mouse hepatocytes. Saturated long chain FFAs induced apoptosis and JNK activation in primary rat hepatocytes, but did not activate the CD95 (Fas, APO-1) system, whereas insulin triggered EGFR activation and hepatocyte proliferation. Coadministration of insulin and FFAs, however, abolished hepatocyte proliferation and triggered CD95-dependent apoptosis due to a JNK-dependent association of the activated EGFR with CD95, subsequent CD95 tyrosine phosphorylation and formation of the death-inducing signaling complex (DISC). JNK inhibition restored the proliferative insulin effect in presence of FFAs and prevented EGFR/CD95 association, CD95 tyrosine phosphorylation and DISC formation. Likewise, in presence of FFAs insulin increased apoptosis in hepatocytes from wild type but not from Alb-Cre-FAS(fl/fl) mice, which lack functional CD95. It is concluded that FFAs can shift insulin-induced hepatocyte proliferation toward hepatocyte apoptosis by triggering a JNK signal, which allows activated EGFR to associate with CD95 and to trigger CD95-dependent apoptosis. Such phenomena may contribute to the pathogenesis of NASH., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Background/aims: Ursodeoxycholic acid, which in vivo is rapidly converted into its taurine conjugate, is frequently used for the treatment of cholestatic liver disease. Apart from its choleretic effects, tauroursodeoxycholate (TUDC) can protect hepatocytes from bile acid-induced apoptosis, but the mechanisms underlying its anti-apoptotic effects are poorly understood., Methods: These mechanisms were investigated in perfused rat liver and isolated rat hepatocytes., Results: It was found that TUDC inhibited the glycochenodeoxycholate (GCDC)-induced activation of the CD95 death receptor at the level of association between CD95 and the epidermal growth factor receptor. This was due to a rapid TUDC-induced β1-integrin-dependent cyclic AMP (cAMP) signal with induction of the dual specificity mitogen-activated protein (MAP) kinase phosphatase 1 (MKP-1), which prevented GCDC-induced phosphorylation of mitogen-activated protein kinase kinase 4 (MKK4) and c-jun-NH2-terminal kinase (JNK) activation. Furthermore, TUDC induced a protein kinase A (PKA)-mediated serine/threonine phosphorylation of the CD95, which was recently identified as an internalization signal for CD95. Furthermore, TUDC inhibited GCDC-induced CD95 targeting to the plasma membrane in a β1-integrin-and PKA-dependent manner. In line with this, the β1-integrin siRNA knockdown in sodium taurocholate cotransporting polypeptide (Ntcp)-transfected HepG2 cells abolished the protective effect of TUDC against GCDC-induced apoptosis., Conclusion: TUDC exerts its anti-apoptotic effect via a β1-integrin-mediated formation of cAMP, which prevents CD95 activation by hydrophobic bile acids at the levels of JNK activation and CD95 serine/threonine phosphorylation., (© 2015 S. Karger AG, Basel.)

The Src family kinases Yes, Fyn, and c-Src play a pivotal role in regulating diverse liver functions such as bile flow, proteolysis, apoptosis, and proliferation and are regulated by anisoosmotic cell volume changes, death receptor ligands, and bile acids. For example, cell swelling leads to an integrin-sensed and focal adhesion kinase-mediated activation of c-Src-triggering choleresis, proteolysis inhibition, regulatory volume decrease via p38MAPK and proliferation via the activation of the epidermal growth factor receptor and extracellular regulated kinases 1 and 2. In contrast, hepatocyte shrinkage generates an almost instantaneous oxidative stress response that triggers the activation of c-Jun N-terminal kinase and the Src family kinases Fyn and Yes. Whereas Fyn activation mediates cholestasis, Yes triggers CD95 activation and apoptosis. This review will discuss the role of Src family kinases in the regulation of liver function with emphasis on their role in osmo-signaling and bile acid signaling.

Unlabelled: Ursodeoxycholic acid, which in vivo is converted to its taurine conjugate tauroursodeoxycholic acid (TUDC), is a mainstay for the treatment of cholestatic liver disease. Earlier work showed that TUDC exerts its choleretic properties in the perfused rat liver in an α5 β1 integrin-mediated way. However, the molecular basis of TUDC-sensing in the liver is unknown. We herein show that TUDC (20 μmol/L) induces in perfused rat liver and human HepG2 cells the rapid appearance of the active conformation of the β1 subunit of α5 β1 integrins, followed by an activating phosphorylation of extracellular signal-regulated kinases. TUDC-induced kinase activation was no longer observed after β1 integrin knockdown in isolated rat hepatocytes or in the presence of an integrin-antagonistic hexapeptide in perfused rat liver. TUDC-induced β1 integrin activation occurred predominantly inside the hepatocyte and required TUDC uptake by way of the Na(+) /taurocholate cotransporting peptide. Molecular dynamics simulations of a 3D model of α5 β1 integrin with TUDC bound revealed significant conformational changes within the head region that have been linked to integrin activation before., Conclusions: TUDC can directly activate intrahepatocytic β1 integrins, which trigger signal transduction pathways toward choleresis. (HEPATOLOGY 2013)., (Copyright © 2012 American Association for the Study of Liver Diseases.)

We studied the downregulation of hepatobiliary transport systems and the effect of pharmacological heme oxygenase-1 (HO-1) preinduction by Hemoglobin-Glutamer 200 (HbG200) in cold ischemia-reperfused rat liver (I/R). Cold I/R reduced bile flow in the reperfusion period from 3.10±0.10 ml/3 h to 0.54±0.20 ml/3 h (p<0.05) and biliary taurocholate excretion from 45.9±13.81 μmol/3 h to 1.87±0.46 μmol/3 h (p<0.05). Mrp2, Bsep and Ntcp peak immunofluorescence in pericentral hepatocytes decreased to 79.0±2.6% (p<0.001), 80.6±8.4% (p<0.05) and 65.8±5.0% (p<0.01), respectively. Pre-induction of HO-1 by HbG200 was largely confined to pericentral hepatocytes. HO-1 induction attenuated the decreased bile flow (0.91±0.16 ml/3 h, p<0.05) and canalicular taurocholate secretion (4.33±1.71 μmol/3 h, p<0.05). Bsep and Mrp2 peak immunofluorescence in pericentral hepatocytes was largely restored. Activation of JNK and Fyn by cold I/R was significantly attenuated by HO-1. Inhibiting HO activity by tin protoporphyrin IX after HbG200 administration reversed the effect on bile flow and canalicular transporter expression. In conclusion, pericentral downregulation of Bsep and Mrp2 following cold I/R is ameliorated by inducing HO-1 and was associated with diminished hepatocellular JNK and Fyn signaling. HO-1 may serve as a therapeutic target to attenuate hepatocellular cholestasis following I/R injury.

Recent evidence suggests that signaling pathways towards cell proliferation and cell death are much more interconnected than previously thought. Whereas not only death receptors such as CD95 (Fas, APO-1) can couple to both, cell death and proliferation, also growth factor receptors such as the epidermal growth factor receptor (EGFR) are involved in these opposing kinds of cell fate. EGFR is briefly discussed as a growth factor receptor involved in liver cell proliferation during liver regeneration. Then the role of EGFR in activating CD95 death receptor in liver parenchymal cells (PC) and hepatic stellate cells (HSC), which represent a liver stem/progenitor cell compartment, is described summarizing different ways of CD95- and EGFR-dependent signaling in the liver. Here, depending on the hepatic cell type (PC vs. HSC) and the respective signaling context (sustained vs. transient JNK activation) CD95-/EGFR-mediated signaling ends up in either liver cell apoptosis or cell proliferation., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Hepatocyte volume regulation has been shown to play an important role in cellular metabolism, proliferation, viability and especially in hepatic functions such as bile formation and proteolysis. Recent studies on liver explants led to the assumption that cell volume changes present a trigger for outside-in signaling via integrins, a protein family involved in mediating cellular response to binding to the extracellular matrix (ECM). However, it remains elusive how these volume change related signaling events are transducted on a single cell level and how these events are influenced and controlled by ECM interactions. One could speculate that an increase in cell volume leads to an increase in integrin/ECM contacts which causes activation of integrins, which act as mechano-sensors. In order to test this idea, it was an important issue to quantify the cell volume-dependence of the contact areas between the cell and the surrounding ECM. In this study we used two wavelength reflection interference contrast microscopy (DW-RICM) to directly observe the dynamics of cell-substrate contacts, mimicking cell-ECM interactions, in response to a controlled and well-defined volume change induced by hypoosmotic stimulation. This is the first time a non-invasive, label-free method is used to uncover a volume change related response of in vitro hepatocytes in real time. The cell cluster analysis we present here agrees well with previous studies on ex vivo whole liver explants. Moreover, we show that the increase in contact area after cell swelling is a reversible process, while the reorganisation of contacts depends on the type of ECM molecules presented to the cells. As our method complements common whole liver studies providing additional insight on a cell cluster level, we expect this technique to be particular suitable for further detailed studies of osmotic stimulation not only in hepatocytes, but also other cell types.

In perfused rat liver, hyperosmolarity induces Mrp2- (Kubitz, R., D'urso, D., Keppler, D., and Häussinger, D. (1997) Gastroenterology 113, 1438-1442) and Bsep retrieval (Schmitt, M., Kubitz, R., Lizun, S., Wettstein, M., and Häussinger, D. (2001) Hepatology 33, 509-518) from the canalicular membrane leading to cholestasis. The aim of this study was to elucidate the underlying signaling events. Hyperosmolarity-induced retrieval of Mrp2 and Bsep from the canalicular membrane in perfused rat liver was accompanied by an activating phosphorylation of the Src kinases Fyn and Yes but not of c-Src. Both hyperosmotic transporter retrieval and Src kinase activation were sensitive to apocynin (300 μmol/liter), N-acetylcysteine (NAC; 10 mmol/liter), and SU6656 (1 μmol/liter). Also PP-2 (250 nmol/liter), which inhibited hyperosmotic Fyn but not Yes activation, prevented hyperosmotic transporter retrieval from the canalicular membrane, suggesting that Fyn but not Yes mediates hyperosmotic Bsep and Mrp2 retrieval. Neither hyperosmotic Fyn activation nor Bsep/Mrp2 retrieval was observed in livers from p47(phox) knock-out mice. Hyperosmotic activation of JNKs was sensitive to apocynin and NAC but insensitive to SU6656 and PP-2, indicating that JNKs are not involved in transporter retrieval, as also evidenced by experiments using the JNK inhibitors L-JNKI-1 and SP6001255, respectively. Hyperosmotic transporter retrieval was accompanied by a NAC and Fyn knockdown-sensitive inhibition of biliary excretion of the glutathione conjugate of 1-chloro-2,4-dinitrobenzene in perfused rat liver and of cholyl-L-lysyl-fluorescein secretion into the pseudocanaliculi formed by hepatocyte couplets. Hyperosmolarity triggered an association between Fyn and cortactin and increased the amount of phosphorylated cortactin underneath the canalicular membrane. It is concluded that the hyperosmotic cholestasis is triggered by a NADPH oxidase-driven reactive oxygen species formation that mediates Fyn-dependent retrieval of the Mrp2 and Bsep from the canalicular membrane, which may involve an increased cortactin phosphorylation.

The phospholipidfloppase MDR3 (gene symbol: ABCB4) is expressed in the canalicular membrane of hepatocytes and mediates the biliary excretion of phosphatidylcholine, which is required for the formation of mixed micelles in bile. Several mutations of ABCB4 have been identified, which cause cholestatic liver diseases of varying severity including progressive familial intrahepatic cholestasis type 3 (PFIC-3), intrahepatic cholestasis of pregnancy (ICP) and the low phospholipid associated cholelithiasis syndrome (LPAC). Here, we report on four new (S1076N; L 23Hfs16X; c.286 + 1G > A; Q 1181E) and one known (S27G) MDR3 mutations in eight patients of three families. The patients presented with a wide spectrum of liver diseases. The clinical presentation and decisive laboratory findings or the association to a trend-setting family history led to the identification of the genetic background in these patients. Even the same mutation may be associated with varying disease progression., (© Georg Thieme Verlag KG Stuttgart · New York.)

The phosphoinositide 3'-kinase (PI3 K)/Akt pathway controls the activity of a number of proteins important in the regulation of apoptosis and cell proliferation. FoxO (forkhead box, class O) transcription factors, substrates of the Ser/Thr kinase Akt, control the expression of several target genes that are crucial to the defense against oxidative stress, the regulation of cell cycle, and apoptosis in mammalian cells. Here, expression of ceruloplasmin (CP), the major copper-containing protein in blood released by the liver, was investigated. We observed a significant downregulation of CP mRNA levels after insulin treatment in H4IIE rat hepatoma cells. The PI3K inhibitor wortmannin counteracted this insulin effect on CP mRNA levels, indicating that the PI3K/Akt cascade is involved in the regulation of CP expression. Stimulation of FoxO1 was induced in H4IIE rat hepatoma cells expressing a conditionally active FoxO1 construct, resulting in significant upregulation of CP mRNA levels. This upregulation was prevented in the presence of insulin. In parallel, mRNAs of established FoxO target genes were analyzed: like CP mRNA, selenoprotein P and glucose 6-phosphatase mRNAs were upregulated by FoxO1, which was prevented by insulin. The same effects of insulin on CP mRNA levels were detected in primary rat hepatocytes. Furthermore, CP release into cell culture media was analyzed with primary hepatocytes and found to be attenuated by insulin. In line with its insulin-mimetic effects on cultured cells, Cu (2+) imitated the effect of insulin on CP expression and caused a downregulation of CP mRNA levels in rat hepatoma cells., (© Georg Thieme Verlag KG Stuttgart · New York.)

Changes in mammalian cell volume as induced by either anisoosmolarity, hormones, nutrients or oxidative stress critically contribute to the regulation of metabolism, membrane transport, gene expression and the susceptibility to cellular stress. Osmosensing, i.e. the registration of cell volume changes, triggers signal transduction pathways towards effector pathways (osmosignaling) which link alterations of cell volume to changes in cell function. This review summarizes our own work on the understanding of how osmosensing and osmosignaling integrate into the overall context of bile acid transport, growth factor signaling and the execution of apoptotic programs., (Copyright © 2011 S. Karger AG, Basel.)

High concentrations of urea were shown to induce a paradoxical regulatory volume decrease response with K(+) channel opening and subsequent hepatocyte shrinkage (Hallbrucker, C., vom Dahl, S., Ritter, M., Lang, F., and Häussinger, D. (1994) Pflügers Arch. 428, 552-560), although the hepatocyte plasma membrane is thought to be freely permeable to urea. The underlying mechanisms remained unclear. As shown in the present study, urea (100 mmol/liter) induced within 1 min an activation of β(1) integrins followed by an activation of focal adhesion kinase, c-Src, p38(MAPK), extracellular signal-regulated kinases, and c-Jun N-terminal kinase. Because α(5)β(1) integrin is known to act as a volume/osmosensor in hepatocytes, which becomes activated in response to hepatocyte swelling, the findings suggest that urea at high concentrations induces a nonosmotic activating perturbation of this osmosensor, thereby triggering a volume regulatory K(+) efflux. In line with this, similar to hypo-osmotic hepatocyte swelling, urea induced an inhibition of hepatic proteolysis, which was sensitive to p38(MAPK) inhibition. Molecular dynamics simulations of a three-dimensional model of the ectodomain of α(5)β(1) integrin in water, urea, or thiourea solutions revealed significant conformational changes of α(5)β(1) integrin in urea and thiourea solutions, in contrast to the simulation of α(5)β(1) in water. These changes lead to an unbending of the integrin structure around the genu, which may suggest activation, whereas the structures of single domains remained essentially unchanged. It is concluded that urea at high concentrations affects hepatic metabolism through direct activation of the α(5)β(1) integrin system.

The aim of the study was to analyze whether the proliferative effects of insulin in rat liver involve cross-signaling toward the epidermal growth factor receptor (EGFR) and whether this is mediated by insulin-induced hepatocyte swelling. Studies were performed in the perfused rat liver and in primary rat hepatocytes. Insulin (35 nmol/liter) induced phosphorylation of the EGFR at position Tyr(845) and Tyr(1173), but not at Tyr(1045), suggesting that EGF is not involved in insulin-induced EGFR activation. Insulin-induced EGFR phosphorylation and subsequent ERK1/2 phosphorylation were sensitive to bumetanide, indicating an involvement of insulin-induced hepatocyte swelling. In line with this, hypoosmotic (225 mosmol/liter) hepatocyte swelling also induced EGFR and ERK1/2 activation. Insulin- and hypoosmolarity-induced EGFR activation were sensitive to inhibition by an integrin-antagonistic RGD peptide, an integrin beta1 subtype-blocking antibody, and the c-Src inhibitor PP-2, indicating the involvement of the recently described integrin-dependent osmosensing/signaling pathway (Schliess, F., Reissmann, R., Reinehr, R., vom Dahl, S., and Häussinger, D. (2004) J. Biol. Chem. 279, 21294-21301). As shown by immunoprecipitation studies, insulin and hypoosmolarity induced a rapid, RGD peptide-, integrin beta1-blocking antibody and PP-2-sensitive association of c-Src with the EGFR. As for control, insulin-induced insulin receptor substrate-1 phosphorylation remained unaffected by the RGD peptide, PP-2, or inhibition of the EGFR tyrosine kinase activity by AG1478. Both insulin and hypoosmolarity induced a significant increase in BrdU uptake in primary rat hepatocytes, which was sensitive to RGD peptide-, integrin beta1-blocking antibody, PP-2, AG1478, and PD098059. It is concluded that insulin- or hypoosmolarity-induced hepatocyte swelling triggers an integrin- and c-Src kinase-dependent EGFR activation, which may explain the proliferative effects of insulin.

Sorafenib and vorinostat interact in a synergistic fashion to kill carcinoma cells by activating CD95; the present studies have determined how sorafenib and vorinostat individually contribute to CD95 activation. Sorafenib (3-6 micromol/L) promoted a dose-dependent increase in Src Y416, ERBB1 Y845 and CD95 Y232/Y291 phosphorylation, and Src Y527 dephosphorylation. Low levels of sorafenib-induced (3 micromol/L) CD95 tyrosine phosphorylation did not promote surface localization whereas sorafenib (6 micromol/L), or sorafenib (3 micromol/L) and vorinostat (500 nmol/L) treatment promoted higher levels of CD95 phosphorylation which correlated with DISC formation, receptor surface localization, and autophagy. CD95 (Y232F, Y291F) was not tyrosine phosphorylated and was unable to localize plasma membrane or induce autophagy. Knockdown/knockout of Src family kinases abolished sorafenib-induced CD95 tyrosine phosphorylation, DISC formation, and the induction of cell death and autophagy. Knockdown of platelet-ived growth factor receptor-beta enhanced Src Y416 and CD95 tyrosine phosphorylation, which correlated with elevated CD95 plasma membrane levels and autophagy, and with a reduced ability of sorafenib to promote CD95 membrane localization. Vorinostat increased reactive oxygen species levels, and in a delayed NF kappa B-dependent fashion, those of FAS ligand and CD95. Neutralization of FAS-L did not alter the initial rapid drug-induced activation of CD95; however, neutralization of FAS-L reduced sorafenib + vorinostat toxicity by approximately 50%. Thus, sorafenib contributes to CD95 activation by promoting receptor tyrosine phosphorylation, whereas vorinostat contributes to CD95 activation via the initial facilitation of reactive oxygen species generation and subsequently of FAS-L expression., ((c) 2010 AACR.)

The targeted therapeutics sorafenib and vorinostat interact in a synergistic fashion to kill carcinoma cells by activating CD95, and this drug combination is entering phase I evaluation. In this study, we determined how CD95 is activated by treatment with this drug combination. Low doses of sorafenib and vorinostat, but not the individual drugs, rapidly increased reactive oxygen species (ROS), Ca(2+), and ceramide levels in gastrointestinal tumor cells. The production of ROS was reduced in Rho zero cells. Quenching ROS blocked drug-induced CD95 surface localization and apoptosis. ROS generation, CD95 activation, and cell killing was also blocked by quenching of induced Ca(2+) levels or by inhibition of PP2A. Inhibition of acidic sphingomyelinase or de novo ceramide generation blocked the induction of ROS; however, combined inhibition of both acidic sphingomyelinase and de novo ceramide generation was required to block the induction of Ca(2+). Quenching of ROS did not affect drug-induced ceramide/dihydro-ceramide levels, whereas quenching of Ca(2+) reduced the ceramide increase. Sorafenib and vorinostat treatment radiosensitized liver and pancreatic cancer cells, an effect that was suppressed by quenching ROS or knockdown of LASS6. Further, sorafenib and vorinostat treatment suppressed the growth of pancreatic tumors in vivo. Our findings show that induction of cytosolic Ca(2+) by sorafenib and vorinostat is a primary event that elevates dihydroceramide levels, each essential steps in ROS generation that promotes CD95 activation.

The present studies determine in greater detail the molecular mechanisms upstream of the CD95 death receptor by which geldanamycin heat shock protein 90 inhibitors and mitogen-activated protein kinase/extracellular signal-regulated kinase kinase 1/2 (MEK1/2) inhibitors interact to kill carcinoma cells. MEK1/2 inhibition enhanced 17-allylamino-17-demethoxygeldanamycin (17AAG) toxicity that was suppressed in cells deleted for mutant active RAS that were nontumorigenic but was magnified in isogenic tumorigenic cells expressing Harvey RAS V12 or Kirsten RAS D13. MEK1/2 inhibitor and 17AAG treatment increased intracellular Ca(2+) levels and reduced GRP78/BiP expression in a Ca(2+)-dependent manner. GRP78/BiP overexpression, however, also suppressed drug-induced intracellular Ca(2+) levels. MEK1/2 inhibitor and 17AAG treatment increased reactive oxygen species (ROS) levels that were blocked by quenching Ca(2+) or overexpression of GRP78/BiP. MEK1/2 inhibitor and 17AAG treatment activated CD95 and inhibition of ceramide synthesis; ROS or Ca(2+) quenching blocked CD95 activation. In SW620 cells that are patient matched to SW480 cells, MEK1/2 inhibitor and 17AAG toxicity was significantly reduced, which correlated with a lack of CD95 activation and lower expression of ceramide synthase 6 (LASS6). Overexpression of LASS6 in SW620 cells enhanced drug-induced CD95 activation and enhanced tumor cell killing. Inhibition of ceramide signaling abolished drug-induced ROS generation but not drug-induced cytosolic Ca(2+) levels. Thus, treatment of tumor cells with MEK1/2 inhibitor and 17AAG induces cytosolic Ca(2+) and loss of GRP78/BiP function, leading to de novo ceramide synthesis pathway activation that plays a key role in ROS generation and CD95 activation.

Recent evidence suggests that epidermal growth factor receptor (EGFR)-dependent signaling contributes to liver cell proliferation, as well as to apoptotic liver cell death, and represents an important regulator of hepatic regeneration. This article summarizes recent findings on the molecular mechanisms involved in EGFR-mediated cell proliferation and apoptosis. The emphasis is on the interplay between EGFR and CD95 (Fas, APO-1) death receptor-signaling, which is determined by the signaling context and liver cell type investigated.

Bile acids have been reported to induce epidermal growth factor receptor (EGFR) activation and subsequent proliferation of activated hepatic stellate cells (HSC), but the underlying mechanisms and whether quiescent HSC are also a target for bile acid-induced proliferation or apoptosis remained unclear. Therefore, primary rat HSC were cultured for up to 48 h and analyzed for their proliferative/apoptotic responses toward bile acids. Hydrophobic bile acids, i.e. taurolithocholate 3-sulfate, taurochenodeoxycholate, and glycochenodeoxycholate, but not taurocholate or tauroursodeoxycholate, induced Yes-dependent EGFR phosphorylation. Simultaneously, hydrophobic bile acids induced phosphorylation of the NADPH oxidase subunit p47(phox) and formation of reactive oxygen species (ROS). ROS production was sensitive to inhibition of acidic sphingomyelinase, protein kinase Czeta, and NADPH oxidases. All maneuvers which prevented bile acid-induced ROS formation also prevented Yes and subsequent EGFR phosphorylation. Taurolithocholate 3-sulfate-induced EGFR activation was followed by extracellular signal-regulated kinase 1/2, but not c-Jun N-terminal kinase (JNK) activation, and stimulated HSC proliferation. When, however, a JNK signal was induced by coadministration of cycloheximide or hydrogen peroxide (H2O2), activated EGFR associated with CD95 and triggered EGFR-mediated CD95-tyrosine phosphorylation and subsequent formation of the death-inducing signaling complex. In conclusion, hydrophobic bile acids lead to a NADPH oxidase-driven ROS generation followed by a Yes-mediated EGFR activation in quiescent primary rat HSC. This proliferative signal shifts to an apoptotic signal when a JNK signal simultaneously comes into play.

We examined whether the multikinase inhibitor sorafenib and histone deacetylase inhibitors (HDACI) interact to kill pancreatic carcinoma cells and determined the impact of inhibiting BCL-2 family function on sorafenib and HDACI lethality. The lethality of sorafenib was enhanced in pancreatic tumor cells in a synergistic fashion by pharmacologically achievable concentrations of the HDACIs vorinostat or sodium valproate. Overexpression of cellular FLICE-like inhibitory protein (c-FLIP-s) or knockdown of CD95 suppressed the lethality of the sorafenib/HDACI combination (sorafenib + HDACI). In immunohistochemical analyses or using expression of fluorescence-tagged proteins, treatment with sorafenib and vorinostat together (sorafenib + vorinostat) promoted colocalization of CD95 with caspase 8 and CD95 association with the endoplasmic reticulum markers calnexin, ATG5, and Grp78/BiP. In cells lacking CD95 expression or in cells expressing c-FLIP-s, the lethality of sorafenib + HDACI exposure was abolished and was restored when cells were coexposed to BCL-2 family inhibitors [ethyl [2-amino-6-bromo-4-(1-cyano-2-ethoxy-2-oxoethyl)]-4H-chromene-3-carboxylate (HA14-1), obatoclax (GX15-070)]. Knockdown of BCL-2, BCL-XL, and MCL-1 recapitulated the effects of GX15-070 treatment. Knockdown of BAX and BAK modestly reduced sorafenib + HDACI lethality but abolished the effects of GX15-070 treatment. Sorafenib + HDACI exposure generated a CD95- and Beclin1-dependent protective form of autophagy, whereas GX15-070 treatment generated a Beclin1-dependent toxic form of autophagy. The potentiation of sorafenib + HDACI killing by GX15-070 was suppressed by knockdown of Beclin1 or of BAX + BAK. Our data demonstrate that pancreatic tumor cells are susceptible to sorafenib + HDACI lethality and that in tumor cells unable to signal death from CD95, use of a BCL-2 family antagonist facilitates sorafenib + HDACI killing via autophagy and the intrinsic pathway.

We studied the inhibitory effect of silibinin on ochratoxin A (OTA) and LPS-mediated tumor necrosis factor alpha (TNF-alpha) release and the leakage of cytotoxic markers glutamate dehydrogenase (GLDH) and lactate dehydrogenase (LDH), from isolated blood-free perfused rat livers, and from isolated pure rat Kupffer cells. In the recirculation perfusion model at the end point 90 min, 2.5 micromol/L OTA released 2600 pg/mL TNF-alpha without effects on liver vitality. LPS at 0.1 microg/mL induced 3000 pg TNF-alpha/mL with slight leakage of GLDH and LDH. Under similar experimental conditions, the addition of silibinin 10 min prior to OTA and LPS showed dose-dependent protection against OTA or LPS-induced hepatic TNF-alpha release. High-dose of silibinin (12.5 microg/mL) also completely restored GLDH and LDH levels in the perfusate. Pretreatment of isolated Kupffer cells with 0.02, 0.1, 0.5, 2.5, and 12.5 microg silibinin/mL 30 min prior to OTA reduced OTA-induced TNF-alpha levels to 90, 70, 25, 25, and 25% at 4 h, respectively, and abrogated any TNF-alpha release at 24 h. Similarly, in the presence of silibinin LPS-induced TNF-alpha levels decreased at 4 h to 71, 57, 18, 22, and 18%, respectively. However, after 24 h of LPS exposition the protection by silibinin vanished and TNF-alpha partially recurred into the incubation medium under LPS. In summary, silibinin had hepatoprotective effects against OTA- or LPS-mediated TNF-alpha release and also reduced the cytotoxicity of both toxins. Isolated Kupffer cells were even more sensitive to the protective effect than perfused livers and responded to very low concentrations of silibinin with a strong inhibition of toxins-mediated TNF-alpha release.

Unlabelled: Selenoprotein P (SeP), the major selenoprotein in plasma, is produced mainly by the liver, although SeP expression is detected in many organs. Recently, we reported stimulation of SeP promoter activity by the forkhead box transcription factor FoxO1a in hepatoma cells and its attenuation by insulin. Here, we demonstrate that this translates into fine-tuning of SeP production and secretion by insulin. Overexpression of peroxisomal proliferator activated receptor-gamma coactivator 1alpha (PGC-1alpha) enhanced the stimulatory effect of FoxO1a on SeP promoter activity. We identified a novel functional binding site for hepatocyte nuclear factor (HNF)-4alpha, termed hepatocyte nuclear factor binding element 1, in the human SeP promoter directly upstream of the FoxO-responsive element daf16-binding element 2 (DBE2). Point mutations in hepatocyte nuclear factor binding element 1 alone or together with DBE2 decreased basal activity and responsiveness of the SeP promoter to PGC-1alpha. Moreover, the PGC-1alpha-inducing glucocorticoid dexamethasone strongly enhanced SeP messenger RNA levels and protein secretion in cultured rat hepatocytes, whereas insulin suppressed the stimulation of both PGC-1alpha and SeP caused by dexamethasone treatment. In a brain-derived neuroblastoma cell line with low basal SeP expression, SeP transcription was stimulated by PGC-1alpha together with FoxO1a, and overexpression of HNF-4alpha potentiated this effect., Conclusion: High-level expression of SeP in liver is ensured by concerted action of the coactivator PGC-1alpha and the transcription factors FoxO1a and HNF-4alpha. Hence, the production of SeP is regulated similarly to that of the gluconeogenic enzyme glucose-6-phosphatase. As hepatic SeP production is crucial for selenium distribution throughout the body, the present study establishes PGC-1alpha as a key regulator of selenium homeostasis.

Whereas ligation of the CD95 death receptor in the plasma membrane of so-called type I cells leads to a direct caspase 8-dependent activation of downstream effector caspases, mitochondrial amplification of caspase 8-derived signals is required in so-called type II cells in order to execute apoptotic cell death. In type I cells CD95L (CD95 ligand) binding to CD95 results in a ceramide-dependent formation of the DISC (death-inducing signalling complex) and caspase 8-dependent CD95 clustering in the plasma membrane, followed by an internalization of these multimeric-receptor-DISC complexes. In contrast, in the hepatocyte, a type II cell, the bulk of CD95 is stored intracellularly under resting conditions and only a few 'sentinel' CD95 receptors are present in the plasma membrane. However, their activation by CD95L is sufficient to trigger a caspase 8-dependent endosomal acidification and a ceramide-dependent trafficking of intracellularly stored CD95 to the plasma membrane, thereby amplifying CD95 activation. Thus, in both type I and type II cells, ceramide and CD95 receptor endo- and exo-cytosis are involved in CD95-mediated apoptosis, but apparently in different ways. This, however, is not the only effect of CD95 ligation on intracellular membrane flow in type II cells, and evidence has been presented that soon after CD95 ligation Golgi elements intermix caspase-dependently with mitochondria. In this issue of the Biochemical Journal, Matarrese et al. report another aspect on endocytosis in response to CD95 ligation in type II cells, namely a caspase-independent endocytosis with vesicle translocation to the mitochondrial compartment, suggestive of an interplay between both organelles in the sense of an 'organelle scrambling'. Thus early effects of CD95 activation on intracellular membrane flow may be much more complex than previously thought, but much has still to be learned about signalling mechanisms and the role they play in apoptosis.

Background & Aims: Despite expression of CD95 (Fas) receptor, hepatic stellate cells (HSCs) are fairly resistant toward CD95 ligand (CD95L)-induced cell death. The underlying mechanisms and the function of the CD95 system in quiescent HSCs, however, are unknown., Methods: The effects of CD95L on quiescent, 1- to 2-day cultured rat HSCs were studied with regard to CD95 activation, signal transduction, proliferation, and apoptosis., Results: In quiescent HSCs, CD95L led to a rapid phosphorylation of the epidermal growth factor receptor (EGFR), extracellular signal-regulated kinase (Erk), and c-Src, but not of c-Jun-N-terminal kinase and p47(phox), an activating subunit of reduced nicotinamide adenine dinucleotide phosphate oxidase. CD95L-induced EGFR and Erk phosphorylation were abolished after proteinase inhibition by GM6001 and in the presence of neutralizing epidermal growth factor antibodies, suggestive of a ligand-dependent EGFR phosphorylation in response to CD95L. In quiescent HSCs, CD95L did not induce apoptotic cell death but stimulated HSC proliferation and triggered a rapid inactivating CD95 tyrosine nitration that was not detected in activated HSCs (10-14 days of culture). EGFR phosphorylation, HSC proliferation, and CD95 tyrosine nitration were also triggered by tumor necrosis factor alpha and tumor necrosis factor-related apoptosis-inducing ligand., Conclusions: In quiescent HSCs, CD95L and other death receptor ligands are mitogens through a ligand-dependent EGFR phosphorylation. Simultaneously, an antiapoptotic signaling is triggered by CD95L-induced CD95 tyrosine nitration. This unusual response to death receptor ligands may help quiescent HSCs to participate in liver regeneration following liver injury.