Palmitate induces integrated stress response and lipoapoptosis in trophoblasts.

Maternal obesity increases the risk of childhood obesity and programs the offspring to develop metabolic syndrome later in their life. Palmitate is the predominant saturated free fatty acid (FFA) that is transported across the placenta to the fetus. We have recently shown that saturated FFA in the maternal circulation as a result of increased adipose tissue lipolysis in third trimester of pregnancy induces trophoblast lipoapoptosis. Here, we hypothesized that palmitate induces integrated stress response by activating mitogen-activated protein kinases (MAPKs), endoplasmic reticulum (ER) stress and granular stress and lipoapoptosis in trophoblasts. Choriocarcinoma-derived third-trimester placental trophoblast-like cells (JEG-3 and JAR) referred as trophoblasts were exposed to various concentrations of palmitate (PA). Apoptosis was assessed by nuclear morphological changes and caspase 3/7 activity. Immunoblot and immunofluorescence analysis was performed to measure the activation of MAPKs, ER stress and granular stress response pathways. Trophoblasts exposed to pathophysiological concentrations of PA showed a concentration-dependent increase in trophoblast lipoapoptosis. PA induces a caspase-dependent trophoblast lipoapoptosis. Further, PA induces MAPK activation (JNK and ERK) via phosphorylation, and activation of ER stress as evidenced by an increased phosphorylation eIF2α & IRE1α. PA also induces the activation of stress granules formation. Two pro-apoptotic transcriptional mediators of PA-induced trophoblast lipoapoptosis, CHOP and FoxO3 have increased nuclear translocation. Mechanistically, PA-induced JNK is critical for trophoblast lipoapoptosis. However, PA-induced activation of ERK and stress granule formation were shown to be cell survival signals to combat subcellular stress due to PA exposure. In conclusion, PA induces the activation of integrated stress responses, among which small molecule inhibition of JNK demonstrated that activation of JNK is critical for PA-induced trophoblast lipoapoptosis and small molecule activation of stress granule formation significantly prevents PA-induced trophoblast lipoapoptosis.

Presentation of preclinical gastrointestinal anatomy via laparoscopic simulation.

In this report, the authors examine the integration of teaching anatomical science with clinical implications in minimally invasive surgery. The authors hypothesized that implementation of integrated laparoscopic simulation during undergraduate medical education would improve student learning of anatomical structures from in situ, laparoscopic orientations; and subsequently improve student preparation for clinical rotations and clerkships. During the fall of 2020 and 2021, 260 (130 students/year) second year medical students at the University of Nebraska Medical Center participated in a six-week gastrointestinal curriculum. Following a traditional anatomy dissection experience, students completed a laparoscopic event consisting of narrated laparoscopic videos and hands-on laparoscopic simulation. To examine the integrated curricular event, outcome measures focused on technical performance using grasping forceps, anatomical knowledge, and perception of the educational innovation. Outcomes were analyzed via timed performance and a pre and post assessment that was designed to assess student anatomical knowledge and perception. Completion of the technical performance assessment ranged from 1 min, 17 s to 6 min. Student knowledge of anatomical structures from in situ, laparoscopic orientations following the laparoscopic simulation sessions was significantly improved (53.3% pre vs 81.0% post), and almost all students (98.9%) agreed that the simulation sessions improved their understanding of laparoscopic anatomy and procedures. This report demonstrates the implementation of a multidisciplinary, integrated simulation that satisfied basic science anatomy teaching objectives, while enhancing student enthusiasm for the content. Future studies will examine the subsequent impact of the innovation on student preparedness for clinical rotations and clerkships.

Glycosylation of FGFR4 in cholangiocarcinoma regulates receptor processing and cancer signaling.

Recent advances in targeted treatment for cholangiocarcinoma have focused on fibroblast growth factor (FGF) signaling. There are four receptor tyrosine kinases that respond to FGFs, and posttranslational processing has been demonstrated for each FGF receptor. Here, we investigated the role of N-linked glycosylation on the processing and function of FGFR4. We altered glycosylation through enzymatic deglycosylation, small molecule inhibition of glycosyltransferases, or through site-directed mutagenesis of selected asparagine residues in FGFR4. Signaling was tested through caspase activation, migration, and subcellular localization of FGFR4. Our data demonstrate that FGFR4 has multiple glycoforms, with predominant bands relating to the full-length receptor that has a high mannose- or hybrid-type form and a complex-type glycan form. We further identified a set of faster migrating FGFR4 bands that correspond to the intracellular kinase domain, termed FGFR4 intracellular domain (R4-ICD). These glycoforms and R4-ICD were detected in human cholangiocarcinoma tumor samples, where R4-ICD was predominant. Removal of glycans in intact cells by enzymatic deglycosylation resulted in increased processing to R4-ICD. Inhibition of glycosylation using NGI-1, an oligosaccharyltransferase inhibitor, reduced both high mannose- or hybrid- and complex-type glycan forms of FGFR4, increased processing and sensitized to apoptosis. Mutation of Asn-112, Asn-258, Asn-290, or Asn-311 to glutamine modestly reduced apoptosis resistance, while mutation of Asn-322 or simultaneous mutation of the other four asparagine residues caused a loss of cytoprotection by FGFR4. None of the glycomutants altered the migration of cancer cells. Finally, mutation of Asn-112 caused a partial localization of FGFR4 to the Golgi. Overall, preventing glycosylation at individual residues reduced the cell survival function of FGFR4 and receptor glycosylation may regulate access to an extracellular protease or proteolytic susceptibility of FGFR4.

Cholangiocarcinoma therapy with nanoparticles that combine downregulation of MicroRNA-210 with inhibition of cancer cell invasiveness.

Cholangiocarcinoma (CCA) is the second most common primary liver malignancy with extremely poor therapeutic outcome due to high drug resistance, widespread metastasis and lack of effective treatment options. CCA progression and metastasis are regulated by multiple biological factors including multiple miRNAs and chemokine receptor CXCR4. The goal of this study was to test if nanotherapeutic blockade of CXCR4 by polymeric CXCR4 antagonist (PCX) combined with inhibition of hypoxia-inducible miR-210 cooperatively enhances therapeutic efficacy in CCA through reducing invasiveness, inducing cell killing, and reversing drug resistance. Methods: We first tested the activity of PCX to inhibit migration of CCA cells. We then prepared PCX/anti-miRNA nanoparticles and analyzed their miRNA delivery efficacy and anticancer activity in vitro. Finally, in vivo biodistribution assay and anticancer activity study were performed in CCA tumor-bearing mice. Results: Our results show that PCX had a broad inhibitory effect on cell migration, effectively delivered anti-miR-210, and downregulated miR-210 expression in CCA cells. Combination PCX/anti-miR-210 nanoparticles showed cytotoxic activity towards CCA cells and reduced the number of cancer stem-like cells. The nanoparticles reversed hypoxia-induced drug resistance and sensitized CCA cells to standard gemcitabine and cisplatin combination treatment. Systemic intravenous treatment with the nanoparticles in a CCA xenograft model resulted in prominent combined antitumor activity. Conclusion: Our findings support PCX-based nanoparticles as a promising delivery platform of therapeutic miRNA in combination CCA therapies.

miR-106b-responsive gene landscape identifies regulation of Kruppel-like factor family.

MicroRNA dysregulation is a common feature of cancer and due to the promiscuity of microRNA binding this can result in a wide array of genes whose expression is altered. miR-106b is an oncomiR overexpressed in cholangiocarcinoma and its upregulation in this and other cancers often leads to repression of anti-tumorigenic targets. The goal of this study was to identify the miR-106b-regulated gene landscape in cholangiocarcinoma cells using a genome-wide, unbiased mRNA analysis. Through RNA-Seq we found 112 mRNAs significantly repressed by miR-106b. The majority of these genes contain the specific miR-106b seed-binding site. We have validated 11 genes from this set at the mRNA level and demonstrated regulation by miR-106b of 7 proteins. Combined analysis of our miR-106b-regulated mRNA data set plus published reports indicate that miR-106b binding is anchored by G:C pairing in and near the seed. Novel targets Kruppel-like factor 2 (KLF2) and KLF6 were verified both at the mRNA and at the protein level. Further investigation showed regulation of four other KLF family members by miR-106b. We have discovered coordinated repression of multiple members of the KLF family by miR-106b that may play a role in cholangiocarcinoma tumor biology.

FoxO3 increases miR-34a to cause palmitate-induced cholangiocyte lipoapoptosis.

Nonalcoholic steatohepatitis (NASH) patients have elevated plasma saturated free fatty acid levels. These toxic fatty acids can induce liver cell death and our recent results demonstrated that the biliary epithelium may be susceptible to lipotoxicity. Here, we explored the molecular mechanisms of cholangiocyte lipoapoptosis in cell culture and in an animal model of NASH. Treatment of cholangiocytes with palmitate (PA) showed increased caspase 3/7 activity and increased levels of cleaved poly (ADP-ribose) polymerase and cleaved caspase 3, demonstrating cholangiocyte lipoapoptosis. Interestingly, treatment with PA significantly increased the levels of microRNA miR-34a, a pro-apoptotic microRNA known to be elevated in NASH. PA induction of miR-34a was abolished in cholangiocytes transduced with forkhead family of transcription factor class O (FoxO)3 shRNA, demonstrating that FoxO3 activation is upstream of miR-34a and suggesting that FoxO3 is a novel transcriptional regulator of miR-34a. Further, anti-miR-34a protected cholangiocytes from PA-induced lipoapoptosis. Direct and indirect targets of miR-34a, such as SIRT1, receptor tyrosine kinase (MET), Kruppel-like factor 4, fibroblast growth factor receptor (FGFR)1, and FGFR4, were all decreased in PA-treated cholangiocytes. SIRT1 and MET were partially rescued by a miR-34a antagonist. Cholangiocyte apoptosis and miR-34a were dramatically increased in the liver of mice with early histologic features of NASH. Our study provides evidence for the pro-apoptotic role of miR-34a in PA-induced cholangiocyte lipoapoptosis in culture and in the liver.

Regulation of miR-29b-1/a transcription and identification of target mRNAs in CHO-K1 cells.

miR-29b and miR-29a transcript levels were reported to increase in exponentially growing CHO-K1 cells. Here, we examine the regulation of miR-29b-1/a in CHO-K1 cells. We observed that 4-hydroxytamoxifen (4-OHT) increased pri-miR-29b-1 and pri-miR-29a transcription in CHO-K1 cells by activating endogenous estrogen receptor α (ERα). DICER, an established, bona fide target of miR-29b-1/a, was shown to be regulated by 4-OHT in CHO-K1 cells. We showed that miR-29b-1 and miR-29a serve a repressive role in cell proliferation, migration, invasion, and colony formation in CHO-K1 cells. To identify other targets of miR-29b-1 and miR-29a, RNA sequencing was performed by transfecting cells with anti-miR-29a, which inhibits both miR-29a and miR-29b-1, pre-miR-29b-1, and/or pre-miR-29a. In silico network analysis in MetaCore™ identified common and unique putative gene targets of miR-29b-1 and miR-29a. Pathway analysis of identified putative miR-29 targets were related to cell adhesion, cytoskeletal remodeling, and development. Further inquiry revealed regulation of pathways mediating responses to growth factor stimulus and cell cycle regulation.

Evidence for Pipecolate Oxidase in Mediating Protection Against Hydrogen Peroxide Stress.

Pipecolate, an intermediate of the lysine catabolic pathway, is oxidized to Δ1 -piperideine-6-carboxylate (P6C) by the flavoenzyme l-pipecolate oxidase (PIPOX). P6C spontaneously hydrolyzes to generate α-aminoadipate semialdehyde, which is then converted into α-aminoadipate acid by α-aminoadipatesemialdehyde dehydrogenase. l-pipecolate was previously reported to protect mammalian cells against oxidative stress. Here, we examined whether PIPOX is involved in the mechanism of pipecolate stress protection. Knockdown of PIPOX by small interference RNA abolished pipecolate protection against hydrogen peroxide-induced cell death in HEK293 cells suggesting a critical role for PIPOX. Subcellular fractionation analysis showed that PIPOX is localized in the mitochondria of HEK293 cells consistent with its role in lysine catabolism. Signaling pathways potentially involved in pipecolate protection were explored by treating cells with small molecule inhibitors. Inhibition of both mTORC1 and mTORC2 kinase complexes or inhibition of Akt kinase alone blocked pipecolate protection suggesting the involvement of these signaling pathways. Phosphorylation of the Akt downstream target, forkhead transcription factor O3 (FoxO3), was also significantly increased in cells treated with pipecolate, further implicating Akt in the protective mechanism and revealing FoxO3 inhibition as a potentially key step. The results presented here demonstrate that pipecolate metabolism can influence cell signaling during oxidative stress to promote cell survival and suggest that the mechanism of pipecolate protection parallels that of proline, which is also metabolized in the mitochondria. J. Cell. Biochem. 118: 1678-1688, 2017. © 2016 Wiley Periodicals, Inc.

Tamoxifen differentially regulates miR-29b-1 and miR-29a expression depending on endocrine-sensitivity in breast cancer cells.

Endocrine-resistance develops in ∼40% of breast cancer patients after tamoxifen (TAM) therapy. Although microRNAs are dysregulated in breast cancer, their contribution to endocrine-resistance is not yet understood. Previous microarray analysis identified miR-29a and miR-29b-1 as repressed by TAM in MCF-7 endocrine-sensitive breast cancer cells but stimulated by TAM in LY2 endocrine-resistant breast cancer cells. Here we examined the mechanism for the differential regulation of these miRs by TAM in MCF-7 versus TAM-resistant LY2 and LCC9 breast cancer cells and the functional role of these microRNAs in these cells. Knockdown studies revealed that ERα is responsible for TAM regulation of miR-29b-1/a transcription. We also demonstrated that transient overexpression of miR-29b-1/a decreased MCF-7, LCC9, and LY2 proliferation and inhibited LY2 cell migration and colony formation but did not sensitize LCC9 or LY2 cells to TAM. Furthermore, TAM reduced DICER1 mRNA and protein in LY2 cells, a known target of miR-29. Supporting this observation, anti-miR-29b-1 or anti-miR-29a inhibited the suppression of DICER by 4-OHT. These results suggest miR-29b-1/a has tumor suppressor activity in TAM-resistant cells and does not appear to play a role in mediating TAM resistance.

Role of apoptotic hepatocytes in HCV dissemination: regulation by acetaldehyde.

Alcohol consumption exacerbates hepatitis C virus (HCV) pathogenesis and promotes disease progression, although the mechanisms are not quite clear. We have previously observed that acetaldehyde (Ach) continuously produced by the acetaldehyde-generating system (AGS), temporarily enhanced HCV RNA levels, followed by a decrease to normal or lower levels, which corresponded to apoptosis induction. Here, we studied whether Ach-induced apoptosis caused depletion of HCV-infected cells and what role apoptotic bodies (AB) play in HCV-alcohol crosstalk. In liver cells exposed to AGS, we observed the induction of miR-122 and miR-34a. As miR-34a has been associated with apoptotic signaling and miR-122 with HCV replication, these findings may suggest that cells with intensive viral replication undergo apoptosis. Furthermore, when AGS-induced apoptosis was blocked by a pan-caspase inhibitor, the expression of HCV RNA was not changed. AB from HCV-infected cells contained HCV core protein and the assembled HCV particle that infect intact hepatocytes, thereby promoting the spread of infection. In addition, AB are captured by macrophages to switch their cytokine profile to the proinflammatory one. Macrophages exposed to HCV(+) AB expressed more IL-1ß, IL-18, IL-6, and IL-10 mRNAs compared with those exposed to HCV(-) AB. The generation of AB from AGS-treated HCV-infected cells even enhanced the induction of aforementioned cytokines. We conclude that HCV and alcohol metabolites trigger the formation of AB containing HCV particles. The consequent spread of HCV to neighboring hepatocytes via infected AB, as well as the induction of liver inflammation by AB-mediated macrophage activation potentially exacerbate the HCV infection course by alcohol and worsen disease progression.

Delivery of miR-200c Mimic with Poly(amido amine) CXCR4 Antagonists for Combined Inhibition of Cholangiocarcinoma Cell Invasiveness.

Cholangiocarcinoma is the second most common primary liver malignancy with extremely poor prognosis due to early invasion and widespread metastasis. The invasion and metastasis are regulated by multiple factors including CXCR4 chemokine receptor and multiple microRNAs. The goal of this study was to test the hypothesis that inhibition of CXCR4 combined with the action of miR-200c mimic will cooperatively enhance the inhibition of the invasion of human cholangiocarcinoma cells. The results show that CXCR4-inhibition polycation PCX can effectively deliver miR-200c mimic and that the combination treatment consisting of PCX and miR-200c results in cooperative antimigration activity, most likely by coupling the CXCR4 axis blockade with epithelial-to-mesenchymal transition inhibition in the cholangiocarcinoma cells. The ability of the combined PCX/miR-200c treatment to obstruct two migratory pathways represents a promising antimetastatic strategy in cholangiocarcinoma.

Ceramide Induces Human Hepcidin Gene Transcription through JAK/STAT3 Pathway.

Changes in lipid metabolism and iron content are observed in the livers of patients with fatty liver disease. The expression of hepcidin, an iron-regulatory and acute phase protein synthesized by the liver, is also modulated. The potential interaction of lipid and iron metabolism is largely unknown. We investigated the role of lipid intermediate, ceramide in the regulation of human hepcidin gene, HAMP. Human hepatoma HepG2 cells were treated with cell-permeable ceramide analogs. Ceramide induced significant up-regulation of HAMP mRNA expression in HepG2 cells. The effect of ceramide on HAMP expression was mediated through transcriptional mechanisms because it was completely blocked with actinomycin D treatment. Reporter assays also confirmed the activation of 0.6 kb HAMP promoter by ceramide. HepG2 cells treated with ceramide displayed increased phosphorylation of STAT3, JNK, and NF-κB proteins. However, ceramide induced the binding of STAT3, but not NF-κB or c-Jun, to HAMP promoter, as shown by the chromatin immunoprecipitation assays. The mutation of STAT3 response element within 0.6 kb HAMP promoter region significantly inhibited the stimulatory effect of ceramide on HAMP promoter activity. Similarly, the inhibition of STAT3 with a pan-JAK kinase inhibitor and STAT3 siRNA pool also diminished the induction of both HAMP promoter activity and mRNA expression by ceramide. In conclusion, we have shown a direct role for ceramide in the activation of hepatic HAMP transcription via STAT3. Our findings suggest a crosstalk between lipid and iron metabolism in the liver, which may contribute to the pathogenesis of obesity-related fatty liver disease.

Saturated fatty acids induce post-transcriptional regulation of HAMP mRNA via AU-rich element-binding protein, human antigen R (HuR).

Iron is implicated in fatty liver disease pathogenesis. The human hepcidin gene, HAMP, is the master switch of iron metabolism. The aim of this study is to investigate the regulation of HAMP expression by fatty acids in HepG2 cells. For these studies, both saturated fatty acids (palmitic acid (PA) and stearic acid (SA)) and unsaturated fatty acid (oleic acid (OA)) were used. PA and, to a lesser extent, SA, but not OA, up-regulated HAMP mRNA levels, as determined by real-time PCR. To understand whether PA regulates HAMP mRNA at the transcriptional or post-transcriptional level, the transcription inhibitor actinomycin D was employed. PA-mediated induction of HAMP mRNA expression was not blocked by actinomycin D. Furthermore, PA activated HAMP 3'-UTR, but not promoter, activity, as shown by reporter assays. HAMP 3'-UTR harbors a single AU-rich element (ARE). Mutation of this ARE abolished the effect of PA, suggesting the involvement of ARE-binding proteins. The ARE-binding protein human antigen R (HuR) stabilizes mRNA through direct interaction with AREs on 3'-UTR. HuR is regulated by phosphorylation-mediated nucleo-cytoplasmic shuttling. PA activated this process. The binding of HuR to HAMP mRNA was also induced by PA in HepG2 cells. Silencing of HuR by siRNA abolished PA-mediated up-regulation of HAMP mRNA levels. PKC is known to phosphorylate HuR. Staurosporine, a broad-spectrum PKC inhibitor, inhibited both PA-mediated translocation of HuR and induction of HAMP expression. Similarly, rottlerin, a novel class PKC inhibitor, abrogated PA-mediated up-regulation of HAMP expression. In conclusion, lipids mediate post-transcriptional regulation of HAMP throughPKC- and HuR-dependent mechanisms.

Mmu-miR-615-3p regulates lipoapoptosis by inhibiting C/EBP homologous protein.

Lipoapoptosis occurring due to an excess of saturated free fatty acids such as palmitate is a key pathogenic event in the initiation of nonalcoholic fatty liver disease. Palmitate loading of cells activates the endoplasmic reticulum stress response, including induction of the proapoptotic transcription factor C/EBP homologous protein (CHOP). Furthermore, the loss of microRNAs is implicated in regulating apoptosis under conditions of endoplasmic reticulum (ER) stress. The aim of this study was to identify specific microRNAs regulating CHOP expression during palmitate-induced ER stress. Five microRNAs were repressed under palmitate-induced endoplasmic reticulum stress conditions in hepatocyte cell lines (miR-92b-3p, miR-328-3p, miR-484, miR-574-5p, and miR-615-3p). We identified miR-615-3p as a candidate microRNA which was repressed by palmitate treatment and regulated CHOP protein expression, by RNA sequencing and in silico analyses, respectively. There is a single miR-615-3p binding site in the 3'untranslated region (UTR) of the Chop transcript. We characterized this as a functional binding site using a reporter gene-based assay. Augmentation of miR-615-3p levels, using a precursor molecule, repressed CHOP expression; and under these conditions palmitate- or tunicamycin-induced cell death were significantly reduced. Our results suggest that palmitate-induced apoptosis requires maximal expression of CHOP which is achieved via the downregulation of its repressive microRNA, miR-615-3p. We speculate that enhancement of miR-615-3p levels may be of therapeutic benefit by inhibiting palmitate-induced hepatocyte lipoapoptosis.

Saturated free fatty acids induce cholangiocyte lipoapoptosis.

UNLABELLED: Recent studies have identified a cholestatic variant of nonalcoholic fatty liver disease (NAFLD) with portal inflammation and ductular reaction. Based on reports of biliary damage, as well as increased circulating free fatty acids (FFAs) in NAFLD, we hypothesized the involvement of cholangiocyte lipoapoptosis as a mechanism of cellular injury. Here, we demonstrate that the saturated FFAs palmitate and stearate induced robust and rapid cell death in cholangiocytes. Palmitate and stearate induced cholangiocyte lipoapoptosis in a concentration-dependent manner in multiple cholangiocyte-derived cell lines. The mechanism of lipoapoptosis relied on the activation of caspase 3/7 activity. There was also a significant up-regulation of the proapoptotic BH3-containing protein, PUMA. In addition, palmitate-induced cholangiocyte lipoapoptosis involved a time-dependent increase in the nuclear localization of forkhead family of transcription factor 3 (FoxO3). We show evidence for posttranslational modification of FoxO3, including early (6 hours) deacetylation and dephosphorylation that coincide with localization of FoxO3 in the nuclear compartment. By 16 hours, nuclear FoxO3 is both phosphorylated and acetylated. Knockdown studies confirmed that FoxO3 and its downstream target, PUMA, were critical for palmitate- and stearate-induced cholangiocyte lipoapoptosis. Interestingly, cultured cholangiocyte-derived cells did not accumulate appreciable amounts of neutral lipid upon FFA treatment. CONCLUSION: Our data show that the saturated FFAs palmitate and stearate induced cholangiocyte lipoapoptosis by way of caspase activation, nuclear translocation of FoxO3, and increased proapoptotic PUMA expression. These results suggest that cholangiocyte injury may occur through lipoapoptosis in NAFLD and nonalcoholic steatohepatitis patients.

XIAP antagonist embelin inhibited proliferation of cholangiocarcinoma cells.

Cholangiocarcinoma cells are dependent on antiapoptotic signaling for survival and resistance to death stimuli. Recent mechanistic studies have revealed that increased cellular expression of the E3 ubiquitin-protein ligase X-linked inhibitor of apoptosis (XIAP) impairs TRAIL- and chemotherapy-induced cytotoxicity, promoting survival of cholangiocarcinoma cells. This study was undertaken to determine if pharmacologic antagonism of XIAP protein was sufficient to sensitize cholangiocarcinoma cells to cell death. We employed malignant cholangiocarcinoma cell lines and used embelin to antagonize XIAP protein. Embelin treatment resulted in decreased XIAP protein levels by 8 hours of treatment with maximal effect at 16 hours in KMCH and Mz-ChA-1 cells. Assessment of nuclear morphology demonstrated a concentration-dependent increase in nuclear staining. Interestingly, embelin induced nuclear morphology changes as a single agent, independent of the addition of TNF-related apoptosis inducing ligand (TRAIL). However, caspase activity assays revealed that increasing embelin concentrations resulted in slight inhibition of caspase activity, not activation. In addition, the use of a pan-caspase inhibitor did not prevent nuclear morphology changes. Finally, embelin treatment of cholangiocarcinoma cells did not induce DNA fragmentation or PARP cleavage. Apoptosis does not appear to contribute to the effects of embelin on cholangiocarcinoma cells. Instead, embelin caused inhibition of cell proliferation and cell cycle analysis indicated that embelin increased the number of cells in S and G2/M phase. Our results demonstrate that embelin decreased proliferation in cholangiocarcinoma cell lines. Embelin treatment resulted in decreased XIAP protein expression, but did not induce or enhance apoptosis. Thus, in cholangiocarcinoma cells the mechanism of action of embelin may not be dependent on apoptosis.

Mechanisms of lysophosphatidylcholine-induced hepatocyte lipoapoptosis.

Isolated hepatocytes undergo lipoapoptosis, a feature of hepatic lipotoxicity, on treatment with saturated free fatty acids (FFA) such as palmitate (PA). However, it is unknown if palmitate is directly toxic to hepatocytes or if its toxicity is indirect via the generation of lipid metabolites such as lysophosphatidylcholine (LPC). PA-mediated hepatocyte lipoapoptosis is associated with endoplasmic reticulum (ER) stress, c-Jun NH(2)-terminal kinase (JNK) activation, and a JNK-dependent upregulation of the potent proapoptotic BH3-only protein PUMA (p53 upregulated modulator of apoptosis). Our aim was to determine which of these mechanisms of lipotoxicity are activated by PA-derived LPC. We employed Huh-7 cells and isolated murine and human primary hepatocytes. Intracellular LPC concentrations increase linearly as a function of the exogenous, extracellular PA, stearate, or LPC concentration. Incubation of Huh-7 cells or primary hepatocytes with LPC induced cell death by apoptosis in a concentration-dependent manner. Substituting LPC for PA resulted in caspase-dependent cell death that was accompanied by activating phosphorylation of JNK with c-Jun phosphorylation and an increase in PUMA expression. LPC also induced ER stress as manifest by eIF2α phosphorylation and CAAT/enhancer binding homologous protein (CHOP) induction. LPC cytotoxicity was attenuated by pharmacological inhibition of JNK or glycogen synthase kinase-3 (GSK-3). Similarly, short-hairpin RNA (shRNA)-targeted knockdown of CHOP protected Huh-7 cells against LPC-induced toxicity. The LPC-induced PUMA upregulation was prevented by JNK inhibition or shRNA-targeted knockdown of CHOP. Finally, genetic deficiency of PUMA rendered murine hepatocytes resistant to LPC-induced apoptosis. We concluded that LPC-induced lipoapoptosis is dependent on mechanisms largely indistinguishable from PA. These data suggest that FFA-mediated cytotoxicity is indirect via the generation of the toxic metabolite, LPC.

miR-25 targets TNF-related apoptosis inducing ligand (TRAIL) death receptor-4 and promotes apoptosis resistance in cholangiocarcinoma.

UNLABELLED: It has been established that microRNA expression and function contribute to phenotypic features of malignant cells, including resistance to apoptosis. Although targets and functional roles for a number of microRNAs have been described in cholangiocarcinoma, many additional microRNAs dysregulated in this tumor have not been assigned functional roles. In this study, we identify elevated miR-25 expression in malignant cholangiocarcinoma cell lines as well as patient samples. In cultured cells, treatment with the Smoothened inhibitor, cyclopamine, reduced miR-25 expression, suggesting Hedgehog signaling stimulates miR-25 production. Functionally, miR-25 was shown to protect cells against TNF-related apoptosis-inducing ligand (TRAIL)-induced apoptosis. Correspondingly, antagonism of miR-25 in culture sensitized cells to apoptotic death. Computational analysis identified the TRAIL Death Receptor-4 (DR4) as a potential novel miR-25 target, and this prediction was confirmed by immunoblot, cell staining, and reporter assays. CONCLUSION: These data implicate elevated miR-25 levels in the control of tumor cell apoptosis in cholangiocarcinoma. The identification of the novel miR-25 target DR4 provides a mechanism by which miR-25 contributes to evasion of TRAIL-induced cholangiocarcinoma apoptosis.

Myofibroblast-derived PDGF-BB promotes Hedgehog survival signaling in cholangiocarcinoma cells.

UNLABELLED: Cholangiocarcinoma (CCA) cells paradoxically express the death ligand, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and, therefore, are dependent upon potent survival signals to circumvent TRAIL cytotoxicity. CCAs are also highly desmoplastic cancers with a tumor microenvironment rich in myofibroblasts (MFBs). Herein, we examine a role for MFB-derived CCA survival signals. We employed human KMCH-1, KMBC, HuCCT-1, TFK-1, and Mz-ChA-1 CCA cells, as well as human primary hepatic stellate and myofibroblastic LX-2 cells, for these studies. In vivo experiments were conducted using a syngeneic rat orthotopic CCA model. Coculturing CCA cells with myofibroblastic human primary hepatic stellate cells or LX-2 cells significantly decreased TRAIL-induced apoptosis in CCA cells, a cytoprotective effect abrogated by neutralizing platelet-derived growth factor (PDGF)-BB antiserum. Cytoprotection by PDGF-BB was dependent upon Hedgehog (Hh) signaling, because it was abolished by the smoothened (SMO; the transducer of Hh signaling) inhibitor, cyclopamine. PDGF-BB induced cyclic adenosine monophosphate-dependent protein kinase-dependent trafficking of SMO to the plasma membrane, resulting in glioma-associated oncogene (GLI)2 nuclear translocation and activation of a consensus GLI reporter gene-based luciferase assay. A genome-wide messenger RNA expression analysis identified 67 target genes to be commonly up- (50 genes) or down-regulated (17 genes) by both Sonic hedgehog and PDGF-BB in a cyclopamine-dependent manner in CCA cells. Finally, in a rodent CCA in vivo model, cyclopamine administration increased apoptosis in CCA cells, resulting in tumor suppression. CONCLUSIONS: MFB-derived PDGF-BB protects CCA cells from TRAIL cytotoxicity by a Hh-signaling-dependent process. These results have therapeutical implications for the treatment of human CCA.

Death receptor 5 signaling promotes hepatocyte lipoapoptosis.

Nonalcoholic steatohepatitis is characterized by hepatic steatosis, elevated levels of circulating free fatty acids (FFA), endoplasmic reticulum (ER) stress, and hepatocyte lipoapoptosis. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) death receptor 5 (DR5) is significantly elevated in patients with nonalcoholic steatohepatitis, and steatotic hepatocytes demonstrate increased sensitivity to TRAIL-mediated cell death. Nonetheless, a role for TRAIL and/or DR5 in mediating lipoapoptotic pathways is unexplored. Here, we examined the contribution of DR5 death signaling to lipoapoptosis by free fatty acids. The toxic saturated free fatty acid palmitate induces an increase in DR5 mRNA and protein expression in Huh-7 human hepatoma cells leading to DR5 localization into lipid rafts, cell surface receptor clustering with subsequent recruitment of the initiator caspase-8, and ultimately cellular demise. Lipoapoptosis by palmitate was not inhibited by a soluble human recombinant DR5-Fc chimera protein suggesting that DR5 cytotoxic signaling is ligand-independent. Hepatocytes from murine TRAIL receptor knock-out mice (DR(-/-)) displayed reduced palmitate-mediated lipotoxicity. Likewise, knockdown of DR5 or caspase-8 expression by shRNA technology attenuated palmitate-induced Bax activation and apoptosis in Huh-7 cells, without altering induction of ER stress markers. Similar observations were verified in other cell models. Finally, knockdown of CHOP, an ER stress-mediated transcription factor, reduced DR5 up-regulation and DR5-mediated caspase-8 activation upon palmitate treatment. Collectively, these results suggest that ER stress-induced CHOP activation by palmitate transcriptionally up-regulates DR5, likely resulting in ligand-independent cytotoxic signaling by this death receptor.