Gores, Gregory J.

We have previously shown that cryopreservation leads to increased apoptotic death of porcine hepatocytes intended for use in a bioartificial liver (BAL). This study was designed to determine if a broad-spectrum caspase inhibitor, IDN–1965, reduced apoptosis and increased function of cryopreserved porcine hepatocytes in static culture or in a BAL. Porcine hepatocytes were studied immediately after isolation and after 2 weeks of cryopreservation in liquid nitrogen using medium supplemented with 25 μmol/L IDN-1965 or vehicle. Both apoptotic and necrotic cells were observed in cultures of fresh and cryopreserved hepatocytes, but the percentage of apoptotic cells increased after cryopreservation. Cryopreservation in IDN-1965 improved hepatocyte viability and reduced apoptotic cell death determined by TUNEL assay. Cryopreservation of hepatocytes in IDN-1965 was also associated with reduced caspase 3-like activity, decreased release of cytochrome c from mitochondria, and a slower decline in mitochondrial membrane potential after thawing. These markers of apoptosis were lowest after cryopreservation when IDN-1965 was added to both the culture and cryopreservation medium. Functional markers of hepatocyte activity (albumin production, diazepam metabolism, urea production) were also increased after cryopreservation and culture of hepatocytes in medium supplemented with 25 μmol/L IDN-1965. Cryopreservation of porcine hepatocytes in the presence of caspase inhibitor IDN-1965 was associated with reduced apoptosis and improved function of porcine hepatocytes in both static culture and a perfused BAL. These data demonstrate that inhibition of apoptosis also preserves cell function.

Abstract Introduction: Cholangiocarcinoma (CCA) is a highly lethal hepatobiliary neoplasm with limited therapeutic options. Thus, new therapeutic strategies are needed to advance our treatment armamentarium for this devastating disease. MicroRNAs represent a new class of endogenous nucleic acids regulating development, cell death, and carcinogenesis. Recent studies have indicated that the miR-106b∼25 cluster, which is upregulated in cancer, has potential antiapoptotic properties. Meanwhile, we have demonstrated that Hedgehog (Hh) signaling is activated in CCA and also has a strong antiapoptotic effect. Based on this information, we posited that Hh signaling may regulate mir-106b∼25 expression. Due to the dependence of CCA on antiapoptotic signals, we conducted a study to determine the role of miR-106b∼25 in CCA apoptosis and unveil its regulatory mechanisms. Experimental procedures: Immortalized benign and malignant human cholangiocyte cell lines were used for the experiments. MicroRNA expression was analyzed using real-time quantitative RT-PCR (qRT-PCR) assays. The effect of the microRNAs was studied using Lipofectamine-based transfection with precursor microRNAs or locked-nucleic acid antagonists (LNAs) to respectively potentiate or decrease microRNAs expression. Functional apoptosis studies employed DAPI staining with fluorescent microscopic analysis of nuclear morphology, or biochemical measurement of caspase-3/7 activity. Western blotting and luciferase assays were conducted to identify potential messenger RNA targets. Results: Expression of miR-106b∼25 was enhanced in malignant vs. benign cholangiocyte cell lines. Treatment with the known antagonist of Hh signaling, cyclopamine, resulted in downregulation of the cluster. Because CCA cells express tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in vivo, we next examined TRAIL-induced apoptosis in CCA cells. TRAIL-mediated apoptosis was attenuated by transfection with miR-25 and enhanced by LNAs. Computational analysis identified the TRAIL death receptor 4 (DR4) as a potential target of this cluster. Indeed, we observed downregulation of DR4 cellular protein levels during overexpression of miR-106b∼25 and upregulation of DR4 upon cluster inhibition with LNAs. The 3’untranslated region (UTR) of the DR4 mRNA was cloned into a luciferase reporter, and the potential direct regulation of DR4 by miR-106b∼25 examined. Indeed, the regulation of DR4 by miR-106b∼25 was confirmed by this luciferase assay demonstrating a direct inhibitory effect of the miR-106b∼25 polycistron on the DR4 3’UTR. Conclusions: The miR-106b∼25 cluster is overexpressed in malignant cholangiocyte cell lines, likely by a Hh signaling pathway. miR-106b∼25 inhibits apoptosis, at least in part, via downregulation of DR4. LNA targeted inhibition of miR-106b∼25 sensitizes CCA cells to apoptosis and may represent a new therapeutic strategy for this cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 199. doi:10.1158/1538-7445.AM2011-199

Objectives: The aim of this was to determine survival after starting neoadjuvant therapy for patients who became ineligible for orthotopic liver transplantation (OLT). Methods and Materials: Since January 1993, 215 patients with unresectable cholangiocarcinoma began treatment with planned OLT. Treatment included external-beam radiation therapy (EBRT) with fluorouracil, bile duct brachytherapy, and postradiotherapy fluorouracil or capecitabine before OLT. Adverse findings at the staging operation, death, and other factors precluded OLT in 63 patients (29%), of whom 61 completed neoadjuvant chemoradiation. Results: By October 2012, 56 (89%) of the 63 patients unable to undergo OLT had died. Twenty-two patients (35%) became ineligible for OLT before the staging operation, 38 (60%) at the staging operation, and 3 (5%) after staging. From the date of diagnosis, median overall survival was 12.3 months. Survival was 17% at 18 months and 7% at 24 months. Median survival after fallout was 6.8 months. Median survival after the staging operation was 6 months. Two patients lived for 3.7 and 8.7 years before dying of cancer or liver failure caused by persistent biliary stricture at the site of the original cancer, respectively. Univariate analysis showed that time from diagnosis to fallout correlated with overall survival (P=0.04). Conclusions: In highly selected patients initially suitable for OLT, the mortality rate for cholangiocarcinoma was high in patients who became ineligible for OLT. Their survival, however, was comparable to expected survival for patients with locally advanced or metastatic disease treated with nontransplant therapies. The most common reason for patient fallout was adverse findings at the staging operation.

AbstractBackground & AimsHepatocyte apoptosis, the hallmark of non‐alcoholic steatohepatitis (NASH) contributes to liver injury and fibrosis. Although, both the intrinsic and extrinsic apoptotic pathways are involved in the pathogenesis ofNASH, the final common step of apoptosis is executed by a family of cysteine‐proteases termed caspases. Thus, our aim was to ascertain if administration of Emricasan, a pan‐caspase inhibitor, ameliorates liver injury and fibrosis in a murine model ofNASH.MethodsC57/BL6J‐mice were fed regular chow or high fat diet (HFD) for 20 weeks. All mice were treated with vehicle or Emricasan.ResultsMice fed a HFD diet demonstrate a five‐fold increase in hepatocyte apoptosis by the TUNEL assay and a 1.5‐fold and 1.3‐fold increase in caspase‐3 and‐8 activities respectively; this increase in apoptosis was substantially attenuated in mice fed a HFD treated with Emricasan (HFD‐Em). Likewise, liver injury and inflammation were reduced in mice fed HFD‐Em as compare to HFD by measuring serum aspartate aminotransferase and alanine aminotransferase levels, NAS histological score and IL 1‐β, TNF‐α, monocyte chemoattractant protein (MCP‐1) and C‐X‐C chemokine ligand‐2 (CXCL2) quantitative reverse‐transcription polymerase chain reaction (qPCR). These differences could not be attributed to differences in hepatic steatosis as liver triglycerides content were similar in both HFD groups. Hepatic fibrosis was reduced by Emricasan in HFD animals by decreasing αSMA (a marker for hepatic stellate cell activation), fibrosis score, Sirius red staining, hydroxyproline liver content and profibrogenic cytokines by qPCR.ConclusionIn conclusion, these data demonstrate that in a murine model ofNASH, liver injury and fibrosis are suppressed by inhibiting hepatocytes apoptosis and suggests that Emricasan may be an attractive antifibrotic therapy inNASH.

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related death and is currently the main event leading to death in patients with cirrhosis. Evolving information suggests that the metabolic syndrome with non-alcoholic liver disease may be an important cause of HCC in addition to viral hepatitis and alcohol-induced liver disease. The molecular pathogenesis is extremely complex and heterogeneous. To date the molecular information has not impacted on treatment decisions. Periodic surveillance imaging of patients with cirrhosis is widely practiced, especially because diagnostic, radiographic criteria for early-stage HCC have been defined (including nodules between 1 and 2 cm) and effective treatment is available for tumours detected at an early stage. Worldwide the approach to resection versus transplantation varies depending upon local resources, expertise and donor availability. The criteria for transplantation are discussed, and the controversial areas highlighted with evidence-based recommendations provided. Several approaches are available for intermediate stage disease, including radiofrequency ablation, transarterial chemoembolisation and radioembolisation; the rationale for these therapies is buttressed by appropriate outcome-based studies. For advanced disease, systemic therapy with sorafenib remains the option best supported by current data. Thus, while several trials have failed to improve the benefits of established therapies, studies assessing the sequential or combined application of those already known to be beneficial are needed. Also, new concepts are provided in regards to selecting and stratifying patients for second-line studies, which may help explain the failure of prior studies.

Regenerative medicine, a paragon of future healthcare, holds unprecedented potential in extending the reach of treatment modalities for individuals across diseases and lifespan. Emerging regenerative technologies, focused on structural repair and functional restoration, signal a radical transformation in medical and surgical practice. Regenerative medicine is poised to provide innovative solutions in addressing major unmet needs for patients, ranging from congenital disease and trauma to degenerative conditions. Realization of the regenerative model of care predicates a stringent interdisciplinary paradigm that will drive validated science into standardized clinical options. Designed as a catalyst in advancing rigorous new knowledge on disease causes and cures into informed delivery of quality care, the Mayo Clinic regenerative medicine blueprint offers a patient-centered, team-based strategy that optimizes the discovery–translation–application roadmap for the express purpose of science-supported practice advancement.

AbstractBackground & AimsC‐Jun N‐terminal kinase (JNK) activation is pivotal in the development of nonalcoholic steatohepatitis (NASH). Mixed lineage kinase 3 (MLK) 3 is one of the mitogen activated protein kinase kinase kinase (MAP3K) that mediates JNK activation in the liver. Despite this concept, the role of MLK3 in modulating liver injury during nutrient excess has not been explored. Our aim was to determine if MLK3 deficient mice were protected against high fat high carbohydrate (HFHC) diet‐induced NASH.MethodsWe employed eight‐week‐old Mlk3−/− male C57BL/6J mice, and wild type (WT) mice C57BL/6J as controls. Mice were fed a HFHC or a chow diet adlib for 16 weeks.ResultsHepatic JNK activating phosphorylation was readily absent in the Mlk3−/− mice fed the HFHC diet, but not in WT mice. This inhibition of JNK activation was hepatoprotective. Despite a comparable increase in weight gain, hepatic steatosis by histological examination and hepatic triglyceride quantification was reduced in HFHC diet‐fed Mlk3−/− mice compared with WT mice. In addition, compared with the WT mice, HFHC diet‐fed Mlk3−/− mice had significantly attenuated liver injury as manifested by reduced ALT levels, hepatocyte apoptosis, markers of hepatic inflammation and indices of hepatic fibrogenesis.ConclusionOur results suggest that loss of MLK3 in mice is protective against HFHC diet‐induced NASH, in a weight‐independent fashion, through attenuation of JNK activation. MLK3 is a potential therapeutic target for the treatment of human NASH.

Hepatocyte apoptosis is a hallmark of nonalcoholic steatohepatitis. We have previously observed that the saturated free fatty acids (FFAs) induce hepatocyte apoptosis in part via a death receptor 5 (DR5)-mediated signaling pathway. Cellular inhibitor of apoptosis protein 1 and 2 (cIAP-1 and cIAP-2) proteins are potent inhibitors of death receptor-mediated apoptosis. However, the role of the cIAPs in FFA-mediated hepatocyte apoptosis is unexplored. Our aim was to determine whether cIAPs are dysregulated during hepatocyte lipoapoptosis. cIAP proteins underwent rapid cellular elimination following treatment with the saturated FFAs palmitate (PA) and stearate. In contrast, PA did not decrease cIAP-1 and cIAP-2 mRNA expression in the cells. Degradation of cIAPs was dependent on their E3-ligase activity, suggesting that cIAPs undergo autoubiquitination that leads to proteasomal degradation. Huh-7 cells stably expressing shRNA targeting cIAP-1, but not cIAP-2, displayed enhanced sensitivity to PA-mediated apoptosis. Incubation with the SMAC mimetic JP1584, which induces rapid degradation of cIAPs, also enhanced PA-mediated apoptosis. Hepatocytes isolated from DR5 knockout mice exhibited reduced apoptosis following treatment with PA plus JP1584, implying that degradation of cIAPs sensitizes to DR5-mediated cell death pathways. A decrease of cIAP-1 was also observed in tissue from patients with nonalcoholic steatohepatitis compared with normal obese subjects. Collectively, these results implicate proteasomal degradation of cIAPs by FFA as a mechanism contributing to hepatocyte lipoapoptosis.