Relacorilant (formerly CORT-125134) is a selective antagonist of glucocorticoid receptors (GR) under investigation for Cushing syndrome (CS), Cushing disease (CD) and solid tumors like ovarian, pancreatic and adrenocortical carcinoma (ACC). Almost all cells of the body have GR. The binding of cortisol with GR activates the translocation of the GR to the nucleus, where it regulates several biological functions. As seen in CD, CS and some tumors, the overproduction of cortisol causes serious conditions with systemic consequences. Relacorilant competes with cortisol for the binding site of the GR. Several in vitro, in vivo and clinical studies established the efficacy and safety of relacorilant Compared to other GR inhibitors like mifepristone, relacorilant has a similar level of effectiveness and a better safety profile. Several clinical trials (phase II and III) on the effects of relacorilant alone or combined with other drugs are expected to complete in the following years.

Neuroblastoma (NB) is a rare pediatric cancer of the nerve tissue that usually arises in the abdomen. The life expectancy of patients with NB has improved in the last 2 decades thanks to better characterization of the risk levels and advancements in therapy. Nevertheless, patients with high-risk NB (HR-NB) are still resistant to current treatments and have a poor prognosis. Disialoganglioside (GD2) has emerged as an exclusive NB marker, and anti-GD2 immunotherapy has improved the overall survival of HR-NB patients. Naxitamab (also named humanized 3F8, hu3F8 or Danyelza) is a humanized anti-GD2 IgG1 antibody with promising features for HR-NB patients that was recently approved by the U.S. Food and Drug Administration. Compared to other anti-GD2 antibodies, naxitamab is more efficient and allows patients to have a better quality of life. Currently, several phase I and II clinical trials are studying naxitamab alone or in combination with cytokines. These trials are expected to finish between 2021 and 2027.

Identification and purification of cancer stem cells (CSCs) could lead to new therapeutic targets, but their heterogeneous expansion is an obstacle to their study. We investigated whether it is possible to monitor pancreatic CSCs in real time, based on their intrinsic low level of proteasome activity.We engineered human pancreatic adenocarcinoma cells (PANC1, MIAPaCa2, BxPC3, and KLM1) to express a green fluorescent molecule fused to the degron of ornithine decarboxylase (Gdeg) from a retroviral vector; the fluorescent Gdeg accumulates in CSCs as a result of low activity of the 26S proteasome. Cells with high and low levels of fluorescence (Gdeghigh and Gdeglow) were isolated by flow cytometry; tumor growth was analyzed in immunocompromised mice. We performed a screen for agents that were specifically toxic to pancreatic CSCs, in a synthetic lethal manner.Gdeghigh cells, but not Gdeglow cells, formed spheres and underwent asymmetric division—features of CSCs. Injection of as few as 10 Gdeghigh cells led to tumor formation in mice. Gemcitabine was toxic to cultured Gdeglow cells, whereas Gdeghigh cells were resistant. We observed that quercetin was toxic to Gdeghigh cells in culture and in pre-established tumors grown from these cells in mice. Nuclear accumulation of β-catenin was detected in Gdeghigh, but not Gdeglow, and lost after exposure to quercetin.We used a fluorescence marker system for level of proteasome activity to identify pancreatic cancer cells with features of cancer stem cells. We identified quercetin as a compound that is specifically toxic to pancreatic CSCs.

e21035 Background: EMT renders neoplastic cancer cells the ability to migrate and to invade distant organs. The hallmark of EMT is the loss of E-cadherin, which is a prerequisite for epithelial tumor cell invasion. In pancreatic cancer, loss of tumor E-cadherin is an independent predictor of poor outcome. Aims: To analyze the effect of pancreatic fibroblasts (PF) on inducing EMT in pancreatic cancer cells and to identify the transcription factors (Snail, Slug, ZEB1, ZEB2) that mediate EMT process. Methods: Human PFs were isolated from human pancreatic specimens obtained from chronic pancreatitis and from unaffected margins of pancreatic adenocarcinoma and serous cistoadenoma. PF were cultured until complete cellular activation, as assessed by expression of α-smooth muscle actin, vimentin and fibronectin. Human pancreatic cancer cells Panc-1 were exposed to PF conditioned medium (PF-CM) and EMT analyzed by cell morphology, migration, and E-cadherin expression (quantitative RT-PCR and immunoblot). Gene expression of Snail, Slug, ZEB1, and ZEB2 was analyzed by quantitative RT-PCR, and their activity modulated by siRNA Results: Conditioned media from all types of activated PFs induced EMT changes in Panc-1 cells, as shown by 1) morphological transition from cobblestone shaped to fibroblast-like cells, 2) stimulation of cell migration, and 3) E-cadherin down–regulation; mRNA expression of Snail transiently increased at 30 min after exposure to PF returning to basal levels afterwards; mRNA levels of ZEB1 were not up-regulated upon exposure to PF-CM. However, ZEB1 protein greatly accumulated after 48h incubation with PF-CM, suggesting that PF prevent ZEB1 degradation in Panc-1 cells. Combined RNA downregulation of ZEB1 and ZEB2, but not of Snail and/or Slug, suppressed E-cadherin repression induced by PF. Conclusions: Activated PFs promote the invasive phenotype of pancreatic cancer cells through ZEB1 and ZEB2 activation.

Einleitung: Die Rolle von Transkriptionsfaktoren ist in der chronischen Pankreatitis (CP) bisher nicht bekannt.

Background: Excessive fibroblast expansion and extracellular matrix (ECM) deposition are key events for the development of bowel stenosis in Crohn's disease (CD) patients. Tocotrienols are vitamin E compounds with proven in vitro antifibrogenic effects on rat pancreatic fibroblasts. We aimed at investigating the effects of tocotrienols on human intestinal fibroblast (HIF) proliferation, apoptosis, autophagy, and synthesis of ECM.Methods: HIF isolated from CD, ulcerative colitis (UC), and normal intestine were treated with tocotrienol-rich fraction (TRF) from palm oil. HIF proliferation was quantified by H-3-thymidine incorporation, apoptosis was studied by DNA fragmentation, propidium iodide staining, caspase activation, and poly(ADP-ribose) polymerase cleavage, autophagy was analyzed by quantification of LC3 protein and identification of autophagic vesicles by immunofluorescence and production of ECM components was measured by Western blot.Results: TRF significantly reduced HIF proliferation and prevented basic fibroblast growth factor-induced proliferation in CD and UC, but not control HIF. TRF enhanced HIF death by promoting apoptosis and autophagy. HIF apoptosis, but not autophagy, was prevented by the pan-caspase inhibitor zVAD-fmk, whereas both types of cell death were prevented when the mitochondrial permeability transition pore was blocked by cyclosporin A, demonstrating a key role of the mitochondria in these processes. TRF diminished procollagen type I and laminin gamma-1 production by HIF.Conclusions: Tocotrienols exert multiple effects on HIF, reducing cell proliferation, enhancing programmed cell death through apoptosis and autophagy, and decreasing ECM production. Considering their in vitro antifibrogenic properties, tocotrienols could be useful to treat or prevent bowel fibrosis in CD patients.

Tocotrienols exhibit anti-inflammatory properties over macrophages and promote cytotoxicity in activated pancreatic stellate cells, suggesting that they may limit chronic pancreatitis progression. We aimed to quantitate the effect of oral tocotrienols on a rat model of chronic pancreatic injury. Chronic-like pancreatitis was induced by repeated arginine pancreatitis. Palm oil tocotrienol-rich fraction (TRF) was given by gavage before and after pancreatitis inductions. Amylase and hydroxyproline were determined in pancreatic homogenates; collagen, fibronectin, α-smooth muscle actin (SMA), glial fibrillary acidic protein (GFAP), and phosphorylated Smad3 were assessed by Western blotting. Transforming growth factor (TGF)-β1 was measured in plasma. Morphological assessment included light microscopy, fibrosis area fraction, and collagen network fractal analysis. Arginine pancreatitis induced pancreatic atrophy and increased hydroxyproline that ameliorated after TRF. Arginine increased TGF-β1 (185 ± 40 vs. 15 ± 2 ng/ml; P <0.01) that was blunted by TRF (53 ± 19; P < 0.01). TRF reduced protease and Smad3 activation, collagen, and fibronectin. α-SMA increased and GFAP diminished in arginine pancreatitis, consistent with long-term stellate cell activation, and TRF reverted these changes to basal. Arginine pancreatitis increased fibrosis area fraction (4.5 ± 0.3% vs. 0.2 ± 0.2%), collagen network complexity (fractal dimension 1.52 ± 0.03 vs. 1.42 ± 0.01; P < 0.001), and inhomogeneity (lacunarity 0.63 ± 0.03 vs. 0.40 ± 0.02; P < 0.001), which were all reduced by TRF (1.3 ± 0.4%, 1.43 ± 0.02%, and 0.51 ± 0.03%, respectively; P < 0.01). Best correlation coefficients were obtained when comparing fibrosis area fraction with lacunarity (r = 0.88) and both parameters with pancreatic weight (r = -0.91 and -0.79, respectively). TRF administered only before pancreatitis best, but not fully, recapitulated the beneficial effects of TRF. Tocotrienols improve quantitative measures of chronic pancreatic damage. They may be of benefit in human chronic pancreatitis.

BACKGROUND & AIMS:Little is known about how transcription factors might regulate pathogenesis of chronic pancreatitis (CP). We analyzed the in vivo role of RelA/p65, a component of the transcription factor nuclear factor (NF)-κB, in different cell types during development of CP in mice.METHODS:RelA/p65 was functionally inactivated in the pancreas (relaΔpanc), in myeloid cells (relaΔmye), or both (relaΔpanc,Δmye) compartments using the Cre-loxP strategy. Experimental CP was induced with repetitive injections of cerulein over 6 weeks. Pancreata were investigated histologically and biochemically. We created an in vitro coculture assay of pancreatic stellate cells (PSC) and macrophages and performed gene arrays from pancreata and macrophages with functionally inactivated RelA/p65. Tissue samples from patients with CP were analyzed for matrix metalloproteinase (MMP) 10 expression.RESULTS:In contrast to their relaF/F littermates, relaΔpanc displayed typical signs of CP after long-term stimulation with cerulein. Numerous macrophages and activated α-smooth muscle actin (SMA)-positive PSCs were detected. Additional inactivation of RelA/p65 in myeloid cells (relaΔpanc,Δmye) attenuated fibrosis. In vitro, RelA/p65-deficient, lipopolysaccharide (LPS)-stimulated macrophages degraded fibronectin in cocultured PSCs. Using gene expression analysis, MMP-10 was identified as a candidate for this process. Recombinant MMP-10 degraded fibronectin in LPS-stimulated PSCs. In tissue samples from patients with CP, MMP-10 was up-regulated in myeloid cells.CONCLUSIONS:RelA/p65 functions in myeloid cells to promote pathogenesis of CP. In acinar cells, RelA/p65 protects against chronic inflammation, whereas myeloid RelA/p65 promotes fibrogenesis. In macrophage, MMP-10 functions as a RelA/p65-dependent, potentially antifibrogenic factor during progression of CP.

Se cree que las células estrelladas inician y mantienen activos los procesos de fibrogénesis que pueden conducir a la destrucción del parénquima en la pancreatitis crónica. Nosotros hemos sugerido que los tocotrienoles pueden utilizarse como agentes antifibrogénicos dada su capacidad de inducir muerte celular programada en células estrelladas activadas in vitro de forma selectiva, sin afectar a células quiescentes o a células acinares.

the 4th Congress of ECCO the European Crohn's and Colitis Organisation S123 When induction of tolerance to TLR and NLR was attempted, HIF showed a lower reduction in IL8 production than monocytes and DCs while HIMEC were tolerized to Nod1 and TLR4 stimulation, although IL8 production remained still elevated.Surprisingly, the attempt to induce tolerance in HIF and HIMEC to TNF-a resulted in a higher IL8 production to TNF-a restimulation.Conclusion: HIF and HIMEC respond to stimulation of several NLRs and TLRs with a substantial production of proinflammatory cytokines at levels comparable or superior to those of monocytes and DCs.They are less tolerizeble to bacterial product stimulation than monocytes and DC.Thus, they are prime pro-inflammatory cells in the gut.This novel concept is further supported by the finding that HIF display an unexpected ability to progressively increase cytokine production upon continuous exposure to TNF-a.Overall, these results suggest that nonimmune cells actively contribute to the innate immune response to gut bacteria and promote chronic intestinal inflammation.

Tocotrienols are a group of natural vitamin E compounds with patent antitumoral effects, mostly based on their ability to induce apoptosis in cancer cells. In activated pancreatic stellate cells (PSCs) we have determined that tocotrienols elicit a dramatic mitochondrial destabilization followed by initiation of non-necrotic forms of programmed cell death, namely apoptosis and autophagy. PSCs are the main cell type involved in the generation of pancreatic fibrosis, and their removal is critical to limit the fibrogenic process. Noteworthy, tocotrienol death-promoting actions are exclusively directed to activated PSCs, but not to their quiescent counterparts nor to terminally differentiated acinar cells. Here, we hypothesize that the transformed phenotype of PSCs may include "activated" mitochondria, which can be used by tocotrienols to trigger autophagic and apoptotic signaling. We propose that mitochondria are the cornerstone of cell sensitivity to tocotrienols, and suggest possible mechanisms, that may be interconnected, on how tocotrienols may govern mitochondrial death pathways.Addendum to:Tocotrienols Induce Apoptosis and Autophagy in Rat Pancreatic Stellate Cells through the Mitochondrial Death PathwayM. Rickmann, E.C. Vaquero, J.R. Malagelada and X. MoleroGastroenterology 2007; 132:2518-32

BACKGROUND & AIMS Selective removal of activated pancreatic stellate cells (PSCs) through induction of their own programmed death is a goal of therapeutic interest in patients with chronic pancreatitis. Here, we investigated the effects of tocotrienols on PSC death outcomes. METHODS Activated and quiescent PSCs and acinar cells from rat pancreas were treated with vitamin E derivatives alpha-tocopherol; individual alpha-, beta-, gamma-, and delta-tocotrienols; and a tocotrienol rich fraction (TRF) from palm oil. RESULTS TRF, but not alpha-tocopherol, reduced viability of activated PSC by setting up a full death program, independent of cell cycle regulation. Activated PSCs died both through apoptosis, as indicated by increased DNA fragmentation and caspase activation, and through autophagy, as denoted by the formation of autophagic vacuoles and LC3-II accumulation. In contrast to alpha-tocopherol, TRF caused an intense and sustained mitochondrial membrane depolarization and extensive cytochrome c release. Caspase inhibition with zVAD-fmk suppressed TRF-induced apoptosis but enhanced autophagy. However, mitochondrial permeability transition pore blockade with cyclosporin A completely abolished the deadly effects of TRF. beta-, gamma-, and delta-tocotrienol, but not alpha-tocotrienol nor alpha-tocopherol, reproduced TRF actions on activated PSCs. TRF death induction was restricted to activated PSCs because it did not cause apoptosis either in quiescent PSCs or in acinar cells. CONCLUSIONS Tocotrienols selectively trigger activated pancreatic stellate cell death by targeting the mitochondrial permeability transition pore. Our findings unveil a novel potential for tocotrienols to ameliorate the fibrogenesis associated with chronic pancreatitis.