Ethanol inhibits pancreatic acinar cell autophagy through upregulation of ATG4B, mediating pathological responses of alcoholic pancreatitis.

Excessive alcohol intake is a major risk factor for pancreatitis, sensitizing the exocrine pancreas to stressors by mechanisms that remain obscure. Impaired autophagy drives nonalcoholic pancreatitis, but the effects of ethanol (EtOH) and alcoholic pancreatitis on autophagy are poorly understood. Here, we find that ethanol reduces autophagosome formation in pancreatic acinar cells, both in a mouse model of alcoholic pancreatitis induced by a combination of EtOH diet and cerulein (a CCK ortholog) and in EtOH+CCK-treated acinar cells (ex vivo model). Ethanol treatments decreased pancreatic level of LC3-II, a key mediator of autophagosome formation. This was caused by ethanol-induced upregulation of ATG4B, a cysteine protease that, cell dependently, regulates the balance between cytosolic LC3-I and membrane-bound LC3-II. We show that ATG4B negatively regulates LC3-II in acinar cells subjected to EtOH treatments. Ethanol raised ATG4B level by inhibiting its degradation, enhanced ATG4B enzymatic activity, and strengthened its interaction with LC3-II. We also found an increase in ATG4B and impaired autophagy in a dissimilar, nonsecretagogue model of alcoholic pancreatitis induced by EtOH plus palmitoleic acid. Adenoviral ATG4B overexpression in acinar cells greatly reduced LC3-II and inhibited autophagy. Furthermore, it aggravated trypsinogen activation and necrosis, mimicking key responses of ex vivo alcoholic pancreatitis. Conversely, shRNA Atg4B knockdown enhanced autophagosome formation and alleviated ethanol-induced acinar cell damage. The results reveal a novel mechanism, whereby ethanol inhibits autophagosome formation and thus sensitizes pancreatitis, and a key role of ATG4B in ethanol's effects on autophagy. Enhancing pancreatic autophagy, particularly by downregulating ATG4B, could be beneficial in mitigating the severity of alcoholic pancreatitis.NEW & NOTEWORTHY Ethanol sensitizes mice and humans to pancreatitis, but the underlying mechanisms remain obscure. Autophagy is important for maintaining pancreatic acinar cell homeostasis, and its impairment drives pancreatitis. This study reveals a novel mechanism, whereby ethanol inhibits autophagosome formation through upregulating ATG4B, a key cysteine protease. ATG4B upregulation inhibits autophagy in acinar cells and aggravates pathological responses of experimental alcoholic pancreatitis. Enhancing pancreatic autophagy, particularly by down-regulating ATG4B, could be beneficial for treatment of alcoholic pancreatitis.

Rab9 Mediates Pancreatic Autophagy Switch From Canonical to Noncanonical, Aggravating Experimental Pancreatitis.

BACKGROUND: Autophagosome, the central organelle in autophagy process, can assemble via canonical pathway mediated by LC3-II, the lipidated form of autophagy-related protein LC3/ATG8, or noncanonical pathway mediated by the small GTPase Rab9. Canonical autophagy is essential for exocrine pancreas homeostasis, and its disordering initiates and drives pancreatitis. The involvement of noncanonical autophagy has not been explored. We examine the role of Rab9 in pancreatic autophagy and pancreatitis severity. METHODS: We measured the effect of Rab9 on parameters of autophagy and pancreatitis responses using transgenic mice overexpressing Rab9 (Rab9TG) and adenoviral transduction of acinar cells. Effect of canonical autophagy on Rab9 was assessed in ATG5-deficient acinar cells. RESULTS: Pancreatic levels of Rab9 and its membrane-bound (active) form decreased in rodent pancreatitis models and in human disease. Rab9 overexpression stimulated noncanonical and inhibited canonical/LC3-mediated autophagosome formation in acinar cells through up-regulation of ATG4B, the cysteine protease that delipidates LC3-II. Conversely, ATG5 deficiency caused Rab9 increase in acinar cells. Inhibition of canonical autophagy in Rab9TG pancreas was associated with accumulation of Rab9-positive vacuoles containing markers of mitochondria, protein aggregates, and trans-Golgi. The shift to the noncanonical pathway caused pancreatitis-like damage in acinar cells and aggravated experimental pancreatitis. CONCLUSIONS: The results show that Rab9 regulates pancreatic autophagy and indicate a mutually antagonistic relationship between the canonical/LC3-mediated and noncanonical/Rab9-mediated autophagy pathways in pancreatitis. Noncanonical autophagy fails to substitute for its canonical counterpart in protecting against pancreatitis. Thus, Rab9 decrease in experimental and human pancreatitis is a protective response to sustain canonical autophagy and alleviate disease severity.

Dysregulation of mannose-6-phosphate-dependent cholesterol homeostasis in acinar cells mediates pancreatitis.

Disordered lysosomal/autophagy pathways initiate and drive pancreatitis, but the underlying mechanisms and links to disease pathology are poorly understood. Here, we show that the mannose-6-phosphate (M6P) pathway of hydrolase delivery to lysosomes critically regulates pancreatic acinar cell cholesterol metabolism. Ablation of the Gnptab gene encoding a key enzyme in the M6P pathway disrupted acinar cell cholesterol turnover, causing accumulation of nonesterified cholesterol in lysosomes/autolysosomes, its depletion in the plasma membrane, and upregulation of cholesterol synthesis and uptake. We found similar dysregulation of acinar cell cholesterol, and a decrease in GNPTAB levels, in both WT experimental pancreatitis and human disease. The mechanisms mediating pancreatic cholesterol dyshomeostasis in Gnptab-/- and experimental models involve a disordered endolysosomal system, resulting in impaired cholesterol transport through lysosomes and blockage of autophagic flux. By contrast, in Gnptab-/- liver the endolysosomal system and cholesterol homeostasis were largely unaffected. Gnptab-/- mice developed spontaneous pancreatitis. Normalization of cholesterol metabolism by pharmacologic means alleviated responses of experimental pancreatitis, particularly trypsinogen activation, the disease hallmark. The results reveal the essential role of the M6P pathway in maintaining exocrine pancreas homeostasis and function, and implicate cholesterol disordering in the pathogenesis of pancreatitis.

Transgenic expression of GFP-LC3 perturbs autophagy in exocrine pancreas and acute pancreatitis responses in mice.

Pancreatitis is a common, sometimes fatal, disease of exocrine pancreas, initiated by damaged acinar cells. Recent studies implicate disordered macroautophagy/autophagy in pancreatitis pathogenesis. ATG8/LC3 protein is critical for autophagosome formation and a widely used marker of autophagic vacuoles. Transgenic GFP-LC3 mice are a valuable tool to investigate autophagy ; however, comparison of homeostatic and disease responses between GFP-LC3 and wild-type (WT) mice has not been done. We examined the effects of GFP-LC3 expression on autophagy, acinar cell function, and experimental pancreatitis. Unexpectedly, GFP-LC3 expression markedly increased endogenous LC3-II level in pancreas, caused by downregulation of ATG4B, the protease that deconjugates/delipidates LC3-II. By contrast, GFP-LC3 expression had lesser or no effect on autophagy in liver, lung and spleen. Autophagic flux analysis showed that autophagosome formation in GFP-LC3 acinar cells increased 3-fold but was not fully counterbalanced by increased autophagic degradation. Acinar cell (ex vivo) pancreatitis inhibited autophagic flux in WT and essentially blocked it in GFP-LC3 cells. In vivo pancreatitis caused autophagy impairment in WT mice, manifest by upregulation of LC3-II and SQSTM1/p62, increased number and size of autophagic vacuoles, and decreased level of TFEB, all of which were exacerbated in GFP-LC3 mice. GFP-LC3 expression affected key pancreatitis responses; most dramatically, it worsened increases in serum AMY (amylase), a diagnostic marker of acute pancreatitis, in several mouse models. The results emphasize physiological importance of autophagy for acinar cell function, demonstrate organ-specific effects of GFP-LC3 expression, and indicate that application of GFP-LC3 mice in disease models should be done with caution.Abbreviations: AP: acute pancreatitis; Arg-AP: L-arginine-induced acute pancreatitis; ATG: autophagy-related (protein); AVs: autophagic vacuoles; CCK: cholecystokinin-8; CDE: choline-deficient, D,L-ethionine supplemented diet; CER: caerulein (ortholog of CCK); CTSB: cathepsin B; CTSD: cathepsin D; CTSL: cathepsin L; ER: endoplasmic reticulum; LAMP: lysosomal-associated membrane protein; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; TEM: transmission electron microscopy; TFEB: transcription factor EB; ZG: zymogen granule(s).

Recent Insights Into the Pathogenic Mechanism of Pancreatitis: Role of Acinar Cell Organelle Disorders.

Acute pancreatitis (AP) is a potentially lethal inflammatory disease that lacks specific therapy. Damaged pancreatic acinar cells are believed to be the site of AP initiation. The primary function of these cells is the synthesis, storage, and export of digestive enzymes. Beginning in the endoplasmic reticulum and ending with secretion of proteins stored in zymogen granules, distinct pancreatic organelles use ATP produced by mitochondria to move and modify nascent proteins through sequential vesicular compartments. Compartment-specific accessory proteins concentrate cargo and promote vesicular budding, targeting, and fusion. The autophagy-lysosomal-endosomal pathways maintain acinar cell homeostasis by removing damaged/dysfunctional organelles and recycling cell constituents for substrate and energy. Here, we discuss studies in experimental and genetic AP models, primarily from our groups, which show that acinar cell injury is mediated by distinct mechanisms of organelle dysfunction involved in protein synthesis and trafficking, secretion, energy generation, and autophagy. These early AP events (often first manifest by abnormal cytosolic Ca signaling) in the acinar cell trigger the inflammatory and cell death responses of pancreatitis. Manifestations of acinar cell organelle disorders are also prominent in human pancreatitis. Our findings suggest that targeting specific mediators of organelle dysfunction could reduce disease severity.

Mitochondrial Dysfunction, Through Impaired Autophagy, Leads to Endoplasmic Reticulum Stress, Deregulated Lipid Metabolism, and Pancreatitis in Animal Models.

BACKGROUND & AIMS: Little is known about the signaling pathways that initiate and promote acute pancreatitis (AP). The pathogenesis of AP has been associated with abnormal increases in cytosolic Ca2+, mitochondrial dysfunction, impaired autophagy, and endoplasmic reticulum (ER) stress. We analyzed the mechanisms of these dysfunctions and their relationships, and how these contribute to development of AP in mice and rats. METHODS: Pancreatitis was induced in C57BL/6J mice (control) and mice deficient in peptidylprolyl isomerase D (cyclophilin D, encoded by Ppid) by administration of L-arginine (also in rats), caerulein, bile acid, or an AP-inducing diet. Parameters of pancreatitis, mitochondrial function, autophagy, ER stress, and lipid metabolism were measured in pancreatic tissue, acinar cells, and isolated mitochondria. Some mice with AP were given trehalose to enhance autophagic efficiency. Human pancreatitis tissues were analyzed by immunofluorescence. RESULTS: Mitochondrial dysfunction in pancreas of mice with AP was induced by either mitochondrial Ca2+ overload or through a Ca2+ overload-independent pathway that involved reduced activity of ATP synthase (80% inhibition in pancreatic mitochondria isolated from rats or mice given L-arginine). Both pathways were mediated by cyclophilin D and led to mitochondrial depolarization and fragmentation. Mitochondrial dysfunction caused pancreatic ER stress, impaired autophagy, and deregulation of lipid metabolism. These pathologic responses were abrogated in cyclophilin D-knockout mice. Administration of trehalose largely prevented trypsinogen activation, necrosis, and other parameters of pancreatic injury in mice with L-arginine AP. Tissues from patients with pancreatitis had markers of mitochondrial damage and impaired autophagy, compared with normal pancreas. CONCLUSIONS: In different animal models, we find a central role for mitochondrial dysfunction, and for impaired autophagy as its principal downstream effector, in development of AP. In particular, the pathway involving enhanced interaction of cyclophilin D with ATP synthase mediates L-arginine-induced pancreatitis, a model of severe AP the pathogenesis of which has remained unknown. Strategies to restore mitochondrial and/or autophagic function might be developed for treatment of AP.

Human Pancreatic Acinar Cells: Proteomic Characterization, Physiologic Responses, and Organellar Disorders in ex Vivo Pancreatitis.

Knowledge of the molecular mechanisms of acute pancreatitis is largely based on studies using rodents. To assess similar mechanisms in humans, we performed ex vivo pancreatitis studies in human acini isolated from cadaveric pancreata from organ donors. Because data on these human acinar preparations are sparse, we assessed their functional integrity and cellular and organellar morphology using light, fluorescence, and electron microscopy; and their proteome by liquid chromatography-tandem mass spectrometry. Acinar cell responses to the muscarinic agonist carbachol (CCh) and the bile acid taurolithocholic acid 3-sulfate were also analyzed. Proteomic analysis of acini from donors of diverse ethnicity showed similar profiles of digestive enzymes and proteins involved in translation, secretion, and endolysosomal function. Human acini preferentially expressed the muscarinic acetylcholine receptor M3 and maintained physiological responses to CCh for at least 20 hours. As in rodent acini, human acini exposed to toxic concentrations of CCh and taurolithocholic acid 3-sulfate responded with trypsinogen activation, decreased cell viability, organelle damage manifest by mitochondrial depolarization, disordered autophagy, and pathological endoplasmic reticulum stress. Human acini also secreted inflammatory mediators elevated in acute pancreatitis patients, including IL-6, tumor necrosis factor-α, IL-1ß, chemokine (C-C motif) ligands 2 and 3, macrophage inhibitory factor, and chemokines mediating neutrophil and monocyte infiltration. In conclusion, human cadaveric pancreatic acini maintain physiological functions and have similar pathological responses and organellar disorders with pancreatitis-causing treatments as observed in rodent acini.

Roles of autophagy and metabolism in pancreatic cancer cell adaptation to environmental challenges.

Pancreatic ductal adenocarcinoma (PDAC) displays extensive and poorly vascularized desmoplastic stromal reaction, and therefore, pancreatic cancer (PaCa) cells are confronted with nutrient deprivation and hypoxia. Here, we investigate the roles of autophagy and metabolism in PaCa cell adaptation to environmental stresses, amino acid (AA) depletion, and hypoxia. It is known that in healthy cells, basal autophagy is at a low level, but it is greatly activated by environmental stresses. By contrast, we find that in PaCa cells, basal autophagic activity is relatively high, but AA depletion and hypoxia activate autophagy only weakly or not at all, due to their failure to inhibit mechanistic target of rapamycin. Basal, but not stress-induced, autophagy is necessary for PaCa cell proliferation, and AA supply is even more critical to maintain PaCa cell growth. To gain insight into the underlying mechanisms, we analyzed the effects of autophagy inhibition and AA depletion on PaCa cell metabolism. PaCa cells display mixed oxidative/glycolytic metabolism, with oxidative phosphorylation (OXPHOS) predominant. Both autophagy inhibition and AA depletion dramatically decreased OXPHOS; furthermore, pharmacologic inhibitors of OXPHOS suppressed PaCa cell proliferation. The data indicate that the maintenance of OXPHOS is a key mechanism through which autophagy and AA supply support PaCa cell growth. We find that the expression of oncogenic activation mutation in GTPase Kras markedly promotes basal autophagy and stimulates OXPHOS through an autophagy-dependent mechanism. The results suggest that approaches aimed to suppress OXPHOS, particularly through limiting AA supply, could be beneficial in treating PDAC.NEW & NOTEWORTHY Cancer cells in the highly desmoplastic pancreatic ductal adenocarcinoma confront nutrient [i.e., amino acids (AA)] deprivation and hypoxia, but how pancreatic cancer (PaCa) cells adapt to these conditions is poorly understood. This study provides evidence that the maintenance of mitochondrial function, in particular, oxidative phosphorylation (OXPHOS), is a key mechanism that supports PaCa cell growth, both in normal conditions and under the environmental stresses. OXPHOS in PaCa cells critically depends on autophagy and AA supply. Furthermore, the oncogenic activation mutation in GTPase Kras upregulates OXPHOS through an autophagy-dependent mechanism.

Analysis of N- and O-Glycosylation of Lysosomal Glycoproteins.

The vast majority of lysosomal proteins are heavily glycosylated. The present protocol describes the method of analyzing N- and O-linked glycans in lysosomal proteins of interest. The method is based on using deglycosylating enzymes, endoglycosidases, and exoglycosidases. Endoglycosidases catalyze the cleavage of an internal bond in an oligosaccharide, while exoglycosidases remove terminal carbohydrates from glycans. Different types of carbohydrate residues or chains can be removed by specific glycosidases. Removing oligosaccharides with glycosidases increases the electrophoretic mobility of a protein. This increase in mobility depends on the size and number of removed carbohydrate chains. Therefore, the treatment of lysosomal proteins with specific glycosidases followed by a western blot analysis of a protein of interest provides a way to determine which types of glycans are present in the protein by comparing the gel mobility before and after treatment.

Acute acinar pancreatitis blocks vesicle-associated membrane protein 8 (VAMP8)-dependent secretion, resulting in intracellular trypsin accumulation.

Zymogen secretory granules in pancreatic acinar cells express two vesicle-associated membrane proteins (VAMP), VAMP2 and -8, each controlling 50% of stimulated secretion. Analysis of secretion kinetics identified a first phase (0-2 min) mediated by VAMP2 and second (2-10 min) and third phases (10-30 min) mediated by VAMP8. Induction of acinar pancreatitis by supramaximal cholecystokinin (CCK-8) stimulation inhibits VAMP8-mediated mid- and late-phase but not VAMP2-mediated early-phase secretion. Elevation of cAMP during supramaximal CCK-8 mitigates third-phase secretory inhibition and acinar damage caused by the accumulation of prematurely activated trypsin. VAMP8-/- acini are resistant to secretory inhibition by supramaximal CCK-8, and despite a 4.5-fold increase in total cellular trypsinogen levels, are fully protected from intracellular trypsin accumulation and acinar damage. VAMP8-mediated secretion is dependent on expression of the early endosomal proteins Rab5, D52, and EEA1. Supramaximal CCK-8 (60 min) caused a 60% reduction in the expression of D52 followed by Rab5 and EEA1 in isolated acini and in in vivo The loss of D52 occurred as a consequence of its entry into autophagic vacuoles and was blocked by lysosomal cathepsin B and L inhibition. Accordingly, adenoviral overexpression of Rab5 or D52 enhanced secretion in response to supramaximal CCK-8 and prevented accumulation of activated trypsin. These data support that acute inhibition of VAMP8-mediated secretion during pancreatitis triggers intracellular trypsin accumulation and loss of the early endosomal compartment. Maintaining anterograde endosomal trafficking during pancreatitis maintains VAMP8-dependent secretion, thereby preventing accumulation of activated trypsin.

Epigenetic control of group V phospholipase A2 expression in human malignant cells.

Secreted phospholipases A2 (sPLA2) are suggested to play an important role in inflammation and tumorigenesis. Different mechanisms of epigenetic regulation are involved in the control of group IIA, III and X sPLA2s expression in cancer cells, but group V sPLA2 (GV-PLA2) in this respect has not been studied. Here, we demonstrate the role of epigenetic mechanisms in regulation of GV-PLA2 expression in different cell lines originating from leukaemia and solid cancers. In blood leukocytes from leukaemic patients, levels of GV-PLA2 transcripts were significantly lower in comparison to those from healthy individuals. Similarly, in DU-145 and PC-3 prostate and CAL-51 and MCF-7 mammary cancer cell lines, levels of GV-PLA2 transcripts were significantly lower in relation to those found in normal epithelial cells of prostate or mammary. By sequencing and methylation-specific high-resolution melting (MS-HRM) analyses of bisulphite-modified DNA, distinct CpG sites in the GV-PLA2 promoter region were identified that were differentially methylated in cancer cells in comparison to normal epithelial and endothelial cells. Spearman rank order analysis revealed a significant negative correlation between the methylation degree and the cellular expression of GV-PLA2 (r = -0.697; p = 0.01). The effects of demethylating agent (5-aza-2'-deoxycytidine) and histone deacetylase inhibitor (trichostatin A) on GV-PLA2 transcription in the analysed cells confirmed the importance of DNA methylation and histone modification in the regulation of the GV-PLA2 gene expression in leukaemic, prostate and mammary cancer cell lines. The exposure of tumour cells to human recombinant GV-PLA2 resulted in a reduced colony forming activity of MCF-7, HepG2 and PC-3 cells, but not of DU-145 cells suggesting a cell-type-dependent effect of GV-PLA2 on cell growth. In conclusion, our results suggest that epigenetic mechanisms such as DNA methylation and histone modification play an important role in downregulation of GV-PLA2 expression in cancer cells.

Mechanism of mitochondrial permeability transition pore induction and damage in the pancreas: inhibition prevents acute pancreatitis by protecting production of ATP.

OBJECTIVE: Acute pancreatitis is caused by toxins that induce acinar cell calcium overload, zymogen activation, cytokine release and cell death, yet is without specific drug therapy. Mitochondrial dysfunction has been implicated but the mechanism not established. DESIGN: We investigated the mechanism of induction and consequences of the mitochondrial permeability transition pore (MPTP) in the pancreas using cell biological methods including confocal microscopy, patch clamp technology and multiple clinically representative disease models. Effects of genetic and pharmacological inhibition of the MPTP were examined in isolated murine and human pancreatic acinar cells, and in hyperstimulation, bile acid, alcoholic and choline-deficient, ethionine-supplemented acute pancreatitis. RESULTS: MPTP opening was mediated by toxin-induced inositol trisphosphate and ryanodine receptor calcium channel release, and resulted in diminished ATP production, leading to impaired calcium clearance, defective autophagy, zymogen activation, cytokine production, phosphoglycerate mutase 5 activation and necrosis, which was prevented by intracellular ATP supplementation. When MPTP opening was inhibited genetically or pharmacologically, all biochemical, immunological and histopathological responses of acute pancreatitis in all four models were reduced or abolished. CONCLUSIONS: This work demonstrates the mechanism and consequences of MPTP opening to be fundamental to multiple forms of acute pancreatitis and validates the MPTP as a drug target for this disease.

Lysosome associated membrane proteins maintain pancreatic acinar cell homeostasis: LAMP-2 deficient mice develop pancreatitis.

BACKGROUND & AIMS: The pathogenic mechanism of pancreatitis is poorly understood. Recent evidence implicates defective autophagy in pancreatitis responses; however, the pathways mediating impaired autophagy in pancreas remain largely unknown. Here, we investigate the role of lysosome associated membrane proteins (LAMPs) in pancreatitis. METHODS: We analyzed changes in LAMPs in experimental models and human pancreatitis, and the underlying mechanisms: LAMP de-glycosylation and degradation. LAMP cleavage by cathepsin B (CatB) was analyzed by mass spectrometry. We used mice deficient in LAMP-2 to assess its role in pancreatitis. RESULTS: Pancreatic levels of LAMP-1 and LAMP-2 greatly decrease across various pancreatitis models and in human disease. Pancreatitis does not trigger LAMPs' bulk de-glycosylation, but induces their degradation via CatB-mediated cleavage of LAMP molecule close to the boundary between luminal and transmembrane domains. LAMP-2 null mice spontaneously develop pancreatitis that begins with acinar cell vacuolization due to impaired autophagic flux, and progresses to severe pancreas damage characterized by trypsinogen activation, macrophage-driven inflammation, and acinar cell death. LAMP-2 deficiency causes a decrease in pancreatic digestive enzymes content, stimulates the basal and inhibits CCK-induced amylase secretion by acinar cells. The effects of LAMP-2 knockout and acute cerulein pancreatitis overlap, which corroborates the pathogenic role of LAMP decrease in experimental pancreatitis models. CONCLUSIONS: The results indicate a critical role for LAMPs, particularly LAMP-2, in maintaining pancreatic acinar cell homeostasis, and provide evidence that defective lysosomal function, resulting in impaired autophagy, leads to pancreatitis. Mice with LAMP-2 deficiency present a novel genetic model of human pancreatitis caused by lysosomal/autophagic dysfunction.

Serum amyloid A, phospholipase A2-IIA and C-reactive protein as inflammatory biomarkers for prostate diseases.

INTRODUCTION: Serum amyloid A (SAA), secreted group IIA phospholipase A2 (sPLA2-IIA), and C-reactive protein (CRP) are acute-phase proteins whose serum concentrations increase not only during inflammatory disorders, but also in the course of malignant diseases. MATERIALS AND METHODS: In this study we analyzed serum levels of these inflammatory markers along with prostate-specific antigens (PSA) in patients with benign prostatic hyperplasia (BPH, n = 55), localized prostate cancers (PCa, n = 55), and metastatic prostate cancers (mPCa, n = 27) using immunological assays. RESULTS: We found that in comparison to healthy individuals (n = 55), patients with BPH, PCa and mPCa have elevated serum levels of SAA, sPLA2-IIA, and CRP, in addition to elevated levels of PSA. Significant differences with respect to inflammatory biomarkers were found between localized and metastatic PCa (p < 0.001), suggesting a prognostic value of these parameters. In addition, serum concentrations of SAA and sPLA2-IIA positively correlate with CRP in BPH patients (p < 0.05) and in patients with PCa and mPCa (p < 0.001), but not with PSA levels, Gleason score, or tumor stage, emphasizing a role of SAA and sPLA2-IIA as circulating biomarkers of inflammation rather than of neoplastic transformation. In contrast to PSA, which differed significantly between BPH and localized PCa patients (p < 0.01), such a difference was not found for SAA, sPLA2-IIA, and CRP. In order to elucidate whether the elevated levels of SAA and sPLA2-IIA can be caused by cancer cell-associated synthesis, in vitro studies were performed. These analyses demonstrated the expression of SAA and sPLA2-IIA in LNCaP and PC-3 prostate cell lines, which can be further upregulated by pro-inflammatory cytokines in a cell type-dependent manner. This might suggest that, in addition to the hepatic origin, SAA and sPLA2-IIA can also be synthesized and secreted by prostatic cancer tissue itself. CONCLUSION: The results of the present study emphasize the utility of SAA, sPLA2-IIA, and CRP as circulating biomarkers of inflammation during BPH development and PCa progression.

Effects of oxidative alcohol metabolism on the mitochondrial permeability transition pore and necrosis in a mouse model of alcoholic pancreatitis.

BACKGROUND & AIMS: Opening of the mitochondrial permeability transition pore (MPTP) causes loss of the mitochondrial membrane potential (ΔΨm) and, ultimately, adenosine triphosphate depletion and necrosis. Cells deficient in cyclophilin D (CypD), a component of the MPTP, are resistant to MPTP opening, loss of ΔΨm, and necrosis. Alcohol abuse is a major risk factor for pancreatitis and is believed to sensitize the pancreas to stressors, by poorly understood mechanisms. We investigated the effects of ethanol on the pancreatic MPTP, the mechanisms of these effects, and their role in pancreatitis. METHODS: We measured ΔΨm in mouse pancreatic acinar cells incubated with ethanol alone and in combination with physiologic and pathologic concentrations of cholecystokinin-8 (CCK). To examine the role of MPTP, we used ex vivo and in vivo models of pancreatitis, induced in wild-type and CypD(-/-) mice by a combination of ethanol and CCK. RESULTS: Ethanol reduced basal ΔΨm and converted a transient depolarization, induced by physiologic concentrations of CCK, into a sustained decrease in ΔΨm, resulting in reduced cellular adenosine triphosphate and increased necrosis. The effects of ethanol and CCK were mediated by MPTP because they were not observed in CypD(-/-) acinar cells. Ethanol and CCK activated MPTP through different mechanisms-ethanol by reducing the ratio of oxidized nicotinamide adenine dinucleotide to reduced nicotinamide adenine dinucleotide, as a result of oxidative metabolism, and CCK by increasing cytosolic Ca(2+). CypD(-/-) mice developed a less-severe form of pancreatitis after administration of ethanol and CCK. CONCLUSIONS: Oxidative metabolism of ethanol sensitizes pancreatic mitochondria to activate MPTP, leading to mitochondrial failure; this makes the pancreas susceptible to necrotizing pancreatitis.

Stability and antioxidant activity of gossypol derivative immobilized on N-polyvinylpyrrolidone.

The objective of this study is analysis of stability and antioxidant and antiradical activities of the gossypol derivative - megosin conjugated with N-polyvinylpyrrolidone (PVP). The results of study have shown the greater stability of megosin+PVP than megosin in aqueous solution of wide range of pH. Here we also demonstrated that megosin+PVP, named rometin, possess high antioxidant activity in the same range as well known antioxidant trolox as determined by its ability to scavenge free ABTS(+) and DPPH radicals in vitro. In addition, megosin+PVP was able to prevent accumulation of products of lipid peroxidation (thiobarbituric acid reactive substances and diene conjugates) and lysophospholipids formation in mitochondria membranes caused by CCl(4)-induced oxidative stress in rat liver in vivo. Furthermore, megosin+PVP rescued mitochondrial functions, such as respiration and oxidative phosphorylation, which declined after CCl(4) administration. Thus we present that the conjugation of megosin to PVP increase its stability and remain antioxidant activity in vivo and in vitro.

Impaired autophagy and organellar dysfunction in pancreatitis.

Recent findings from our group, obtained on experimental in vivo and ex vivo models of pancreatitis, reveal that this disease causes a profound dysfunction of key cellular organelles, lysosomes and mitochondria. We found that autophagy, the main cellular degradative, lysosome-driven process, is activated but also impaired in acute pancreatitis because of its' inefficient progression/resolution (flux) resulting from defective function of lysosomes. One mechanism underlying the lysosomal dysfunction in pancreatitis is abnormal processing (maturation) and activation of cathepsins, major lysosomal hydrolases; another is a decrease in pancreatic levels of key lysosomal membrane proteins LAMP-1 and LAMP-2. Our data indicate that lysosomal dysfunction plays an important initiating role in pancreatitis pathobiology. The impaired autophagy mediates vacuole accumulation in acinar cells; furthermore, the abnormal maturation and activation of cathepsins leads to increase in intra-acinar trypsin, the hallmark of pancreatitis; and LAMP-2 deficiency causes inflammation and acinar cell necrosis. Thus, the autophagic and lysosomal dysfunctions mediate key pathologic responses of pancreatitis. On the other hand, we showed that pancreatitis causes acinar cell mitochondria depolarization, mediated by the permeability transition pore (PTP). Genetic (via deletion of cyclophilin D) inactivation of PTP prevents mitochondrial depolarization and greatly ameliorates the pathologic responses of pancreatitis. Further, our data suggest that mitochondrial damage, by stimulating autophagy, increases the demand for efficient lysosomal degradation and therefore aggravates the pathologic consequences of lysosomal dysfunction. Thus, the combined autophagic, lysosomal and mitochondrial dysfunctions are key to the pathogenesis of pancreatitis.

Serum levels of secreted group IIA phospholipase A(2) in benign prostatic hyperplasia and prostate cancer: a biomarker for inflammation or neoplasia?

Secreted group IIA phospholipase A(2) (sPLA(2)-IIA) is markedly up-regulated in human prostate cancer (PCa) specimens and in some PCa-derived cell lines, indicating an important role of this enzyme in tumourigenesis. In this study, we measured levels of sPLA(2)-IIA, C-reactive protein (CRP), and prostate-specific antigen (PSA) in serum samples obtained from patients with benign prostatic hyperplasia (BPH) and with PCa of different stages. We found that serum levels of sPLA(2)-IIA and CRP in BPH and PCa patients were significantly elevated compared to those of healthy individuals, but the concentrations of these inflammatory biomarkers did not differ between patients with BPH or PCa. Furthermore, serum levels of sPLA(2)-IIA correlated with concentrations of CRP, but not with PSA, Gleason grade or tumour stage. In conclusion, these findings suggest that cancer-related changes are not exclusive factors contributing to elevated serum sPLA(2)-IIA levels and emphasize the utility of sPLA(2)-IIA as a circulating marker of inflammation in patients with BPH and PCa.

Investigating the pathobiology of alcoholic pancreatitis.

Alcohol abuse is one of the most common causes of pancreatitis. The risk of developing alcohol-induced pancreatitis is related to the amount and duration of drinking. However, only a small portion of heavy drinkers develop disease, indicating that other factors (genetic, environmental, or dietary) contribute to disease initiation. Epidemiologic studies suggest roles for cigarette smoking and dietary factors in the development of alcoholic pancreatitis. The mechanisms underlying alcoholic pancreatitis are starting to be understood. Studies from animal models reveal that alcohol sensitizes the pancreas to key pathobiologic processes that are involved in pancreatitis. Current studies are focussed on the mechanisms responsible for the sensitizing effect of alcohol; recent findings reveal disordering of key cellular organelles including endoplasmic reticulum, mitochondria, and lysosomes. As our understanding of alcohol's effects continue to advance to the level of molecular mechanisms, insights into potential therapeutic strategies will emerge providing opportunities for clinical benefit.

Grp78 heterozygosity regulates chaperone balance in exocrine pancreas with differential response to cerulein-induced acute pancreatitis.

The endoplasmic reticulum (ER) is abundant in the acinar cells of the exocrine pancreas. To test the role of ER homeostasis in acute pancreatitis, we manipulated GRP78 levels, a major ER chaperone, in mice. Grp78(+/+) and (+/-) littermates were fed either a regular diet (RD) or a high-fat diet. Acinar cells were examined for ER structure by electron microscopy, and ER chaperone levels were assessed by immunoblotting. Pancreatitis was induced by cerulein injection, and multiple pathological parameters were analyzed. Grp78(+/-) mice showed decreased GRP78 expression in acinar cells. Exocrine pancreata of RD-fed Grp78(+/-) mice in an outbred C57BL/6 × 129/sv genetic background exhibited ER lumen dilation, a reduction in chaperones calnexin (CNX) and calreticulin (CRT), and exacerbated pancreatitis associated with high CHOP induction. With the high-fat diet regimen, Grp78 heterozygosity triggered GRP94 up-regulation and restoration of GRP78, CNX, and CRT to wild-type levels, corresponding with mitigated pancreatitis on cerulein insult. Interestingly, after backcrossing into the C57BL/6 background, RD-fed Grp78(+/-) mice exhibited an increase in GRP94 and levels of CNX and CRT equivalent to wild type, associated with decreased experimental pancreatitis severity. Administration of a chemical chaperone, 4-phenolbutyrate, was protective against cerulein-induced death. Thus, in exocrine pancreata, Grp78 heterozygosity regulates ER chaperone balance, in dietary- and genetic background-dependent manners, and improved ER protein folding capacity might be protective against pancreatitis.