Anti-tumor effect of cisplatin in human oral squamous cell carcinoma was enhanced by andrographolide via upregulation of phospho-p53 in vitro and in vivo.

Oral squamous cell carcinoma is one of the most common neoplasm in the world. Despite the improvements in diagnosis and treatment, the outcome is still poor now. Thus, the development of novel therapeuticapproaches is needed. The aim of this study is to assess the synergistic anti-tumor effect of andrographolide with cisplatin (DDP) in oral squamous cell carcinoma CAL-27 cells in vitro and in vivo. We performed Cell Counting Kit-8 proliferation assay, apoptosis assay, and western blotting on CAL-27 cells treated with andrographolide, DDP or the combination in vitro. In vivo, we also treated CAL-27 xenografts with andrographolide or the combination, and performed terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling assay and immunohistochemical analysis of Ki-67. The results showed the combination of andrographolide and DDP synergistically inhibited CAL-27 cell proliferation in vitro and caused tumor regression in vivo in the CAL-27 xenografts. In addition, the synergistic anti-tumor effect of andrographolide with synergistic was due to an enhanced apoptosis. Moreover, the combination therapy upregulated the expression level of p-p53 in vitro and decreased Ki-67 expression in vivo. Our data indicate that the combination treatment of andrographolide and DDP results in synergistic anti-tumor growth activity against oral squamous cell carcinoma CAL-27 in vitro and in vivo. These results demonstrated that combination of andrographolide with DDP was likely to represent a potential therapeutic strategy for oral squamous cell carcinoma.

Cellular FLICE-like inhibitory protein secures intestinal epithelial cell survival and immune homeostasis by regulating caspase-8.

BACKGROUND & AIMS: The intestinal epithelium generates a barrier that protects mammals from potentially harmful intestinal contents, such as pathogenic bacteria. Dysregulation of epithelial cell death has been implicated in barrier dysfunction and in the pathogenesis of intestinal inflammation. We investigated mechanisms of cell-death regulation in the intestinal epithelium of mice. METHODS: Conditional knockout mice (either inducible or permanent) with deletion of cellular FLICE-inhibitory protein (cFlip) or caspase-8 in the intestinal epithelium were analyzed by histology and high-resolution endoscopy. We assessed the effects of cFlip or caspase-8 deficiency on intestinal homeostasis. RESULTS: Expression of cFlip in the intestinal epithelium was required for constitutive activation of caspase-8 under steady-state conditions. Intestinal expression of cFlip was required for development; disruption of the gene encoding cFlip from the intestinal epithelium (cFlip(fl/fl) VillinCre(+) mice) resulted in embryonic lethality. When cFlip was deleted from the intestinal epithelium of adult mice (cFlip(iΔIEC) mice), the animals died within a few days from severe tissue destruction, epithelial cell death, and intestinal inflammation. Death of cFlip-depleted intestinal epithelial cells was regulated extrinsically and required the presence of death receptor ligands, such as tumor necrosis factor-α and CD95 ligand, but was independent of receptor-interacting protein 3. cFlip deficiency was associated with strong up-regulation of caspase-8 and caspase-3 activity and excessive apoptosis in intestinal crypts. CONCLUSIONS: cFlip is required for intestinal tissue homeostasis in mice. It controls the level of activation of caspase-8 to promote survival of intestinal epithelial cells.

Incomplete Caspase 3 Activation and Mitigation of Apoptosis in Hibernating Ground Squirrels, Spermophilus lateralis.

AbstractHibernating golden-mantled ground squirrels, Spermophilus [Callospermophilus] lateralis, tolerate proapoptotic conditions, such as low body temperature, anorexia, acidosis, and ischemia/reperfusion. Avoiding widespread apoptosis is critical for hibernator survival. Caspase 3, the key executioner of apoptosis, cleaves a majority of apoptotic targets. Under proapoptotic conditions, inactive procaspase 3 (32 kDa) is activated when cleaved into 17- and 12-kDa fragments (p32, p17, and p12, respectively). Caspase 3 activation results in extreme enzymatic activation. Activity increases >10,000-fold followed by apoptotic execution. Is widespread apoptosis occurring during the proapoptotic hibernation season? Western blots showed p17 increased ∼2-fold during hibernation, indicating caspase 3 activation. However, in vitro caspase 3 activity assays found no extreme increases in activity. Downstream caspase 3 targets ICAD (inhibitor of caspase-activated deoxyribonuclease) and PARP (poly (ADP-ribose) polymerase) did not experience elevated cleavage during hibernation, which is inconsistent with caspase 3 activation. TUNEL (terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling) assays from multiple tissues found only 0.001%-0.009% of cells were TUNEL positive during winter, indicating negligible apoptosis during hibernation. Typically, caspase 3 activation generates a strong commitment toward apoptosis. We found that despite a ∼2-fold increase in active caspase 3, hibernators experience no downstream caspase 3 activity or widespread apoptosis. A systems-level approach suggests an incomplete signaling cascade wherein some caspase 3 activation during hibernation does not necessarily lead to bona fide apoptosis.

The FGF1/CPP-C chimera protein protects against intestinal adverse effects of C-ion radiotherapy without exacerbating pancreatic carcinoma.

BACKGROUND AND PURPOSE: Carbon ion (C-ion) beams are concentrated to irradiate pancreatic carcinoma in the upper abdomen; however, this radiotherapy potentially causes adverse reactions in the gastrointestinal tract. FGF1 is a candidate radioprotector for radiation-induced intestinal damage, but may promote the malignancy of pancreatic cancer. An FGF1/CPP-C chimeric protein was created to enhance the intracellular signaling mode of FGF1 instead of FGFR signaling. The present study investigated the effects of FGF1/CPP-C on the intestinal adverse reactions of C-ion radiotherapy as well as its influence on the malignancy of pancreatic cancer. MATERIALS AND METHODS: FGF1/CPP-C was administered intraperitoneally to BALB/c mice without heparin 12 h before total body irradiation (TBI) with low-LET C-ion (17 keV/µm) at 6-8 Gy. Several radioprotective effects were examined in the jejunum. The invasion and migration of the human pancreatic carcinoma cell lines MIAPaCa-2 and PANC-1 were assessed using Boyden chambers after cultures with FGF1/CPP-C. RESULTS: The FGF1/CPP-C treatment promoted crypt survival after C-ion irradiation at 7-8 Gy significantly more than the FGF1 treatment. FGF1/CPP-C also inhibited C-ion radiotherapy-induced apoptosis and reduced γH2AX foci in crypt cells more than FGF1. However, FGF1/CPP-C inhibited the downstream signaling pathways of FGFRs and suppressed the activation of cell-cycle regulatory molecules in the intestine until 4 h after TBI. Furthermore, IEC6 cells were arrested in G2M after cultures with FGF1/CPP-C or FGF1, suggesting that DNA repair after irradiation is promoted by FGF1/CPP-C-induced G2M arrest. In contrast, FGF1/CPP-C appeared to be internalized into MIAPaCa-2 and PANC-1 cells more efficiently than FGF1. Therefore, FGF1/CPP-C reduced the in vitro proliferation, invasion, and migration of MIAPaCa-2 and PANC-1 cells significantly more than FGF1 through the cellular internalization of FGF1. CONCLUSION: These results suggest that the intracellular signaling mode of FGF1/CPP-C attenuates the intestinal adverse effects of C-ion radiotherapy without enhancing the malignancy of pancreatic carcinoma.

Interleukin-17A Promotes Parietal Cell Atrophy by Inducing Apoptosis.

BACKGROUND & AIMS: Atrophic gastritis caused by chronic inflammation in the gastric mucosa leads to the loss of gastric glandular cells, including acid-secreting parietal cells. Parietal cell atrophy in a setting of chronic inflammation induces spasmolytic polypeptide expressing metaplasia, a critical step in gastric carcinogenesis. However, the mechanisms by which inflammation causes parietal cell atrophy and spasmolytic polypeptide expressing metaplasia are not well defined. We investigated the role of interleukin-17A (IL-17A) in causing parietal cell atrophy. METHODS: A mouse model of autoimmune atrophic gastritis was used to examine IL-17A production during early and late stages of disease. Organoids derived from corpus glands were used to determine the direct effects of IL-17A on gastric epithelial cells. Immunofluorescent staining was used to examine IL-17A receptors and the direct effect of signaling on parietal cells. Mice were infected with an IL-17A-producing adenovirus to determine the effects of IL-17A on parietal cells in vivo. Finally, IL-17A neutralizing antibodies were administered to mice with active atrophic gastritis to evaluate the effects on parietal cell atrophy and metaplasia. RESULTS: Increased IL-17A correlated with disease severity in mice with chronic atrophic gastritis. IL-17A caused caspase-dependent gastric organoid degeneration, which could not be rescued with a necroptosis inhibitor. Parietal cells expressed IL-17A receptors and IL-17A treatment induced apoptosis in parietal cells. Overexpressing IL-17A in vivo induced caspase-3 activation and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling staining in parietal cells. Finally, IL-17A neutralizing antibody decreased parietal cell atrophy and metaplasia in mice with chronic atrophic gastritis. CONCLUSIONS: These data identify IL-17A as a cytokine that promotes parietal cell apoptosis during atrophic gastritis, a precursor lesion for gastric cancer.

Organic Solute Transporter α-ß Protects Ileal Enterocytes From Bile Acid-Induced Injury.

BACKGROUND & AIMS: Ileal bile acid absorption is mediated by uptake via the apical sodium-dependent bile acid transporter (ASBT), and export via the basolateral heteromeric organic solute transporter α-ß (OSTα-OSTß). In this study, we investigated the cytotoxic effects of enterocyte bile acid stasis in Ostα-/- mice, including the temporal relationship between intestinal injury and initiation of the enterohepatic circulation of bile acids. METHODS: Ileal tissue morphometry, histology, markers of cell proliferation, gene, and protein expression were analyzed in male and female wild-type and Ostα-/- mice at postnatal days 5, 10, 15, 20, and 30. Ostα-/-Asbt-/- mice were generated and analyzed. Bile acid activation of intestinal Nrf2-activated pathways was investigated in Drosophila. RESULTS: As early as day 5, Ostα-/- mice showed significantly increased ileal weight per length, decreased villus height, and increased epithelial cell proliferation. This correlated with premature expression of the Asbt and induction of bile acid-activated farnesoid X receptor target genes in neonatal Ostα-/- mice. Expression of reduced nicotinamide adenine dinucleotide phosphate oxidase-1 and Nrf2-anti-oxidant responsive genes were increased significantly in neonatal Ostα-/- mice at these postnatal time points. Bile acids also activated Nrf2 in Drosophila enterocytes and enterocyte-specific knockdown of Nrf2 increased sensitivity of flies to bile acid-induced toxicity. Inactivation of the Asbt prevented the changes in ileal morphology and induction of anti-oxidant response genes in Ostα-/- mice. CONCLUSIONS: Early in postnatal development, loss of Ostα leads to bile acid accumulation, oxidative stress, and a restitution response in ileum. In addition to its essential role in maintaining bile acid homeostasis, Ostα-Ostß functions to protect the ileal epithelium against bile acid-induced injury. NCBI Gene Expression Omnibus: GSE99579.

Neutrophil-Hepatic Stellate Cell Interactions Promote Fibrosis in Experimental Steatohepatitis.

BACKGROUND & AIMS: Hepatic infiltration of neutrophils is a hallmark of steatohepatitis; however, the role of neutrophils in the progression of steatohepatitis remains unknown. METHODS: A clinically relevant mouse model of steatohepatitis induced by high-fat diet (HFD) plus binge ethanol feeding was used. Liver fibrosis was examined. In vitro cell culture was used to analyze the interaction of hepatic stellate cells (HSCs) and neutrophils. RESULTS: HFD plus one binge ethanol (HFD+1B) feeding induced significant hepatic neutrophil infiltration, liver injury, and fibrosis. HFD plus multiple binges of ethanol (HFD+mB) caused more pronounced liver fibrosis. Microarray analyses showed that the most highly activated signaling pathway in this HFD+1B model was related to liver fibrosis and HSC activation. Blockade of chemokine (C-X-C motif) ligand 1 or intercellular adhesion molecule-1 expression reduced hepatic neutrophil infiltration and ameliorated liver injury and fibrosis. Disruption of the p47phox gene (also called neutrophil cytosolic factor 1), a critical component of reactive oxygen species producing nicotinamide adenine dinucleotide phosphate-oxidase in neutrophils, diminished HFD+1B-induced liver injury and fibrosis. Co-culture of HSCs with neutrophils, but not with neutrophil apoptotic bodies, induced HSC activation and prolonged neutrophil survival. Mechanistic studies showed that activated HSCs produce granulocyte-macrophage colony-stimulating factor and interleukin-15 to prolong the survival of neutrophils, which may serve as a positive forward loop to promote liver damage and fibrosis. CONCLUSIONS: The current data from a mouse model of HFD plus binge ethanol feeding suggest that obesity and binge drinking synergize to promote liver fibrosis, which is partially mediated via the interaction of neutrophils and HSCs. Microarray data in this article have been uploaded to NCBI's Gene Expression Omnibus (GEO accession number: GSE98153).

Upregulation of miR-216a exerts neuroprotective effects against ischemic injury through negatively regulating JAK2/STAT3-involved apoptosis and inflammatory pathways.

OBJECTIVE: Ischemic stroke remains a significant cause of death and disability in industrialized nations. Janus tyrosine kinase (JAK) and signal transducer and activator of transcription (STAT) of the JAK2/STAT3 pathway play important roles in the downstream signal pathway regulation of ischemic stroke-related inflammatory neuronal damage. Recently, microRNAs (miRNAs) have emerged as major regulators in cerebral ischemic injury; therefore, the authors aimed to investigate the underlying molecular mechanism between miRNAs and ischemic stroke, which may provide potential therapeutic targets for ischemic stroke. METHODS: The JAK2- and JAK3-related miRNA (miR-135, miR-216a, and miR-433) expression levels were detected by real-time quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) and Western blot analysis in both oxygen-glucose deprivation (OGD)-treated primary cultured neuronal cells and mouse brain with middle cerebral artery occlusion (MCAO)-induced ischemic stroke. The miR-135, miR-216a, and miR-433 were determined by bioinformatics analysis that may target JAK2, and miR-216a was further confirmed by 3' untranslated region (3'UTR) dual-luciferase assay. The study further detected cell apoptosis, the level of lactate dehydrogenase, and inflammatory mediators (inducible nitric oxide synthase [iNOS], matrix metalloproteinase-9 [MMP-9], tumor necrosis factor-α [TNF-α], and interleukin-1ß [IL-1ß]) after cells were transfected with miR-NC (miRNA negative control) or miR-216a mimics and subjected to oxygen-glucose deprivation/reoxygenation (OGD/R) damage with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, annexin V-FITC/PI, Western blots, and enzyme-linked immunosorbent assay detection. Furthermore, neurological deficit detection and neurological behavior grading were performed to determine the infarction area and neurological deficits. RESULTS: JAK2 showed its highest level while miR-216a showed its lowest level at day 1 after ischemic reperfusion. However, miR-135 and miR-433 had no obvious change during the process. The luciferase assay data further confirmed that miR-216a can directly target the 3'UTR of JAK2, and overexpression of miR-216a repressed JAK2 protein levels in OGD/R-treated neuronal cells as well as in the MCAO model ischemic region. In addition, overexpression of miR-216a mitigated cell apoptosis both in vitro and in vivo, which was consistent with the effect of knockdown of JAK2. Furthermore, the study found that miR-216a obviously inhibited the inflammatory mediators after OGD/R, including inflammatory enzymes (iNOS and MMP-9) and cytokines (TNF-α and IL-1ß). Upregulating miR-216a levels reduced ischemic infarction and improved neurological deficit. CONCLUSIONS: These findings suggest that upregulation of miR-216a, which targets JAK2, could induce neuroprotection against ischemic injury in vitro and in vivo, which provides a potential therapeutic target for ischemic stroke.

Increased Bcl-xL Expression in Pancreatic Neoplasia Promotes Carcinogenesis by Inhibiting Senescence and Apoptosis.

BACKGROUND & AIMS: Bcl-xL, an anti-apoptotic Bcl-2 family protein, is overexpressed in 90% of pancreatic ductal adenocarcinoma (PDAC) cases. However, Bcl-xL expression in pancreatic intraepithelial neoplasias (PanINs) and its significance in PDAC carcinogenesis remain unclear. The aim of this study was to elucidate the significance of Bcl-xL expression in PanINs. METHODS: We investigated the expression levels of Bcl-xL in pancreas-specific KrasG12D (P-KrasG12D) mice and human PanINs and PDAC. We examined the impact of Bcl-xL expression on Kras-mutated pancreatic neoplasia using Bcl-xL-overexpressing P-KrasG12D mice and Bcl-xL-knockout P-KrasG12D mice. RESULTS: In P-KrasG12D mice, the number of PanINs increased and their grades progressed with age. In total, 55.6% of these mice developed PDAC at 12-14 months. According to the immunohistochemistry of mouse pancreas and human resected specimens, Bcl-xL expression was increased significantly in PanIN-1 compared with that in normal pancreatic ducts, and augmented further with the progression of pancreatic neoplasia in PanIN-2/3 and PDAC. Oncogene-induced senescence was observed frequently in PanIN-1, but rarely was detected in PanIN-2/3 and PDAC. Bcl-xL overexpression significantly accelerated the progression to high-grade PanINs and PDAC and reduced the survival of P-KrasG12D mice. Bcl-xL overexpression in P-KrasG12D mice suppressed oncogene-induced senescence in PanIN-1 and inhibited apoptosis in PanIN-3. Bcl-xL deficiency in P-KrasG12D mice induced cellular senescence in PanIN-2/3. CONCLUSIONS: Bcl-xL expression increases with the progression from PanIN-1 to PDAC, whereas oncogene-induced senescence decreases. Bcl-xL overexpression increases PDAC incidence rates by inhibiting oncogene-induced senescence and apoptosis in PanINs. Conversely, Bcl-xL deficiency induced senescence in PanINs. Anti-Bcl-xL treatments may have the potency to suppress the progression from PanINs to PDAC.

Lithium posttreatment confers neuroprotection through glycogen synthase kinase-3ß inhibition in intracerebral hemorrhage rats.

OBJECTIVE Inflammation and apoptosis are two key factors contributing to secondary brain injury after intracerebral hemorrhage (ICH). The objective of this study was to evaluate the effects of lithium posttreatment on behavior, brain atrophy, inflammation, and perihematomal cell death. Furthermore, the authors aimed to determine the role of the pro-apoptotic glycogen synthase kinase-3ß (GSK-3ß) after experimental ICH. METHODS Male Sprague-Dawley rats (n = 108) were subjected to intracerebral infusion of semicoagulated autologous blood. Window of opportunity and dose optimization studies of lithium on ICH-induced injury were performed by measuring neurological deficits. Animals with ICH received vehicle administration or lithium posttreatment (60 mg/kg) for up to 21 days. Hemispheric atrophy was evaluated. Perihematomal cell death was quantified through terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL). The number of myeloperoxidase (MPO)-positive neutrophils and OX42-positive microglia in the perihematomal areas were calculated. Western blotting was used for the quantification of GSK-3ß, heat shock protein 70 (HSP70), nuclear factor-κB p65 (NF-κB p65), and cy-clooxygenase-2 (COX-2). RESULTS Lithium, at a dose of 60 mg/kg initiated from 2 hours after injury, exhibited the best effects of improving neurological outcomes 3, 5, 7, 14, 21, and 28 days after ICH, reduced the hemispheric atrophy at 42 days after surgery, and reduced the number of TUNEL-positive cells, MPO-positive neutrophils, and OX42-positive microglia in the perihematomal areas. Furthermore, lithium posttreatment modulated GSK-3ß, increased HSP70, and decreased NF-κB p65 and COX-2 expression in the ipsilateral hemisphere. CONCLUSIONS Lithium posttreatment at a dose of 60 mg/kg, initiated beginning 2 hours after injury, improves functional and morphological outcomes, and inhibits inflammation and perihematomal cell death in a rat model of semicoagulated autologous blood ICH through inactivation of GSK-3ß.

Interdependency of EGF and GLP-2 Signaling in Attenuating Mucosal Atrophy in a Mouse Model of Parenteral Nutrition.

BACKGROUND & AIMS: Total parenteral nutrition (TPN), a crucial treatment for patients who cannot receive enteral nutrition, is associated with mucosal atrophy, barrier dysfunction, and infectious complications. Glucagon-like peptide-2 (GLP-2) and epidermal growth factor (EGF) improve intestinal epithelial cell (IEC) responses and attenuate mucosal atrophy in several TPN models. However, it remains unclear whether these 2 factors use distinct or overlapping signaling pathways to improve IEC responses. We investigated the interaction of GLP-2 and EGF signaling in a mouse TPN model and in patients deprived of enteral nutrition. METHODS: Adult C57BL/6J, IEC-Egfrknock out (KO) and IEC-pik3r1KO mice receiving TPN or enteral nutrition were treated with EGF or GLP-2 alone or in combination with reciprocal receptor inhibitors, GLP-2(3-33) or gefitinib. Jejunum was collected and mucosal atrophy and IEC responses were assessed by histologic, gene, and protein expression analyses. In patients undergoing planned looped ileostomies, fed and unfed ileum was analyzed. RESULTS: Enteral nutrient deprivation reduced endogenous EGF and GLP-2 signaling in mice and human beings. In the mouse TPN model, exogenous EGF or GLP-2 attenuated mucosal atrophy and restored IEC proliferation. The beneficial effects of EGF and GLP-2 were decreased upon Gefitinib treatment and in TPN-treated IEC-EgfrKO mice, showing epidermal growth factor-receptor dependency for these IEC responses. By contrast, in TPN-treated IEC-pi3kr1KO mice, the beneficial actions of EGF were lost, although GLP-2 still attenuated mucosal atrophy. CONCLUSIONS: Upon enteral nutrient deprivation, exogenous GLP-2 and EGF show strong interdependency for improving IEC responses. Understanding the differential requirements for phosphatidylinositol 3-kinase/phosphoAKT (Ser473) signaling may help improve future therapies to prevent mucosal atrophy.

Requirement of Gαq/Gα11 Signaling in the Preservation of Mouse Intestinal Epithelial Homeostasis.

BACKGROUND & AIMS: Proliferation, differentiation, and morphogenesis of the intestinal epithelium are tightly regulated by a number of molecular pathways. Coordinated action of intestine is achieved by gastrointestinal hormones, most of which exert these actions through G-protein-coupled receptors. We herein investigated the role of Gαq/11-mediated signaling in intestinal homeostasis. METHODS: Intestinal tissues from control (Gnaqflox/floxGna11+/+ ), Int-Gq knock-out (KO) (VilCre+/-Gnaqflox/floxGna11+/+ ), G11 KO (Gnaqflox/floxGna11-/- ), and Int-Gq/G11 double knock-out (DKO) (VilCre+/-Gnaqflox/floxGna11-/- ) mice were examined by microscopy, transmission electron microscopy, and immunohistochemistry. The effect of Gαq/11-mediated signaling was studied in the cell lineage, proliferation, and apoptosis. Dextran sodium sulfate (DSS) colitis was induced to study the role of Gαq/11 in colon. RESULTS: Paneth cells were enlarged, increased in number, and mislocalized in Int-Gq/G11 DKO small intestine. Paneth cells also reacted with PAS and Muc2 antibody, indicating an intermediate character of Paneth and goblet cells. The nuclear ß-catenin, T-cell factor 1, and Sox9 expression were reduced severely in the crypt base of Int-Gq/G11 DKO intestine. Proliferation was activated in the crypt base and apoptosis was enhanced along the crypt. Int-Gq/G11 DKO mice were susceptible to DSS colitis. Proliferation was inhibited in the crypt of unaffected and regenerative areas. Cystic crypts, periodic acid-Schiff-positive cells, and Muc2-positive cells were unusually observed in the ulcerative region. CONCLUSIONS: The Gαq/11-mediated pathway plays a pivotal role in the preservation of intestinal homeostasis, especially in Paneth cell maturation and positioning. Wnt/ß-catenin signaling was reduced significantly in the crypt base in Gαq/G11-deficient mice, resulting in the defective maturation of Paneth cells, induction of differentiation toward goblet cells, and susceptibility to DSS colitis.

Acrolein Is a Pathogenic Mediator of Alcoholic Liver Disease and the Scavenger Hydralazine Is Protective in Mice.

BACKGROUND & AIMS: Alcoholic liver disease (ALD) remains a major cause of morbidity and mortality, with no Food and Drug Administration-approved therapy. Chronic alcohol consumption causes a pro-oxidant environment and increases hepatic lipid peroxidation, with acrolein being the most reactive/toxic by-product. This study investigated the pathogenic role of acrolein in hepatic endoplasmic reticulum (ER) stress, steatosis, and injury in experimental ALD, and tested acrolein elimination/scavenging (using hydralazine) as a potential therapy in ALD. METHODS: In vitro (rat hepatoma H4IIEC cells) and in vivo (chronic+binge alcohol feeding in C57Bl/6 mice) models were used to examine alcohol-induced acrolein accumulation and consequent hepatic ER stress, apoptosis, and injury. In addition, the potential protective effects of the acrolein scavenger, hydralazine, were examined both in vitro and in vivo. RESULTS: Alcohol consumption/metabolism resulted in hepatic accumulation of acrolein-protein adducts, by up-regulation of cytochrome P4502E1 and alcohol dehydrogenase, and down-regulation of glutathione-s-transferase-P, which metabolizes/detoxifies acrolein. Alcohol-induced acrolein adduct accumulation led to hepatic ER stress, proapoptotic signaling, steatosis, apoptosis, and liver injury; however, ER-protective/adaptive responses were not induced. Notably, direct exposure to acrolein in vitro mimicked the in vivo effects of alcohol, indicating that acrolein mediates the adverse effects of alcohol. Importantly, hydralazine, a known acrolein scavenger, protected against alcohol-induced ER stress and liver injury, both in vitro and in mice. CONCLUSIONS: Our study shows the following: (1) alcohol consumption triggers pathologic ER stress without ER adaptation/protection; (2) alcohol-induced acrolein is a potential therapeutic target and pathogenic mediator of hepatic ER stress, cell death, and injury; and (3) removal/clearance of acrolein by scavengers may have therapeutic potential in ALD.

Netrin-1 Protects Hepatocytes Against Cell Death Through Sustained Translation During the Unfolded Protein Response.

BACKGROUND & AIMS: Netrin-1, a multifunctional secreted protein, is up-regulated in cancer and inflammation. Netrin-1 blocks apoptosis induced by the prototypical dependence receptors deleted in colorectal carcinoma and uncoordinated phenotype-5. Although the unfolded protein response (UPR) triggers apoptosis on exposure to stress, it first attempts to restore endoplasmic reticulum homeostasis to foster cell survival. Importantly, UPR is implicated in chronic liver conditions including hepatic oncogenesis. Netrin-1's implication in cell survival on UPR in this context is unknown. METHODS: Isolation of translational complexes, determination of RNA secondary structures by selective 2'-hydroxyl acylation and primer extension/dimethyl sulfate, bicistronic constructs, as well as conventional cell biology and biochemistry approaches were used on in vitro-grown hepatocytic cells, wild-type, and netrin-1 transgenic mice. RESULTS: HepaRG cells constitute a bona fide model for UPR studies in vitro through adequate activation of the 3 sensors of the UPR (protein kinase RNA-like endoplasmic reticulum kinase (PERK)), inositol requiring enzyme 1α (IRE1α), and activated transcription factor 6 (ATF6). The netrin-1 messenger RNA 5'-end was shown to fold into a complex double pseudoknot and bear E-loop motifs, both of which are representative hallmarks of related internal ribosome entry site regions. Cap-independent translation of netrin 5' untranslated region-driven luciferase was observed on UPR in vitro. Unlike several structurally related oncogenic transcripts (l-myc, c-myc, c-myb), netrin-1 messenger RNA was selected for translation during UPR both in human hepatocytes and in mice livers. Depletion of netrin-1 during UPR induces apoptosis, leading to cell death through an uncoordinated phenotype-5A/C-mediated involvement of protein phosphatase 2A and death-associated protein kinase 1 in vitro and in netrin transgenic mice. CONCLUSIONS: UPR-resistant, internal ribosome entry site-driven netrin-1 translation leads to the inhibition of uncoordinated phenotype-5/death-associated protein kinase 1-mediated apoptosis in the hepatic context during UPR, a hallmark of chronic liver disease.

Consistent delayed unilateral neuronal death after modified transient focal cerebral ischemia in mice that mimics neuronal injury after transient global cerebral ischemia.

OBJECT: Numerous studies have attempted to reveal the pathophysiology of ischemic neuronal injury using a representative transient global cerebral ischemia (tGCI) model in rodents; however, most of them have used gerbil or rat models. Recent advances in transgene and gene-knockout technology have enabled the precise molecular mechanisms of ischemic brain injury to be investigated. Because the predominant species for the study of genetic mutations is the mouse, a representative mouse model of tGCI is of particular importance. However, simple mouse models of tGCI are less reproducible; therefore, a more complex process or longer duration of ischemia, which causes a high mortality rate, has been used in previous tGCI models in mice. In this study, the authors aimed to overcome these problems and attempted to produce consistent unilateral delayed hippocampal CA1 neuronal death in mice. METHODS: C57BL/6 mice were subjected to short-term unilateral cerebral ischemia using a 4-mm silicone-coated intraluminal suture to obstruct the origin of the posterior cerebral artery (PCA), and regional cerebral blood flow (rCBF) of the PCA territory was measured using laser speckle flowmetry. The mice were randomly assigned to groups of different ischemic durations and histologically evaluated at different time points after ischemia. The survival rate and neurological score of the group that experienced 15 minutes of ischemia were also evaluated. RESULTS: Consistent neuronal death was observed in the medial CA1 subregion 4 days after 15 minutes of ischemia in the group of mice with a reduction in rCBF of < 65% in the PCA territory during ischemia. Morphologically degenerated cells were mostly positive for terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling and cleaved caspase 3 staining 4 days after ischemia. The survival rates of the mice 24 hours (n = 24), 4 days (n = 15), and 7 days (n = 7) after being subjected to 15 minutes of ischemia were 95.8%, 100%, and 100%, respectively, and the mice had slight motor deficits. CONCLUSIONS: The authors established a model of delayed unilateral hippocampal neuronal death in C57BL/6 mice by inducing ischemia in the PCA territory using an intraluminal suture method and established inclusion criteria for PCAterritory rCBF monitored by laser speckle flowmetry. This model may be useful for investigating the precise molecular mechanisms of ischemic brain injury.

Role of oxidized LDL and lectin-like oxidized LDL receptor-1 in cerebral vasospasm after subarachnoid hemorrhage.

OBJECT: Cerebral vasospasm after subarachnoid hemorrhage (SAH) is a serious complication. Free radicals derived from subarachnoid clotting are recognized to play an important role. Oxidized low-density lipoprotein (ox-LDL) and lectin-like oxidized LDL receptor-1 (LOX-1) have been shown to be related to the pathogenesis of atherosclerosis and may increase in cerebral arteries after SAH, due to the action of free radicals derived from a subarachnoid clot. These molecules may also affect the pathogenesis of vasospasm, generating intracellular reactive oxygen species and downregulating the expression of endothelial NO synthase (eNOS). If so, apple polyphenol might be effective in the prevention of vasospasm due to an abundant content of procyanidins, which exhibit strong radical scavenging effects, and the ability to suppress ox-LDL and LOX-1. The purposes of this study were to investigate changes in levels of ox-LDL and LOX-1 after SAH and whether administering apple polyphenol can modify cerebral vasospasm. METHODS: Forty Japanese white rabbits were assigned randomly to 4 groups: an SAH group (n = 10); a shamoperation group (n = 10), which underwent intracisternal saline injection; a low-dose polyphenol group (n = 10) with SAH and oral administration of apple polyphenol at 10 mg/kg per day from Day 0 to Day 3; and a high-dose polyphenol group (n = 10) with SAH and oral administration of apple polyphenol at 50 mg/kg per day. At Day 4, the basilar artery and brain was excised from each rabbit. The degree of cerebral vasospasm was evaluated by measuring the cross-sectional area of each basilar artery, and the expression of ox-LDL, LOX-1, and eNOS was examined for each basilar artery by immunohistochemical staining and reverse transcriptase polymerase chain reaction. In addition, neuronal apoptosis in the cerebral cortex was evaluated by TUNEL. RESULTS: Compared with the sham group, the expression of ox-LDL and LOX-1 in the basilar arterial wall was significantly increased in the SAH group, the expression of eNOS was significantly decreased, and the cross-sectional area of basilar artery was significantly decreased. Compared with the SAH group, the cross-sectional area of basilar artery was increased in the polyphenol groups, together with the decreased expression of ox-LDL and LOX-1 and the increased expression of eNOS. In the high-dose polyphenol group, those changes were statistically significant compared with the SAH group. In the low-dose polyphenol group, those changes were smaller than in the high-dose polyphenol group. No apoptosis and no changes were seen in the cerebral cortex in all groups. CONCLUSIONS: This is the first study suggesting that ox-LDL and LOX-1 increase due to SAH and that they may play a role in the pathogenesis of vasospasm. It is assumed that procyanidins in apple polyphenol may inhibit a vicious cycle of ox-LDL, LOX-1, and ROS in a dose-dependent manner. Apple polyphenol is a candidate for preventive treatment of cerebral vasospasm.

Wnt/ß-catenin signaling regulates follicular development by modulating the expression of Foxo3a signaling components.

Wnt signaling is an evolutionarily conserved pathway that regulates cell proliferation, differentiation and apoptosis. To investigate the possible role of Wnt signaling in the regulation of ovarian follicular development, secondary follicles were isolated and cultured in vitro in the presence or absence of its activator (LiCl or Wnt3a) or inhibitor (IWR-1). We have demonstrated that activation of ß-catenin signals by activators dramatically suppressed follicular development by increasing granulosa cell apoptosis and inhibiting follicle steroidogenesis. In contrast, inhibition of Wnt signaling by IWR-1 was observed with better developed follicles and increased steroidogenesis. Further studies have shown that the transcription factor Forkhead box O3a (Foxo3a) and its downstream target molecules were modulated by the activators or the inhibitor. These findings provide evidence that Wnt signaling might negatively regulate follicular development potentially through Foxo3a signaling components.

Protective effect of lycopene against ochratoxin A induced renal oxidative stress and apoptosis in rats.

This study was designed to investigate the possible protective effect of lycopene against the renal toxic effects of OTA. Male Sprague-Dawley rats (<200 g, n=6) were treated with OTA (0.5 mg/kg/day) and/or lycopene (5 mg/kg/day) by gavage for 14 days. Histopathological examinations were performed and apoptotic cell death in both cortex and medulla was evaluated by TUNEL assay. Besides, biochemical parameters and activities of renal antioxidant selenoenzymes [glutathione peroxidase 1 (GPx1), thioredoxin reductase (TrxR)], catalase (CAT), superoxide dismutase (SOD); concentrations of total glutathione (GSH), and malondialdehyde (MDA) levels were measured. OTA treatment was found to induce oxidative stress in rat kidney, as evidenced by marked decreases in CAT (35%) activity and GSH levels (44%) as well as increase in SOD activity (22%) vs control group. Furthermore, TUNEL analysis revealed a significant increase in the number of TUNEL-positive cells in cortex (49%) and medulla (75%) in OTA administrated group compared to control (p<0.05). Lycopene supplementation with OTA increased GPx1 activity and GSH levels, and decreased apoptotic cell death in both cortex and medulla vs. control. The results of this study showed that at least one of the mechanisms underlying the renal toxicity of OTA is oxidative stress and apoptosis is the major form of cell death caused by OTA. Besides, our data indicate that the natural antioxidant lycopene might be partially protective against OTA-induced nephrotoxicity and oxidative stress in rat.