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The phosphatidylinositol (PI3K)/AKT/mTOR axis represents an important therapeutic target to treat human cancers. A well-described downstream target of the PI3K pathway is the forkhead box O (FOXO) transcription factor family. FOXOs have been implicated in many cellular responses, including drug-induced resistance in cancer cells. However, FOXO-dependent acute phase resistance mediated by pictilisib, a potent small molecule PI3K inhibitor (PI3Ki), has not been studied. Here, we report that pictilisib-induced adaptive resistance is regulated by the FOXO-dependent rebound activity of receptor tyrosine kinases (RTKs) in mucinous colorectal adenocarcinoma (MCA) cells. The resistance mediated by PI3K inhibition involves the nuclear localization of FOXO and the altered expression of RTKs, including ErbB2, ErbB3, EphA7, EphA10, IR, and IGF-R1 in MCA cells. Further, in the presence of FOXO siRNA, the pictilisib-induced feedback activation of RTK regulators (pERK and pAKT) was altered in MCA cells. Interestingly, the combinational treatment of pictilisib (Pi3Ki) and FOXO1i (AS1842856) synergistically reduced MCA cell viability and increased apoptosis. These results demonstrate that pictilisib used as a single agent induces acute resistance, partly through FOXO1 inhibition. Therefore, overcoming PI3Ki single-agent adaptive resistance by rational design of FOXO1 and PI3K inhibitor combinations could significantly enhance the therapeutic efficacy of PI3K-targeting drugs in MCA cells.
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Adenocarcinoma , Neoplasias Colorrectales , Humanos , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Tirosina Quinasas Receptoras , Factores de Transcripción Forkhead/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/genética , Tirosina , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteína Forkhead Box O1/genéticaRESUMEN
Alcohol consumption exacerbates hepatitis C virus (HCV) pathogenesis and promotes disease progression, although the mechanisms are not quite clear. We have previously observed that acetaldehyde (Ach) continuously produced by the acetaldehyde-generating system (AGS), temporarily enhanced HCV RNA levels, followed by a decrease to normal or lower levels, which corresponded to apoptosis induction. Here, we studied whether Ach-induced apoptosis caused depletion of HCV-infected cells and what role apoptotic bodies (AB) play in HCV-alcohol crosstalk. In liver cells exposed to AGS, we observed the induction of miR-122 and miR-34a. As miR-34a has been associated with apoptotic signaling and miR-122 with HCV replication, these findings may suggest that cells with intensive viral replication undergo apoptosis. Furthermore, when AGS-induced apoptosis was blocked by a pan-caspase inhibitor, the expression of HCV RNA was not changed. AB from HCV-infected cells contained HCV core protein and the assembled HCV particle that infect intact hepatocytes, thereby promoting the spread of infection. In addition, AB are captured by macrophages to switch their cytokine profile to the proinflammatory one. Macrophages exposed to HCV(+) AB expressed more IL-1ß, IL-18, IL-6, and IL-10 mRNAs compared with those exposed to HCV(-) AB. The generation of AB from AGS-treated HCV-infected cells even enhanced the induction of aforementioned cytokines. We conclude that HCV and alcohol metabolites trigger the formation of AB containing HCV particles. The consequent spread of HCV to neighboring hepatocytes via infected AB, as well as the induction of liver inflammation by AB-mediated macrophage activation potentially exacerbate the HCV infection course by alcohol and worsen disease progression.
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Acetaldehído/metabolismo , Apoptosis , Hepacivirus/fisiología , Hepatocitos/metabolismo , Replicación Viral , Línea Celular , Células Cultivadas , Hepacivirus/patogenicidad , Hepatocitos/virología , Humanos , Interleucinas/genética , Interleucinas/metabolismo , Macrófagos/metabolismo , Macrófagos/virología , MicroARNs/genética , MicroARNs/metabolismo , ARN Viral/genética , Proteínas Virales/genética , Proteínas Virales/metabolismoRESUMEN
BACKGROUND: Alcohol-induced reduction in the hepatocellular S-adenosylmethionine (SAM):S-adenosylhomocysteine (SAH) ratio impairs the activities of many SAM-dependent methyltransferases. These impairments ultimately lead to the generation of several hallmark features of alcoholic liver injury including steatosis. Guanidinoacetate methyltransferase (GAMT) is an important enzyme that catalyzes the final reaction in the creatine biosynthetic process. The liver is a major site for creatine synthesis which places a substantial methylation burden on this organ as GAMT-mediated reactions consume as much as 40% of all the SAM-derived methyl groups. We hypothesized that dietary creatine supplementation could potentially spare SAM, preserve the hepatocellular SAM:SAH ratio, and thereby prevent the development of alcoholic steatosis and other consequences of impaired methylation reactions. METHODS: For these studies, male Wistar rats were pair-fed the Lieber-DeCarli control or ethanol (EtOH) diet with or without 1% creatine supplementation. At the end of 4 to 5 weeks of feeding, relevant biochemical and histological analyses were performed. RESULTS: We observed that creatine supplementation neither prevented alcoholic steatosis nor attenuated the alcohol-induced impairments in proteasome activity. The lower hepatocellular SAM:SAH ratio seen in the EtOH-fed rats was also not normalized or SAM levels spared when these rats were fed the creatine-supplemented EtOH diet. However, a >10-fold increased level of creatine was observed in the liver, serum, and hearts of rats fed the creatine-supplemented diets. CONCLUSIONS: Overall, dietary creatine supplementation did not prevent alcoholic liver injury despite its known efficacy in preventing high-fat-diet-induced steatosis. Betaine, a promethylating agent that maintains the hepatocellular SAM:SAH, still remains our best option for treating alcoholic steatosis.
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Creatina/uso terapéutico , Hígado Graso Alcohólico/prevención & control , Amidinotransferasas/metabolismo , Animales , Suplementos Dietéticos , Guanidinoacetato N-Metiltransferasa/metabolismo , Riñón/enzimología , Hígado/enzimología , Masculino , Miocardio/metabolismo , Ratas Wistar , S-Adenosilhomocisteína/metabolismo , S-Adenosilmetionina/metabolismoRESUMEN
BACKGROUND: Non-alcoholic and alcoholic fatty liver disease (NAFLD and AFLD, respectively) are major health problems, as patients with either condition can progress to hepatitis, fibrosis, and cirrhosis. Although histologically similar, key differences likely exist in these two models. For example, altered content of several vesicle trafficking proteins have been identified in AFLD, but their content in NAFLD is unknown. In this study, we compared select parameters in NAFLD and AFLD in a rat model. METHODS: We fed either Lieber- DeCarli liquid control or alcohol-containing (35 % as calories) diet (AFLD model) or lean or high-fat (12 or 60 % derived from fat, respectively) pellets (NAFLD model) for 8-10 weeks, n = 8 in each model. Serum, hepatocytes and liver tissue were analyzed. Liver injury markers were measured in serum, triglyceride content and endocytosis (binding and internalization of (125)I- asialoorosomucoid) was measured in isolated hepatocytes, and content of selected trafficking proteins (Rab3D, Rab7 and Rab18) were determined in whole liver tissue. RESULTS: Although liver injury markers and triglyceride content were similar in both models, binding and internalization of (125)I- asialoorosomucoid was significantly impaired in the hepatocytes from AFLD, but not NAFLD, animals. In addition, protein content of the asialoglycoprotein receptor (ASGPR) and three trafficking proteins, Rab3D, Rab7and Rab18, were significantly decreased after alcohol, but not high-fat feeding. Levels of protein carbonylation, amount of glutathione stores, and lipid peroxidation were similar irrespective of the insult to the livers that resulted in fatty liver. CONCLUSION: Impairments in protein trafficking in AFLD are likely a direct result of alcohol administration, and not a function of fatty liver.
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Endocitosis/fisiología , Hígado Graso Alcohólico/metabolismo , Hepatocitos/metabolismo , Enfermedad del Hígado Graso no Alcohólico/metabolismo , ARN Mensajero/metabolismo , Vesículas Transportadoras/metabolismo , Alanina Transaminasa/metabolismo , Fosfatasa Alcalina/metabolismo , Animales , Receptor de Asialoglicoproteína/genética , Receptor de Asialoglicoproteína/metabolismo , Ácidos y Sales Biliares/metabolismo , Western Blotting , Colesterol/metabolismo , Dieta Alta en Grasa , Modelos Animales de Enfermedad , Etanol/toxicidad , Hígado Graso Alcohólico/etiología , Inmunohistoquímica , Proteínas de la Membrana/metabolismo , Perilipina-2 , Ratas , Albúmina Sérica/metabolismo , Solventes/toxicidad , Triglicéridos/metabolismo , Proteínas de Unión al GTP rab/genética , Proteínas de Unión al GTP rab/metabolismo , Proteínas de Unión al GTP rab3/genética , Proteínas de Unión al GTP rab3/metabolismo , Proteínas de Unión a GTP rab7RESUMEN
BACKGROUND: Alcoholic liver disease is manifested by the presence of fatty liver, primarily due to accumulation of hepatocellular lipid droplets (LDs). The presence of membrane-trafficking proteins (e.g., Rab GTPases) with LDs indicates that LDs may be involved in trafficking pathways known to be altered in ethanol (EtOH) damaged hepatocytes. As these Rab GTPases are crucial regulators of protein trafficking, we examined the effect EtOH administration has on hepatic Rab protein content and association with LDs. METHODS: Male Wistar rats were pair-fed Lieber-DeCarli diets for 5 to 8 weeks. Whole liver and isolated LD fractions were analyzed. Identification of LDs and associated Rab proteins was performed in frozen liver or paraffin-embedded sections followed by immunohistochemical analysis. RESULTS: Lipid accumulation was characterized by larger LD vacuoles and increased total triglyceride content in EtOH-fed rats. Rabs 1, 2, 3d, 5, 7, and 18 were analyzed in postnuclear supernatant (PNS) as well as LDs. All of the Rabs were found in the PNS, and Rabs 1, 2, 5, and 7 did not show alcohol-altered content, while Rab 3d content was reduced by over 80%, and Rab 18 also showed EtOH-induced reduction in content. Rab 3d was not found to associate with LDs, while all other Rabs were found in the LD fractions, and several showed an EtOH-related decrease (Rabs 2, 5, 7, 18). Immunohistochemical analysis revealed the enhanced content of a LD-associated protein, perilipin 2 (PLIN2) that was paralleled with an associated decrease of Rab 18 in EtOH-fed rat sections. CONCLUSIONS: Chronic EtOH feeding was associated with increased PLIN2 and altered Rab GTPase content in enriched LD fractions. Although mechanisms driving these changes are not established, further studies on intracellular protein trafficking and LD biology after alcohol administration will likely contribute to our understanding of fatty liver disease.
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Depresores del Sistema Nervioso Central/química , Etanol/química , Metabolismo de los Lípidos/efectos de los fármacos , Lípidos/química , Solventes/química , Proteínas de Unión al GTP rab/química , Alanina Transaminasa/sangre , Animales , Aspartato Aminotransferasas/sangre , Western Blotting , Dieta , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Inmunohistoquímica , Hígado/citología , Hígado/efectos de los fármacos , Hígado/metabolismo , Masculino , Proteínas de la Membrana/metabolismo , Tamaño de la Partícula , Perilipina-2 , Ratas , Ratas Wistar , Triglicéridos/metabolismoRESUMEN
Excessive alcohol consumption presents considerable health risks in humans. A variety of morphologic and functional changes contribute to hepatic injury produced by heavy drinking. The present review summarizes the current knowledge of alcohol-induced liver disease and describes preclinical experimental approaches used to understand alcoholic liver disease (ALD), with a particular emphasis on impaired protein and lipid trafficking, disruption of proteolysis and autophagy, alterations in methionine metabolism and perturbations in metabolic signaling that cause dysfunctional gene expression and the eventual formation of aggresomal Mallory-Denk bodies (MDB) in liver cells. These changes eventually lead to some of the more severe hepatic impairments, including alcoholic hepatitis and fibrosis. Moreover the misuse of alcohol contributes to immune dysfunction and inadequate immune response to viral infections.
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Modelos Animales de Enfermedad , Hepatopatías Alcohólicas/patología , Investigación Biomédica Traslacional , Animales , Humanos , Hepatopatías Alcohólicas/metabolismoRESUMEN
Peroxisome-proliferator-activated receptor gamma (PPARγ) is a transcription factor with adipogenic, insulin-sensitizing, and antifibrotic properties. Strong PPARγ activators, such as the thiazolidinediones, can induce unwanted effects such as edema, weight gain, and bone loss, and therefore selective modulators of PPARγ are in development. We previously reported that one selective PPARγ modulator, SR1664, reduced toxin-induced hepatic fibrosis and the activation of hepatic stellate cells (HSCs), the main collagen-producing liver cell in fibrosis. In this study, we used a high fat and high carbohydrate (HFHC) model of hepatic steatosis and fibrosis to determine the effect of SR1664. Mice were placed on a standard chow or HFHC diet for 16 weeks, with SR1664 or control treatment for the final 4 weeks. SR1664 did not alter weight gain or fasting insulin or glucose levels. The size of lipid droplets in the HFHC group was reduced by SR1664, but there was no effect on total liver triglyceride levels. The degree of fibrosis was significantly reduced by SR1664 in mice on the HFHC diet, and this was accompanied by a decrease in activated HSC. In summary, SR1664 improved insulin sensitivity and reduced fibrosis in the HFHC diet, suggesting selective PPARγ modulation is effective in obesity-related liver fibrosis.
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BACKGROUND: Chronic ethanol exposure leads to enhanced protein acetylation and acetaldehyde adduction. Of the multitude of proteins that are modified on ethanol administration, tubulin is among the best studied. However, an open question is whether these modifications are observed in patient samples. Both modifications have also been implicated in promoting alcohol-induced defects in protein trafficking, but whether they do so directly is also unanswered. METHODS AND RESULTS: We first confirmed that tubulin was hyperacetylated and acetaldehyde-adducted in the livers from ethanol-exposed individuals to a similar extent as observed in the livers from ethanol-fed animals and hepatic cells. Livers from individuals with nonalcohol-associated fatty liver showed modest increases in tubulin acetylation, whereas nonalcohol-associated fibrotic human and mouse livers showed virtually no tubulin modifications. We also asked whether tubulin acetylation or acetaldehyde adduction can directly explain the known alcohol-induced defects in protein trafficking. Acetylation was induced by overexpressing the α-tubulin-specific acetyltransferase, αTAT1, whereas adduction was induced by directly adding acetaldehyde to cells. Both αTAT1 overexpression and acetaldehyde treatment significantly impaired plus-end (secretion) and minus-end (transcytosis)-directed microtubule-dependent trafficking and clathrin-mediated endocytosis. Each modification led to similar levels of impairment as observed in ethanol-treated cells. The levels of impairment by either modification showed no dose dependence or no additive effects suggesting that substoichiometric tubulin modifications lead to altered protein trafficking and that lysines are not selectively modified. CONCLUSIONS: These results not only confirm that enhanced tubulin acetylation is observed in human livers but that it is most relevant to alcohol-induced injury. Because these tubulin modifications are associated with altered protein trafficking that alters proper hepatic function, we propose that changing the cellular acetylation levels or scavenging free aldehydes are feasible strategies for treating alcohol-associated liver disease.
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Hepatopatías Alcohólicas , Tubulina (Proteína) , Ratones , Animales , Humanos , Tubulina (Proteína)/metabolismo , Etanol/farmacología , Hepatopatías Alcohólicas/metabolismo , Acetaldehído/metabolismo , Procesamiento Proteico-Postraduccional , Transporte de ProteínasRESUMEN
Alcohol-associated liver disease (AALD) is a major cause of liver disorders worldwide. Current treatment options are limited, especially for AALD-associated fibrosis. Promising approaches include RNA interference for miR-155 overexpression in Kupffer cells (KCs), as well as the use of CXCR4 antagonists that inhibit the activation of hepatic stellate cells (HSCs) through the CXCL12/CXCR4 axis. The development of dual-functioning nanoparticles for the effective delivery of antifibrotic RNA together with a CXCR4 inhibitor thus promises to improve the treatment of AALD fibrosis. In this study, cholesterol-modified polymeric CXCR4 inhibitor (Chol-PCX) was synthesized and used to encapsulate anti-miR-155 or non-coding (NC) miRNA in the form of Chol-PCX/miRNA nanoparticles. The results indicate that the nanoparticles induce a significant miR-155 silencing effect both in vitro and in vivo. Treatment with the Chol-PCX/anti-miR-155 particles in a model of moderate alcohol consumption with secondary liver insult resulted in a significant reduction in aminotransferase enzymes as well as collagen content in the liver parenchyma. Overall, our data support the use of Chol-PCX as a carrier for anti-miR-155 for the combined therapeutic inhibition of CXCR4 and miR-155 expression as a way to improve fibrotic damage in the liver.
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BACKGROUND: Excessive alcohol consumption leads to the increased extracellular matrix deposition of cellular fibronectin (cFn) in the liver, which is also implicated as an initiating event in the fibrogenic process. We propose that cFn directly stimulates Kupffer cells (KCs), which are involved in the early response to tissue damage, to produce factors that enhance the progression of alcohol-induced liver injury toward inflammation and fibrosis. METHOD: KCs were isolated from rats fed a control or ethanol liquid diet for 4 to 6 weeks. The effect of exogenous cFn on KC viability and the secretion of the cytokines, TNF-α and IL-6, as well as of matrix remodeling factors, MMP-2 and TIMP-2, was determined after 20 hours of cell culture. RESULTS: For KCs from both control- and ethanol-fed rats, viability remained unaffected by treatment with cFn. TNF-α and IL-6 production were increased in KCs exposed to cFn, with cells treated with 1, 2.5, and 5 µg/ml cFn secreting significantly higher levels of both cytokines compared with untreated cells (p < 0.05). Chronic ethanol administration resulted in a significantly enhanced secretion of IL-6 by KCs regardless of treatment with cFn. When MMP-2 protein and activity levels were measured by western blot analysis and gelatin zymography, respectively, we found that cFn stimulated a dramatic increase in both cells from ethanol- and control-fed rats, with the KCs from ethanol animals being more responsive to cFn at higher concentrations (p < 0.05). Significantly higher levels of TIMP-2, which inhibits both the activation and activity of MMP-2, were secreted by KCs treated with 5 µg/ml cFn. Correspondingly, more pro-MMP-2 than active-MMP-2 was detected. CONCLUSION: Altogether, these results show that cFn stimulates KCs to produce factors that may enhance the promotion of tissue damage and that ethanol administration increases these responses.
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Depresores del Sistema Nervioso Central/toxicidad , Etanol/toxicidad , Fibronectinas/fisiología , Inflamación/inducido químicamente , Macrófagos del Hígado/efectos de los fármacos , Macrófagos del Hígado/fisiología , Hígado/fisiopatología , Animales , Caspasa 3/metabolismo , Técnicas de Cultivo de Célula , Supervivencia Celular/efectos de los fármacos , Depresores del Sistema Nervioso Central/metabolismo , Depresores del Sistema Nervioso Central/farmacología , Etanol/metabolismo , Etanol/farmacología , Matriz Extracelular/fisiología , Inflamación/fisiopatología , Interleucina-6/metabolismo , Macrófagos del Hígado/citología , Macrófagos del Hígado/patología , Hígado/citología , Masculino , Metaloproteinasa 2 de la Matriz , Ratas , Ratas Wistar , Inhibidor Tisular de Metaloproteinasa-2/metabolismo , Factor de Necrosis Tumoral alfaRESUMEN
The aberrant use of alcohol is a major factor in cancer progression and metastasis. Contributing mechanisms include the systemic effects of alcohol and the exchange of bioactive molecules between cancerous and non-cancerous cells along the brain-gut-liver axis. Such interplay leads to changes in molecular, cellular, and biological functions resulting in cancer progression. Recent investigations have examined the role of extracellular vesicles (EVs) in cancer mechanisms in addition to their contribution as diagnostic biomarkers. Also, EVs are emerging as novel cell-free mediators in pathophysiological scenarios including alcohol-mediated gut microbiome dysbiosis and the release of nanosized EVs into the circulatory system. Interestingly, EVs in cancer patients are enriched with oncogenes, miRNA, lipids, and glycoproteins whose delivery into the hepatic microenvironment may be enhanced by the detrimental effects of alcohol. Proof-of-concept studies indicate that alcohol-associated liver disease is impacted by the effects of exosomes, including altered immune responses, reprogramming of stromal cells, and remodeling of the extracellular matrix. Moreover, the culmination of alcohol-related changes in the liver likely contributes to enhanced hepatic metastases and poor outcomes for cancer patients. This review summarizes the numerous aspects of exosome communications between organs with emphasis on the relationship of EVs in alcohol-associated diseases and cancer metastasis. The potential impact of EV cargo and release along a multi-organ axis is highly relevant to the promotion of tumorigenic mechanisms and metastatic disease. It is hypothesized that EVs target recipient tissues to initiate the formation of prometastatic niches and cancer progression. The study of alcohol-associated mechanisms in metastatic cancers is expected to reveal a better understanding of factors involved in the growth of secondary malignancies as well as novel approaches for therapeutic interventions.
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Neoplasias Hepáticas , MicroARNs , Comunicación Celular , Comunicación , Humanos , Oncogenes , Microambiente TumoralRESUMEN
Hepatic fibrosis is the accumulation of excess collagen as a result of chronic liver injury. If left unabated, hepatic fibrosis can lead to the disruption of the liver architecture, portal hypertension, and increased risk of progression to cirrhosis and hepatocellular carcinoma. The thiazolidinedione class of antidiabetic drugs, through their target peroxisome proliferator-activated receptor γ (PPARγ), have protective effects against liver fibrosis, and can inhibit the profibrotic activity of hepatic stellate cells, the major collagen-producing liver cells. However, these drugs have been ineffective in the treatment of established fibrosis, possibly due to side effects such as increased weight and adiposity. Recently, selective PPARγ modulators that lack these side effects have been identified, but their role in treating fibrosis has not been studied. In this study, we tested the effectiveness of one of these selective modulators, SR1664, in the mouse carbon tetrachloride model of established hepatic fibrosis. Treatment with SR1664 reduced the total and type 1 collagen content without increasing body weight. The abundance of activated hepatic stellate cells was also significantly decreased. Finally, SR1664 inhibited the profibrotic phenotype of hepatic stellate cells. In summary, a selective PPARγ modulator was effective in the reduction of established hepatic fibrosis and the activated phenotype of hepatic stellate cells. This may represent a new treatment approach for hepatic fibrosis.
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BACKGROUND: Work from our laboratory has shown that the ethanol-induced increase in apoptotic hepatocellular death is closely related to the impairment in the ability of the asialoglycoprotein receptor (ASGP-R) to remove neighboring apoptotic cells. In this study, we assessed the role of ASGP-R in fulminant liver failure and investigated whether prior treatment with betaine (a naturally occurring tertiary amine) is protective. METHODS: Lipopolysaccharide (LPS; 50 µg/kg BW) and galactosamine (GalN; 350 mg/kg BW) were injected together to wild-type and ASGP-R-deficient mice that were treated for two weeks prior with or without 2% betaine in drinking water. The mice were sacrificed 1.5, 3, or 4.5 h post-injection, and tissue samples were collected. RESULTS: LPS/GalN injection generate distinct molecular processes, which includes increased production of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), thus causing apoptosis as evident by increased caspase-3 activity. ASGP-R deficient animals showed increased liver caspase activities, serum TNF-α and IL-6 levels, as well as more pronounced liver damage compared with the wild-type control animals after intraperitoneal injection of LPS/GalN. In addition, prior administration of betaine was found to significantly attenuate the LPS/GalN-induced increases in liver injury parameters. CONCLUSION: Our work underscores the importance of normal functioning of ASGP-R in preventing severe liver damage and signifies a therapeutic role of betaine in prevention of liver injuries from toxin-induced fulminant liver failure.
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We employed a proteomic profiling strategy to examine the effects of ethanol and betaine diet supplementation on major liver protein level changes. Male Wistar rats were fed control, ethanol or betaine supplemented diets for 4 weeks. Livers were removed and liver cytosolic proteins resolved by one-dimensional and two-dimensional separation techniques. Significant upregulation of betaine homocysteine methyltransferase-1, methionine adenosyl transferase-1, and glycine N-methyltransferase were the most visually prominent protein changes observed in livers of rats fed the betaine supplemented ethanol diet. We hypothesise that this concerted upregulation of these methionine metabolic pathway enzymes is the protective mechanism by which betaine restores a normal metabolic ratio of liver S-adenosylmethionine to S-adenosylhomocysteine. Ethanol also induced significant downregulation of carbonic anhydrase-III protein levels which was not restored by betaine supplementation. Carbonic anhydrase-III can function to resist oxidative stress, and we therefore hypothesise that carbonic anhydrase-III protein levels compromised by ethanol consumption, contribute to ethanol-induced redox stress.
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Betaína/administración & dosificación , Etanol/toxicidad , Hepatopatías Alcohólicas/enzimología , Hígado/efectos de los fármacos , Metionina/metabolismo , Proteómica , Animales , Anhidrasa Carbónica III/metabolismo , Regulación hacia Abajo , Etanol/antagonistas & inhibidores , Glicina N-Metiltransferasa/metabolismo , Homocisteína S-Metiltransferasa/metabolismo , Hígado/enzimología , Masculino , Metionina Adenosiltransferasa/metabolismo , Estrés Oxidativo/efectos de los fármacos , Ratas , Ratas Wistar , Regulación hacia ArribaRESUMEN
Hepatocyte apoptosis, inflammation, and fibrosis are prominent features of liver disease in general and of alcoholic liver injury in particular. Although the link between these processes remains unclear, one universal characteristic of liver injury is the induction of hepatocellular damage, which results in the generation of apoptotic bodies. Work from our laboratory over the last several years has studied the effect of ethanol administration on the process of apoptosis and a role for altered endocytosis in alcoholic apoptosis. We initially focused our research on the hepatocyte by examining endocytosis using the asialoglycoprotein receptor (ASGP-R) pathway as a model and we identified multiple ethanol-induced impairments in receptor function. We also showed that uptake of apoptotic bodies is impaired in hepatocytes isolated from ethanol-fed animals compared to controls, and that this impairment is linked to altered ASGP-R function. Recent work from our laboratory is examining a link between ethanol-impaired ASGP-R function, apoptotic body accumulation, and inflammation in the liver. We are particularly interested in data showing that factors produced by Kupffer cells incubated with apoptotic bodies can lead to production of tumor necrosis factor-alpha and interleukin-6, and that this effect is exacerbated in the setting of alcohol administration. In addition, we have preliminary data showing that media from Kupffer cell cultures incubated with apoptotic bodies can induce hepatocyte killing. The goal of our future work is to show that inadequate removal of apoptotic cells, in part via altered receptor-mediated endocytosis, plays a role in the course of pathogenesis of alcoholic liver injury.
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Apoptosis/efectos de los fármacos , Endocitosis/efectos de los fármacos , Endocitosis/fisiología , Etanol/farmacología , Hepatocitos/efectos de los fármacos , Hepatocitos/fisiología , Macrófagos del Hígado/efectos de los fármacos , Animales , Macrófagos del Hígado/fisiologíaRESUMEN
Chronic ethanol consumption is associated with serious and potentially fatal alcohol-related liver injuries such as hepatomegaly, alcoholic hepatitis and cirrhosis. Moreover, it has been documented that the clinical progression of alcohol-induced liver damage may be associated with an increase in hepatocellular death that involves apoptotic mechanisms. Although much information has been learned about the clinical manifestations associated with alcohol-related diseases, the search continues for a better understanding of the molecular and/or cellular mechanisms by which ethanol exerts its deleterious effects such as the induction of pro-apoptotic mechanisms and related cell damaging events. As part of the effort to enhance our understanding of those particular cellular pathways and mechanisms associated with ethanol toxicity, researchers over the years have utilized a variety of model systems. Recently, work has come forth demonstrating the utility of a hybrid cell line (WIF-B) as a cell culture model system for the study of alcohol-associated alterations in hepatocellular mechanisms. Success with such emerging model systems could aid in the development of potential therapeutic treatments for the prevention of alcohol-induced apoptotic cell death that may ultimately serve as a significant target in delaying the onset and/or progression of clinical symptoms of alcohol-mediated liver disease. This review article summarizes the current understanding of ethanol-mediated modifications in cell survival and thus the promotion of pro-apoptotic events with emphasis on analyses made in various experimental model systems, particularly the more recently characterized WIF-B cell system.
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Apoptosis/efectos de los fármacos , Depresores del Sistema Nervioso Central/farmacología , Etanol/farmacología , Animales , Línea Celular , Quimera , Humanos , Hígado/efectos de los fármacos , Hígado/patología , Modelos Biológicos , RatasRESUMEN
Fibrosis is a major player in cardiovascular disease, both as a contributor to the development of disease, as well as a post-injury response that drives progression. Despite the identification of many mechanisms responsible for cardiovascular fibrosis, to date no treatments have emerged that have effectively reduced the excess deposition of extracellular matrix associated with fibrotic conditions. Novel treatments have recently been identified that hold promise as potential therapeutic agents for cardiovascular diseases associated with fibrosis, as well as other fibrotic conditions. The purpose of this review is to provide an overview of emerging antifibrotic agents that have shown encouraging results in preclinical or early clinical studies, but have not yet been approved for use in human disease. One of these agents is bone morphogenetic protein-7 (BMP7), which has beneficial effects in multiple models of fibrotic disease. Another approach discussed involves altering the levels of micro-RNA (miR) species, including miR-29 and miR-101, which regulate the expression of fibrosis-related gene targets. Further, the antifibrotic potential of agonists of the peroxisome proliferator-activated receptors will be discussed. Finally, evidence will be reviewed in support of the polypeptide hormone relaxin. Relaxin is long known for its extracellular remodeling properties in pregnancy, and is rapidly emerging as an effective antifibrotic agent in a number of organ systems. Moreover, relaxin has potent vascular and renal effects that make it a particularly attractive approach for the treatment of cardiovascular diseases. In each case, the mechanism of action and the applicability to various fibrotic diseases will be discussed.
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For more than 30 years, lipid droplets (LDs) were considered as an inert bag of lipid for storage of energy-rich fat molecules. Following a paradigm shift almost a decade ago, LDs are presently considered an active subcellular organelle especially designed for assembling, storing and subsequently supplying lipids for generating energy and membrane synthesis (and in the case of hepatocytes for VLDL secretion). LDs also play a central role in many other cellular functions such as viral assembly and protein degradation. Here, we have explored the structural and functional changes that occur in hepatic and adipose tissue LDs following chronic ethanol consumption in relation to their role in the pathogenesis of alcoholic liver injury.
Asunto(s)
Tejido Adiposo/metabolismo , Gotas Lipídicas/metabolismo , Hepatopatías Alcohólicas/metabolismo , Tejido Adiposo/patología , Animales , Autofagia , Hepatocitos/metabolismo , Hepatocitos/patología , Humanos , Metabolismo de los Lípidos , Hígado/metabolismo , Hígado/patología , Hepatopatías Alcohólicas/patología , Proteolisis , Transducción de SeñalRESUMEN
Metastatic liver disease is a major cause of mortality in colorectal cancer (CRC) patients. Alcohol consumption is a noted risk factor for secondary cancers yet the role of alcoholic liver disease (ALD) in colorectal liver metastases (CRLM) is not defined. This work evaluated tumor cell colonization in the alcoholic host liver using a novel preclinical model of human CRC liver metastases. Immunocompromised Rag1-deficient mice were fed either ethanol (E) or isocaloric control (C) diets for 4 weeks prior to intrasplenic injection of LS174T human CRC cells. ALD and CRLM were evaluated 3 or 5 weeks post-LS174T cell injection with continued C/E diet administration. ALD was confirmed by increased serum transaminases, hepatic steatosis and expression of cytochrome P4502E1, a major ethanol-metabolizing enzyme. Alcohol-mediated liver dysfunction was validated by impaired endocytosis of asialoorosomucoid and carcinoembryonic antigen (CEA), indicators of hepatocellular injury and progressive CRC disease, respectively. Strikingly, the rate and burden of CRLM was distinctly enhanced in alcoholic livers with metastases observed earlier and more severely in E-fed mice. Further, alcohol-related increases (1.5-3.0 fold) were observed in the expression of hepatic cytokines (TNF-α, IL-1 beta, IL-6, IL-10) and other factors noted to be involved in the colonization of CRC cells including ICAM-1, CCL-2, CCL-7, MMP-2, and MMP-9. Also, alcoholic liver injury was associated with altered hepatic localization as well as increased circulating levels of CEA released from CRC cells. Altogether, these findings indicate that the alcoholic liver provides a permissive environment for the establishment of CRLM, possibly through CEA-related inflammatory mechanisms.
Asunto(s)
Neoplasias Colorrectales/patología , Hepatopatías Alcohólicas/complicaciones , Neoplasias Hepáticas Experimentales/secundario , Animales , Línea Celular Tumoral , Citocromo P-450 CYP2E1/biosíntesis , Citocromo P-450 CYP2E1/genética , Citocinas/biosíntesis , Citocinas/genética , Endocitosis , Inducción Enzimática , Etanol/toxicidad , Hepatocitos/metabolismo , Hepatocitos/patología , Xenoinjertos , Proteínas de Homeodominio/genética , Humanos , Huésped Inmunocomprometido , Neoplasias Hepáticas Experimentales/etiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/genética , Trasplante de NeoplasiasRESUMEN
A potential in vitro model for studying the mechanisms of alcohol-induced hepatocyte injury is the WIF-B cell line. It has many hepatocyte-like features, including a differentiated, polarized phenotype resulting in formation of bile canaliculi. The aim of this study was to examine the effects of ethanol treatment on this cell line. WIF-B cells were cultured up to 96 h in the absence or presence of 25 mM ethanol and subsequently were analyzed for ethanol-induced physiological and morphological changes. Initial studies revealed WIF-B cells exhibited alcohol dehydrogenase (ADH) activity, expressed cytochrome p4502E1 (CYP2E1), and efficiently metabolized ethanol in culture. This cell line also produced the ethanol metabolite acetaldehyde and exhibited low K(m) aldehyde dehydrogenase (ALDH) activity, comparable to hepatocytes. Ethanol treatment of the WIF-B cells for 48 h led to significant increases in the lactate/pyruvate redox ratio and cellular triglyceride levels. Ethanol treatment also significantly altered WIF-B morphology, decreasing the number of bile canaliculi, increasing the number of cells exhibiting finger-like projections, and increasing cell diameter. The ethanol-induced changes occurring in this cell line were negated by addition of the ADH inhibitor, 4-methylpyrazole (4-MP), indicating the effects were due to ethanol metabolism. In summary, the WIF-B cell line metabolizes ethanol and exhibits many ethanol-induced changes similar to those found in hepatocytes. Because of these similarities, WIF-B cells appear to be a suitable model for studying ethanol-induced hepatocyte injury.