Murine cirrhosis induces hepatocyte epithelial mesenchymal transition and alterations in survival signaling pathways.

UNLABELLED: Hepatocytes that reside in a chronically-injured liver have altered growth responses compared to hepatocytes in normal liver. Transforming growth factor beta (TGFbeta) is upregulated in the cirrhotic liver, and cirrhotic hepatocytes, unlike normal hepatocytes exposed to this cytokine, exhibit decreased apoptosis. In fetal hepatocytes, TGFbeta also induces epithelial-mesenchymal transition (EMT) and signaling changes in cell survival pathways. Here, chronic murine liver injury was induced by twice-weekly carbon tetrachloride administration for 8 weeks. Normal liver-derived hepatocytes (NLDH) and cirrhotic liver-derived hepatocytes (CLDH) were examined for EMT and the small mothers against decapentaplegic homolog (Smad), phosphatidylinositol-3-kinase (PI3K/Akt), and mitogen activated protein kinase (MAPK) pathways were investigated. Immunofluorescence imaging of cirrhotic livers demonstrated increased vimentin expression, which was confirmed by immunoblot analysis. In vitro, CLDH exhibited increased vimentin and type 1 collagen expression within cellular extensions consistent with EMT. Treatment with TGFbeta augmented the EMT response in CLDH. In contrast, untreated NLDH did not display features of EMT but responded to TGFbeta with increased vimentin expression and EMT characteristics. In response to PI3K/Akt inhibition, CLDH had decreased basal and insulin-stimulated p-Akt expression and decreased apoptosis compared to NLDH. In both NLDH and CLDH, vimentin expression was dependent on PI3K/Akt activity. CLDH demonstrated increased basal p-extracellular signal-regulated kinase expression that was independent of Smad and PI3K/Akt signaling. Inhibition of the MAPK pathway produced a marked increase in CLDH apoptosis. CONCLUSION: CLDH have increased vimentin and type 1 collagen expression and morphologic features consistent with EMT. In addition, compared to NLDH, the cellular signaling phenotype of CLDH changes from a MAPK-independent pathway to a MAPK-dependent cell survival pathway. These findings may have clinical implications for chemoprevention of hepatocellular carcinoma in the cirrhotic liver.

Impaired autophagy: A mechanism of mitochondrial dysfunction in anoxic rat hepatocytes.

UNLABELLED: Autophagy selectively removes abnormal or damaged organelles such as dysfunctional mitochondria. The mitochondrial permeability transition (MPT) is a marker of impaired mitochondrial function that is evident in hepatic ischemia/reperfusion (I/R) injury. However, the relationship between mitochondrial dysfunction and autophagy in I/R injury is unknown. Cultured rat hepatocytes and mouse livers were exposed to anoxia/reoxygenation (A/R) and I/R, respectively. Expression of autophagy-related protein 7 (Atg7), Beclin-1, and Atg12, autophagy regulatory proteins, was analyzed by western blots. Some hepatocytes were incubated with calpain 2 inhibitors or infected with adenoviruses encoding green fluorescent protein (control), Atg7, and Beclin-1 to augment autophagy. To induce nutrient depletion, a condition stimulating autophagy, hepatocytes were incubated in an amino acid-free and serum-free medium for 3 hours prior to onset of anoxia. For confocal imaging, hepatocytes were coloaded with calcein and tetramethylrhodamine methyl ester to visualize onset of the MPT and mitochondrial depolarization, respectively. To further examine autophagy, hepatocytes were infected with an adenovirus expressing green fluorescent protein-microtubule-associated protein light chain 3 (GFP-LC3) and subjected to A/R. Calpain activity was fluorometrically determined with succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin. A/R markedly decreased Atg7 and Beclin-1 concomitantly with a progressive increase in calpain activity. I/R of livers also decreased both proteins. However, inhibition of calpain isoform 2, adenoviral overexpression, and nutrient depletion all substantially suppressed A/R-induced loss of autophagy proteins, prevented onset of the MPT, and decreased cell death after reoxygenation. Confocal imaging of GFP-LC3 confirmed A/R-induced depletion of autophagosomes, which was reversed by nutrient depletion and adenoviral overexpression. CONCLUSION: Calpain 2-mediated degradation of Atg7 and Beclin-1 impairs mitochondrial autophagy, and this subsequently leads to MPT-dependent hepatocyte death after A/R.

RNAi-mediated knockdown of aldehyde dehydrogenase class-1A1 and class-3A1 is specific and reveals that each contributes equally to the resistance against 4-hydroperoxycyclophosphamide.

PURPOSE: Aldehyde dehydrogenases class-1A1 (ALDH1A1) and class-3A1 (ALDH3A1) have been associated with resistance to cyclophosphamide (CP) and its derivatives. We have previously reported the downregulation of these enzymes by all-trans retinoic acid (ATRA). METHODS: In this study, we used siRNA duplexes as well as retrovirally expressed siRNA to knockdown one or both enzymes together in A549 lung cancer cell line in order to investigate the role of each one in mediating the resistance and the effect of the addition of ATRA. RESULTS: The results show that significant and specific knockdown of each enzyme can be achieved and that each one contributes similarly to cell resistance to 4-hydroperoxycyclophosphamide (4-HC), an active derivative of CP. Added effects were seen when both enzymes were inhibited. The addition of ATRA also exhibited additional inhibitory effects on ALDH activity and increased 4-HC toxicity when added to single siRNA aimed at one of the enzymes. On the other hand, ATRA had minimal and insignificant additional inhibitory effects on ALDH enzyme activity when added to a combination of siRNAs against both enzymes, but still increased 4-HC toxicity beyond that seen with RNAi-mediated inhibition of both enzymes together. CONCLUSIONS: We conclude that both enzymes, ALDH1A1 and ALDH3A1 will need to be blocked in order to achieve the highest sensitivity to 4-HC. Furthermore, ATRA increases 4-HC toxicity even when added to a combination of siRNAs against both enzymes, thus suggesting additional mechanisms by which ATRA can increase drug toxicity.

Influence of coagulation factor, vitamin K epoxide reductase complex subunit 1, and cytochrome P450 2C9 gene polymorphisms on warfarin dose requirements.

INTRODUCTION: The primary objective of this study was to determine whether variability in warfarin dose requirements is determined by common polymorphisms in genes whose products are involved in the pharmacodynamics and pharmacokinetics of warfarin, namely, the coagulation factors, vitamin K epoxide reductase complex subunit 1 (VKORC1), and cytochrome P450 (CYP) 2C9. METHODS: Patients (N = 350) receiving stable doses of warfarin at 3 consecutive visits were enrolled, and a deoxyribonucleic acid sample was collected. Samples were genotyped for polymorphisms in the factor II, factor VII, factor X, VKORC1, and CYP2C9 genes. A stepwise linear regression analysis was used to determine the independent effects of genetic and nongenetic factors on mean warfarin dose requirements. RESULTS: Variables associated with lower warfarin dose requirements were VKORC1 3673 AA genotype (P < .0001), VKORC1 3673 GA genotype (P < .0001), 1 variant CYP2C9 allele (P < .0001), 2 variant CYP2C9 alleles (P = .0004), increasing age (P = .0005), concomitant CYP2C9 inhibitors (P = .0005), and goal international normalized ratio (P = .01). Variables associated with higher warfarin dose requirements were weight (P < .0001), current smoker status (P = .0009), mean international normalized ratio (P = .001), concomitant CYP2C9 inducers (P = .006), factor X insertion/deletion genotype (P = .01), factor X insertion/insertion genotype (P = .04), factor VII deletion/deletion genotype (P = .04), and calculated vitamin K intake (P = .05). The linear regression model explained 51.4% of the variability in warfarin dose requirements. CONCLUSION: Polymorphisms in warfarin drug target and metabolizing enzyme genes, in addition to nongenetic factors, were important determinants of warfarin dose requirements.

Basal reactive oxygen species determine the susceptibility to apoptosis in cirrhotic hepatocytes.

Hepatocytes from cirrhotic murine livers exhibit increased basal ROS activity and resistance to TGFbeta-induced apoptosis, yet when ROS levels are decreased by antioxidant pretreatment, these cells recover susceptibility to apoptotic stimuli. To further study these redox events, hepatocytes from cirrhotic murine livers were pretreated with various antioxidants prior to TGFbeta treatment and the ROS activity, apoptotic response, and mitochondrial ROS generation were assessed. In addition, normal hepatocytes were treated with low-dose H(2)O(2) and ROS and apoptotic responses determined. Treatment of cirrhotic hepatocytes with various antioxidants decreased basal ROS and rendered them susceptible to apoptosis. Examination of normal hepatocytes by confocal microscopy demonstrated colocalization of ROS activity and respiring mitochondria. Basal assessment of cirrhotic hepatocytes showed nonfocal ROS activity that was abolished by antioxidants. After pretreatment with an adenovirus expressing MnSOD, basal cirrhotic hepatocyte ROS were decreased and TGFbeta-induced colocalization of ROS and mitochondrial respiration was present. Treatment of normal hepatocytes with H(2)O(2) resulted in a sustained increase in ROS and resistance to TGFbeta apoptosis that was reversed when these cells were pretreated with an antioxidant. In conclusion, cirrhotic hepatocytes have a nonfocal distribution of ROS. However, normal and cirrhotic hepatocytes exhibit mitochondrial localization of ROS that is necessary for apoptosis.

Presenilins in the heart: presenilin-2 expression is increased by low glucose and by hypoxia in cardiac cells.

Cardiac cells are subjected to hypoxia in many cardiovascular diseases. We studied a broad spectrum of genes using a macroarrays-based method to analyze RNA of rat cardiac fetal cell line H9c2 after 4 h of hypoxic conditions in the incubator-1% oxygen concentration, as compared to normoxic conditions (21% oxygen). The cDNAs were prepared from total RNAs using Atlas Rat 1.2 Array (Clontech Laboratories) and hybridized to the membrane containing 1176 rat cDNAs and 9 housekeeping control cDNAs. Genes expression was analyzed using AtlasImage 1.01 software. We found over 45 genes up-regulated in a range of 1.5-2.9 times and 9 genes down-regulated to a range of 0.4-0.7 times, under hypoxia versus normoxia. Presenilin-2 (PS2) was detected in the cultured heart cells. RT-PCR confirmed the presence of PS2 in the heart of adult rats. Using quantitative real-time RT-PCR, we further studied the expression of presenilin-2 mRNA under conditions of low oxygen supply and glucose starvation. Glucose deprivation itself caused significant up-regulation of the presenilin-2 (to 160%) and with low oxygen increased presenilin-2 level to over 200% of the control. Presenilin-2 has previously been associated with intercellular signaling in the central nervous system, in Alzheimer's disease. The finding of presenilin-2 in the heart and the responsiveness to low glucose and hypoxia suggests that PS2 may be regulated by conditions of ischemia, a condition which both the heart and brain may experience.

Liver regeneration and the atrophy-hypertrophy complex.

The atrophy-hypertrophy complex (AHC) refers to the controlled restoration of liver parenchyma following hepatocyte loss. Different types of injury (e.g., toxins, ischemia/reperfusion, biliary obstruction, and resection) elicit the same hypertrophic response in the remnant liver. The AHC involves complex anatomical, histological, cellular, and molecular processes. The signals responsible for these processes are both intrinsic and extrinsic to the liver and involve both physical and molecular events. In patients in whom resection of large liver malignancies would result in an inadequate functional liver remnant, preoperative portal vein embolization may increase the remnant liver sufficiently to permit aggressive resections. Through continued basic science research, the cellular mechanisms of the AHC may be maximized to permit curative resections in patients with potentially prohibitive liver function.

Retinoic acid down-regulates aldehyde dehydrogenase and increases cytotoxicity of 4-hydroperoxycyclophosphamide and acetaldehyde.

Multiple prior studies have identified aldehyde dehydrogenases (ALDH) that are capable of oxidizing retinal to retinoic acid. In this study, we test the hypothesis that the accumulation of intracellular retinoic acid may lead to the suppression of ALDH expression and thus increase cytotoxicity to 4-hydroperoxycyclophosphamide (4-HC) in vitro. Mainly A549, but also other lung cancer cell lines, were used in our experiments, with the former having high levels of two ALDH isozymes expressed. Dose-response and time-course experiments were performed by incubating the cells with all-trans retinoic acid (ATRA) as well as other commercially available retinoids. The results show that incubation of A549 cells with any of the retinoids at pharmacologic doses for > or =48 h results in a significant decrease in ALDH-1A1 and ALDH-3A1 enzyme activity and protein levels but not the corresponding mRNAs. Such a decrease in ALDH activity was seen in all cell lines tested and results in a significant increase in toxicity of 4-HC and acetaldehyde, both of which are substrates for the enzymes. Prior incubation with ATRA also results in increased cytotoxicity, although to a lesser degree, of phenylketophosphamide and melphalan, neither of which is a substrate for ALDHs. These results suggest a post-translational mechanism through which retinoids decrease both ALDH expression, which results in increased cytotoxicity of 4-HC and acetaldehyde, although other previously described effects of these retinoids may contribute to the slight increase in cytotoxicity seen with other chemotherapy agents. These results may have clinical implications in regard to the use of retinoids in lung cancer prevention and treatment.