Zoltan Derdak

The Mitochondrial Uncoupling Protein-2 Promotes Chemoresistance in Cancer Cells

Cancer cells acquire drug resistance as a result of selection pressure dictated by unfavorable microenvironments. This survival process is facilitated through efficient control of oxidative stress originating from mitochondria that typically initiates programmed cell death. We show this critical adaptive response in cancer cells to be linked to uncoupling protein-2 (UCP2), a mitochondrial suppressor of reactive oxygen species (ROS). UCP2 is present in drug-resistant lines of various cancer cells and in human colon cancer. Overexpression of UCP2 in HCT116 human colon cancer cells inhibits ROS accumulation and apoptosis after exposure to chemotherapeutic agents. Tumor xenografts of UCP2-overexpressing HCT116 cells retain growth in nude mice receiving chemotherapy. Augmented cancer cell survival is accompanied by altered NH(2)-terminal phosphorylation of the pivotal tumor suppressor p53 and induction of the glycolytic phenotype (Warburg effect). These findings link UCP2 with molecular mechanisms of chemoresistance. Targeting UCP2 may be considered a novel treatment strategy for cancer.

Inhibition of p53 attenuates steatosis and liver injury in a mouse model of non-alcoholic fatty liver disease

BACKGROUND & AIMS p53 and its transcriptional target miRNA34a have been implicated in the pathogenesis of fatty liver. We tested the efficacy of a p53 inhibitor, pifithrin-α p-nitro (PFT) in attenuating steatosis, associated oxidative stress and apoptosis in a murine model of non-alcoholic fatty liver disease (NAFLD). METHODS C57BL/6 mice were fed a high-fat (HFD) or control diet for 8 weeks; PFT or DMSO (vehicle) was administered three times per week. Markers of oxidative stress and apoptosis as well as mediators of hepatic fatty acid metabolism were assessed by immunohistochemistry, Western blot, real-time PCR, and biochemical assays. RESULTS PFT administration suppressed HFD-induced weight gain, ALT elevation, steatosis, oxidative stress, and apoptosis. PFT treatment blunted the HFD-induced upregulation of miRNA34a and increased SIRT1 expression. In the livers of HFD-fed, PFT-treated mice, activation of the SIRT1/PGC1α/PPARα axis increased the expression of malonyl-CoA decarboxylase (MLYCD), an enzyme responsible for malonyl-CoA (mCoA) degradation. Additionally, the SIRT1/LKB1/AMPK pathway (upstream activator of MLYCD) was promoted by PFT. Thus, induction of these two pathways by PFT diminished the hepatic mCoA content by enhancing MLYCD expression and function. Since mCoA inhibits carnitine palmitoyltransferase 1 (CPT1), the decrease of hepatic mCoA in the PFT-treated, HFD-fed mice increased CPT1 activity, favored fatty acid oxidation, and decreased steatosis. Additionally, we demonstrated that PFT abrogated steatosis and promoted MLYCD expression in palmitoleic acid-treated human HepaRG cells. CONCLUSIONS The p53 inhibitor PFT diminished hepatic triglyceride accumulation and lipotoxicity in mice fed a HFD, by depleting mCoA and favoring the β-oxidation of fatty acids.

Uncoupling protein‐2 deficiency promotes oxidant stress and delays liver regeneration in mice

The control of liver regeneration remains elusive. Because reactive oxygen species (ROS) are able to mediate cell growth arrest and activate proteins that inhibit the cell cycle, ROS production may have a negative impact on liver regeneration. We examined how liver regeneration is affected by uncoupling protein‐2 (UCP2), an inner mitochondrial membrane carrier that senses and negatively regulates superoxide production. Liver regeneration was monitored up to 5 days and was found to be significantly delayed in UCP2−/− mice after partial hepatectomy. Apoptosis rates in UCP2+/+ and UCP2− /− liver remnants were similar, while parameters of cell proliferation indicated a diminished response in UCP2− /− mice with corresponding changes in the expression of key cell cycle regulatory proteins and prolonged activation of stress‐responsive protein kinase p38. Levels of malondialdehyde, a marker of ROS generation and oxidant stress, were elevated in UCP2− /− livers at every examined time point. Liver remnants of UCP2+ /+ mice 48 hours post‐hepatectomy showed a fourfold increase in the expression of UCP2 protein primarily detected in hepatocytes. In conclusion, our results suggest that absent or insufficient UCP2 function in the regenerating liver results in increased ROS production and negatively modulates the control of cell cycle. (HEPATOLOGY 2004;39:386–392.)

Obesity-associated mechanisms of hepatocarcinogenesis

Obesity has been recognized as a key component of the metabolic syndrome, a cluster of risk factors associated with diabetes and cardiovascular morbidity. In addition, obesity has been linked to higher frequency of cancers in a variety of tissues including the liver. Liver cancer most often occurs as hepatocellular carcinoma (HCC) complicating cirrhosis due to chronic viral infection or toxic injury and remains the third leading cause of cancer death in the world. However, HCC is increasingly diagnosed among individuals with obesity and related disorders. As these metabolic conditions have become globally prevalent, they coexist with well-established risk factors of HCC and create a unique challenge for the liver as a chronically diseased organ. Obesity-associated HCC has recently been attributed to molecular mechanisms such as chronic inflammation due to adipose tissue remodeling and pro-inflammatory adipokine secretion, ectopic lipid accumulation and lipotoxicity, altered gut microbiota, and disrupted senescence in stellate cells, as well as insulin resistance leading to increased levels of insulin and insulin-like growth factors. These mechanisms synergize with those occurring in chronic liver disease resulting from other etiologies and accelerate the development of HCC before or after the onset of cirrhosis. Increasingly common interactions between oncogenic pathways linked to obesity and chronic liver disease may explain why HCC is one of the few malignancies with rising incidence in developed countries. Better understanding of this complex process will improve our strategies of cancer prevention, prediction, and surveillance.

Activation of p53 enhances apoptosis and insulin resistance in a rat model of alcoholic liver disease

BACKGROUND & AIMS Chronic ethanol consumption in the Long-Evans (LE) rat has been associated with hepatic p53 activation, and inhibition of the insulin/PI3K/AKT signal transduction cascade due to increased expression of PTEN. We hypothesize that p53 activation and altered insulin signaling may influence the susceptibility of rats to ethanol-induced liver damage. Furthermore, p53 not only activates programmed cell death pathways and suppresses hepatocellular survival signals, but also promotes gluconeogenesis to increase systemic insulin resistance due to a novel metabolic function. METHODS Fischer (F), Sprague-Dawley (SD) and LE rats were fed ethanol-containing or control liquid diet for 8 weeks. Histopathological and biochemical changes were assessed. RESULTS Here, we demonstrate that chronic ethanol feeding in rats promotes p53 activation, hepatic steatosis, oxidative stress, PUMA, and PTEN expression, which contribute to hepatocellular death and diminished insulin signaling in the liver. Such changes are pronounced in the LE, less prominent in SD, and virtually absent in the F rat strain. More importantly, there is activation of Tp53-induced glycolysis and apoptosis regulator (TIGAR) in the ethanol-fed LE rat. This event generates low hepatic fructose-2,6-bisphosphate (Fru-2,6-P₂) levels, reduced lactate/pyruvate ratio and may contribute to increased basal glucose turnover and high residual hepatic glucose production during euglycemic hyperinsulinemic clamp. CONCLUSIONS p53 activation correlates with the susceptibility to ethanol-induced liver damage in different rat strains. p53 not only orchestrates apoptosis and suppresses cell survival, but by activating TIGAR and decreasing hepatic Fru-2,6-P₂) levels it promotes insulin resistance and therefore, contributes to the metabolic abnormalities associated with hepatic steatosis.

Nanomaterials for X-ray Imaging: Gold Nanoparticle-Enhancement of X-ray Scatter Imaging of Hepatocellular Carcinoma

We present the development of a new imaging technique for the early diagnosis of hepatocellular carcinoma that utilizes surface-modified gold nanoparticles in combination with X-ray imaging. Tissues labeled with these electron-dense particles show enhanced X-ray scattering over normal tissues, distinguishing cells containing gold nanoparticles from cells without gold in X-ray scatter images. Our results suggest that this novel approach could enable the in vivo detection of tumors as small as a few millimeters in size.

Alcohol, insulin resistance and the liver-brain axis.

Chronic alcohol exposure inhibits insulin and insulin‐like growth factor signaling in the liver and brain by impairing the signaling cascade at multiple levels. These alterations produced by alcohol cause severe hepatic and central nervous system insulin resistance as the cells fail to adequately transmit signals downstream through Erk/mitogen‐activated protein kinase (MAPK), which is needed for DNA synthesis and liver regeneration, and phosphatidylinositol 3‐kinase (PI3K), which promotes growth, survival, cell motility, glucose utilization, plasticity, and energy metabolism. The robust inhibition of insulin signaling in liver and brain is augmented by additional factors involving the activation of phosphatases such as phosphatase and tensin homologue (PTEN), which further impairs insulin signaling through PI3K/Akt. Thus, intact insulin signaling is important for neuronal survival. Chronic alcohol consumption produces steatohepatitis, which also promotes hepatic insulin resistance, oxidative stress and injury, with the attendant increased generation of “toxic lipids” such as ceramides that increase insulin resistance. The PI3K/Akt signaling cascade is altered by direct interaction with ceramides as well as through PTEN upregulation as a downstream target gene of enhanced p53 transcriptional activity. Cytotoxic ceramides transferred from the liver to the blood can enter the brain due to their lipid‐soluble nature, and thereby exert neurodegenerative effects via a liver–brain axis. We postulate that the neurotoxic and neurodegenerative effects of liver‐derived ceramides activate pro‐inflammatory cytokines and increase lipid adducts and insulin resistance in the brain to impair cognitive and motor function. These observations are discussed in the context of insulin sensitizers as potential cytoprotective agents against liver and brain injury induced by alcohol.

Lack of UCP2 reduces Fas-mediated liver injury in ob/ob mice and reveals importance of cell-specific UCP2 expression

Fatty liver is vulnerable to conditions that challenge hepatocellular energy homeostasis. Lipid‐laden hepatocytes highly express uncoupling protein‐2 (UCP2), a mitochondrial carrier that competes with adenosine triphosphate (ATP) synthesis by mediating proton leak. However, evidence for a link between UCP2 expression and susceptibility of liver to acute injury is lacking. We asked whether absence of UCP2 protects ob/ob mice from Fas‐mediated acute liver damage. UCP2‐deficient ob/ob mice (ob/ob:ucp2−/−) and UCP2‐competent littermates (ob/ob:ucp2+/+) received a single dose of agonistic anti‐Fas antibody (Jo2). Low‐dose Jo2 (0.15 mg/kg intraperitoneally) caused less serum alanine aminotransferase (ALT) elevation and lower apoptosis rates in ob/ob:ucp2−/− mice. High‐dose Jo2 (0.40 mg/kg intraperitoneally) proved uniformly fatal; however, ob/ob:ucp2−/− mice survived longer with less depletion of liver ATP stores, indicating that fatty hepatocytes may benefit from lack of UCP2 during Jo2 challenge. Although UCP2 reportedly controls mitochondrial oxidant production, its absence had no apparent effect on fatty liver tissue malondialdehyde levels augmented by Jo2. This finding prompted us to determine UCP2 expression in Kupffer cells, a major source of intrahepatic oxidative stress. UCP2 expression was found diminished in Kupffer cells of untreated ob/ob:ucp2+/+ mice, conceivably contributing to increased oxidative stress in fatty liver and limiting the impact of UCP2 ablation. In conclusion, whereas UCP2 abundance in fatty hepatocytes exacerbates Fas‐mediated injury by compromising ATP stores, downregulation of UCP2 in Kupffer cells may account for persistent oxidative stress in fatty liver. Our data support a cell‐specific approach when considering the therapeutic effects of mitochondrial uncoupling in fatty liver disease. (HEPATOLOGY 2006;44:592–601.)

Low density contrast agents for x-ray phase contrast imaging: the use of ambient air for x-ray angiography of excised murine liver tissue

We report a new preparative method for providing contrast through reduction in electron density that is uniquely suited for propagation-based differential x-ray phase contrast imaging. The method, which results in an air or fluid filled vasculature, makes possible visualization of the smallest microvessels, roughly down to 15 microm, in an excised murine liver, while preserving the tissue for subsequent histological workup. We show the utility of spatial frequency filtering for increasing the visibility of minute features characteristic of phase contrast imaging, and the capability of tomographic reconstruction to reveal microvessel structure and three-dimensional visualization of the sample. The effect of water evaporation from livers during x-ray imaging on the visibility of blood vessels is delineated. The deformed vascular tree in a cancerous murine liver is imaged.

Atorvastatin effect on the distribution of high-density lipoprotein subfractions and human paraoxonase activity

Human serum paraoxonase-1 (PON1) protects lipoproteins against oxidation by hydrolyzing lipid peroxides in oxidized low-density lipoprotein (LDL); therefore, it may protect against atherosclerosis. Changes in the ratio of high-density lipoprotein (HDL) subfractions may alter the stability and the antioxidant capacity of PON1. The aim of the study was to examine the effect of atorvastatin treatment on the distribution of HDL subfractions, LDL size, cholesteryl ester transfer protein (CETP), lecithin-cholesterol acyltransferase (LCAT), and PON1 activity. In all, 33 patients with type IIa and IIb hypercholesterolemia were involved in the study. LDL sizes and HDL subfractions were determined by gradient gel electrophoresis. CETP, LCAT, and PON1 activities were measured spectrophotometrically. Three months of treatment with atorvastatin 20 mg daily significantly increased the HDL3 (+8.13%) and decreased the HDL2a and HDL2b subfractions (-1.57% and -6.55%, respectively). The mean LDL size was significantly increased (+3.29%). The level of oxidized LDL was significantly decreased (-46.0%). The PON1 activity was augmented by the atorvastatin treatment (+5.0%). The CETP activity positively correlated with the HDL2b level and negatively correlated with the HDL3 and HDL2a levels. Atorvastatin alters the HDL subfractions, which may improve its antiatherogenic effect via enhancement of the PON1 activity.