Claire Legendre

Drug-metabolising enzymes are down-regulated by hypoxia in differentiated human hepatoma HepaRG cells: HIF-1α involvement in CYP3A4 repression

Weak blood irrigation within solid tumours including hepatocellular carcinomas (HCCs) plays an important role in resistance to anticancer drugs by decreasing accessibility of cytotoxic agents to tumour cells. Reduced oxygen levels, or hypoxia, also contribute to drug resistance because many anticancer drugs require molecular oxygen to be cytotoxic. Our aim was to develop a new in vitro model mimicking hypoxic cells within HCCs in order to further explore the molecular responses to hypoxia, including regulation of drug-metabolising enzymes (DMEs) expression. For this purpose, we used the highly differentiated human hepatoma HepaRG cells cultured under either normoxic or hypoxic (24h at 1% O(2)) conditions. Gene and protein expressions were investigated by quantitative PCR and immunoblotting, respectively. We showed that HepaRG cells adapt to prolonged moderate hypoxia by a switch from aerobic to anaerobic glycolysis and a repression of critical genes involved in amino acid, lipid and ethanol metabolisms. Importantly, expression of several DMEs (particularly cytochromes P450 (CYPs) and phase II enzymes) and xenosensors (CAR, PXR and AhR) was down-regulated and CYPs activities (using testosterone and paclitaxel as substrates) were decreased during hypoxia. In addition, a new role for HIF-1alpha in the repression of CYP3A4 is demonstrated in cells treated with chemical inducers of HIF-1alpha, cobalt chloride or desferrioxamine, and by transfecting untreated HepaRG cells with HIF-1alpha expression vector. In conclusion, HepaRG cells cultured under hypoxia might mimic metabolic changes occurring within poorly irrigated differentiated HCCs. Furthermore, hypoxia down-regulates hepatic DMEs, a phenomenon that might compromise chemotherapy effectiveness in HCC treatment. Thus, HepaRG cells might represent a new in vitro model to test anticancer agents in hypoxic versus normoxic conditions.

Reperfusion stress induced during intermittent selective clamping accelerates rat liver regeneration through JNK pathway.

BACKGROUND & AIMS Liver resection includes temporal vascular inflow occlusion resulting in ischemia/reperfusion injury in the remnant liver. Here, we developed a rat model of selective lobe occlusion to isolate reperfusion stress from ischemia and to analyze its effect on liver regeneration. METHODS Left lateral and median lobes of liver were either mobilized or subjected twice for 10min to ischemia followed by 5min reperfusion prior to resection while the regenerative lobes were only subjected to reperfusion. RESULTS Although intermittent reperfusion stress induced higher levels of serum transaminases, analysis of cell cycle regulators revealed accelerated regenerative response compared to standard partial hepatectomy. The G0/G1 transition occurred before tissue resection, as evidenced by c-fos, junB, and IL-6 induction. Following hepatectomy, Cyclin D1 up-regulation, G1/S transition, and cell division occurred earlier than normal. Unexpectedly, liver mobilization, a component of the clamping procedure, also resulted in earlier G1/S transition. The shortened G1-phase was driven by the c-Jun N-terminal Kinase pathway and was associated with an oxidative stress response as evidenced by the expression of inducible nitric oxide synthase. CONCLUSION Intermittent selective clamping of lobes to be resected induced reperfusion stress on remnant liver that was beneficial for liver regeneration, suggesting this procedure could be applied in clinical practice.

Glutamine metabolism and neuropathologic disorders in experimental hepatic encephalopathy: Effect of transplanted hepatocytes

BACKGROUND Physiopathology of hepatic encephalopathy remains unclear. Recent studies have suggested that ammonia would not act by itself but through an increase in glutamine in the brain. We have previously demonstrated that transplantation of syngeneic hepatocytes into the spleen was able to correct both behavioral deficits and plasma amino acid changes observed in portacaval shunted rats. The aim of the present work was to show a correlation between the correction of chronic hepatic encephalopathy by means of intrasplenic hepatocyte transplantation and two parameters, brain glutamine concentration and ultrastructural aspects of astrocytes. METHODS Inbred male Wistar Furth rats were divided into three groups: sham-operated rats (n = 10), rats subjected to portacaval shunt (n = 10), and rats subjected to portacaval shunt and intrasplenic hepatocellular transplantation of 10(7) hepatocytes isolated from livers of syngeneic rats (n = 10). Chronic hepatic encephalopathy was quantified 30 and 60 days after operation by means of nose-poke exploration and spontaneous activity. Pathologic examination and measurement of glutamine concentrations in the corpus striatus and in the cerebral cortex were performed 60 days after operation. RESULTS Portacaval shunt rats showed reduced spontaneous activity and nose-poke exploration scores. After portacaval shunt a significant glutamine increase occurred in the corpus striatus and in the cerebral cortex when compared with sham rats (p < 0.05). Ultrastructural examination showed modification of astrocytes named Alzheimer type II after portacaval shunt. Correction of behavioral abnormalities by means of intrasplenic hepatocyte transplantation was associated with partial correction of striatal glutamine increase and with decrease in astrocyte alterations. Cortex glutamine concentration in portacaval shunt-intrasplenic hepatocyte transplantation group and in portacaval shunt rats did not differ significantly. CONCLUSIONS These data show that intrasplenic hepatocyte transplantation not only prevents neurologic disorders of hepatic encephalopathy but can also decrease glutamine and ultrastructural alterations in the corpus striatus in an experimental model of chronic liver failure. These data are in favor of the involvement of glutamine in chronic hepatic encephalopathy. These results suggest that intrasplenic hepatocyte transplantation might be of therapeutic interest in chronic liver failure.

Anticancer Drug Metabolism: Chemotherapy Resistance and New Therapeutic Approaches

Over the last decades, several studies have demonstrated that cancer cells have a unique metabolism compared to normal cells (Herling et al., 2011). Metabolic changes occurring in cancer cells are considered to be fundamental for the transformation of normal cells into cancer cells and are also responsible for the resistance to different types of chemotherapeutic drugs (Cree, 2011). Therefore, resistance to chemotherapy represents a major problem in the treatment of several tumor types. Among the different metabolic and signalling pathways that are altered in cancer cells, variations in the expression and activity of several drugmetabolizing enzymes play a critical role in drug resistance (Rochat, 2009). Resistance can occur prior to drug treatment (primary or innate resistance) or may develop over time following exposure to the drug (acquired resistance). In some patients, prolonged exposure to a single chemotherapeutic agent may lead to the development of resistance to multiple other structurally unrelated compounds, known as cross resistance or multidrug resistance.

Effect of bile acids on ischemia-reperfusion liver injury

We investigated whether stimulation of bile flow by taurocholic acid (TCA), ursodeoxycholic acid (UDCA) or its taurine conjugate (TUDCA) could protect the liver from ischemia-reperfusion injury. The isolated perfused rat liver model was used. In livers perfused without bile acids (n = 8), 60 min of ischemia induced a significant reduction in bile flow and in portal flow, together with a marked increase in LDH, AST and uric acid release in the perfusate. These alterations were maximal at the beginning of reperfusion. In livers perfused with TCA (n = 6), UDCA (n = 7) or TUDCA (n = 6), bile flow was significantly increased as compared to controls during the pre-ischemic phase, as well as during the reperfusion phase. However, no significant improvement was observed in any of the biochemical, hemodynamic or histologic parameters studied. The results show that stimulation of bile flow either by TCA, UDCA or TUDCA does not reduce ischemia-reperfusion liver injury. Furthermore, the results do not provide evidence for a cytoprotective effect of UDCA or TUDCA in this model of liver injury.

Iron metabolism: a double-edged sword in the resistance of glioblastoma to therapies.

Glioblastoma (GBM), the deadliest primary tumor of the central nervous system (CNS), is a clear illustration of the resistance of cancer cells to conventional therapies. Application of combinatorial strategies able to overcome pivotal factors of GBM resistance, particularly within the resection margins, represents an essential issue. This review focuses on the role of iron metabolism in GBM progression and resistance to therapy, and the impact of its pharmaceutical modulation on the disease. Iron, through its involvement in many biological processes, is a key factor in the control of cell behavior and cancer biology. Therefore, targeting cellular iron signaling or taking advantage of its dysregulation in cancer cells may lead to new opportunities for improving treatments and drug delivery in GBM.

Locoregional Confinement and Major Clinical Benefit of 188Re-Loaded CXCR4-Targeted Nanocarriers in an Orthotopic Human to Mouse Model of Glioblastoma

Purpose: Gold standard beam radiation for glioblastoma (GBM) treatment is challenged by resistance phenomena occurring in cellular populations well prepared to survive or to repair damage caused by radiation. Among signals that have been linked with radio-resistance, the SDF1/CXCR4 axis, associated with cancer stem-like cell, may be an opportune target. To avoid the problem of systemic toxicity and blood-brain barrier crossing, the relevance and efficacy of an original system of local brain internal radiation therapy combining a radiopharmaceutical with an immuno-nanoparticle was investigated. Experiment design: The nanocarrier combined lipophilic thiobenzoate complexes of rhenium-188 loaded in the core of a lipid nanocapsule (LNC188Re) with a function-blocking antibody, 12G5 directed at the CXCR4, on its surface. The efficiency of 12G5-LNC188Re was investigated in an orthotopic and xenogenic GBM model of CXCR4-positive U87MG cells implanted in the striatum of Scid mice. Results: We demonstrated that 12G5-LNC188Re single infusion treatment by convection-enhanced delivery resulted in a major clinical improvement in median survival that was accompanied by locoregional effects on tumor development including hypovascularization and stimulation of the recruitment of bone marrow derived CD11b- or CD68-positive cells as confirmed by immunohistochemistry analysis. Interestingly, thorough analysis by spectral imaging in a chimeric U87MG GBM model containing CXCR4-positive/red fluorescent protein (RFP)-positive- and CXCR4-negative/RFP-negative-GBM cells revealed greater confinement of DiD-labeled 12G5-LNCs than control IgG2a-LNCs in RFP compartments. Main conclusion: These findings on locoregional impact and targeting of disseminated cancer cells in tumor margins suggest that intracerebral active targeting of nanocarriers loaded with radiopharmaceuticals may have considerable benefits in clinical applications.

Choledocho-ureteral anastomosis in the rat: A new experimental model of long-term, total, internal bile diversion

Choledocho-ureteral anastomosis is a new experimental model of total bile diversion in the rat. The anastomosis is carried out using a polyethylene tutor drain and requires neither an external drain nor restraint of the animal. After 14 days of diversion, bile flow fell by 53% and there were no biological signs of cholestasis. The rats survived for up to 2 months. Choledocho-ureteral anastomosis is a reliable and simple experimental model which can be used to study the effects of prolonged interruption of enterohepatic bile-acid circulation.