Annelies Peeters

Chemical Inhibition of Acetyl-CoA Carboxylase Induces Growth Arrest and Cytotoxicity Selectively in Cancer Cells

Development and progression of cancer is accompanied by marked changes in the expression and activity of enzymes involved in the cellular homeostasis of fatty acids. One class of enzymes that play a particularly important role in this process are the acetyl-CoA carboxylases (ACC). ACCs produce malonyl-CoA, an intermediate metabolite that functions as substrate for fatty acid synthesis and as negative regulator of fatty acid oxidation. Here, using the potent ACC inhibitor soraphen A, a macrocyclic polyketide from myxobacteria, we show that ACC activity in cancer cells is essential for proliferation and survival. Even at nanomolar concentrations, soraphen A can block fatty acid synthesis and stimulate fatty acid oxidation in LNCaP and PC-3M prostate cancer cells. As a result, the phospholipid content of cancer cells decreased, and cells stopped proliferating and ultimately died. LNCaP cells predominantly died through apoptosis, whereas PC-3M cells showed signs of autophagy. Supplementation of the culture medium with exogenous palmitic acid completely abolished the effects of soraphen A and rescued the cells from cell death. Interestingly, when added to cultures of premalignant BPH-1 cells, soraphen A only slightly affected cell proliferation and did not induce cell death. Together, these findings indicate that cancer cells have become dependent on ACC activity to provide the cell with a sufficient supply of fatty acids to permit proliferation and survival, introducing the concept of using small-molecule ACC inhibitors as therapeutic agents for cancer.

Gene-Targeting of Phd2 Improves Tumor Response to Chemotherapy and Prevents Side-Toxicity

The success of chemotherapy in cancer treatment is limited by scarce drug delivery to the tumor and severe side-toxicity. Prolyl hydroxylase domain protein 2 (PHD2) is an oxygen/redox-sensitive enzyme that induces cellular adaptations to stress conditions. Reduced activity of PHD2 in endothelial cells normalizes tumor vessels and enhances perfusion. Here, we show that tumor vessel normalization by genetic inactivation of Phd2 increases the delivery of chemotherapeutics to the tumor and, hence, their antitumor and antimetastatic effect, regardless of combined inhibition of Phd2 in cancer cells. In response to chemotherapy-induced oxidative stress, pharmacological inhibition or genetic inactivation of Phd2 enhances a hypoxia-inducible transcription factor (HIF)-mediated detoxification program in healthy organs, which prevents oxidative damage, organ failure, and tissue demise. Altogether, our study discloses alternative strategies for chemotherapy optimization.

Determination of OATP-, NTCP- and OCT-mediated substrate uptake activities in individual and pooled batches of cryopreserved human hepatocytes

While the utility of cryopreserved human hepatocyte suspensions (CHHS) for in vitro drug metabolism assays has been established, less is known about the effects of cryopreservation on transporter activity in human hepatocytes. In the present study, the activities of NTCP (sodium taurocholate co-transporting polypeptide; SLC10A1), as well as of the hepatic OATP (organic anion transporting polypeptide; SLCO gene family) and OCT (organic cation transporter; SLC22A) isoforms were assessed in 14 individual and four pooled batches of CHHS. For comparative purposes, substrate accumulation rates were also measured in sandwich-cultured human hepatocytes. In CHHS, the mean accumulation clearance of the NTCP substrate taurocholate (1 μM) was 27.5 (±15.0) μl/min/million cells and decreased by 10-fold when extracellular sodium was replaced by choline. The accumulation clearance of digoxin and of the OATP substrates estrone-3-sulfate and estradiol-17β-D-glucuronide (E(2)-17β-G; 1 μM) amounted to 9.5 (±4.9), 99 (±67) and 5.2 (±2.6) μl/min/million cells, respectively. Presence of the known OATP inhibitor rifampicin (25 μM) significantly (p<0.01) decreased the accumulation of estrone-3-sulfate and E(2)-17β-G to 48% and 70% of the control value, respectively, while no significant effect on digoxin accumulation was observed. The mean accumulation clearance of the OCT substrate 1-methyl-4-phenylpyridinium amounted to 19.8 (±10.9) μl/min/million cells. Co-incubation with the OCT1 inhibitor prazosin (3 μM) and the OCT3 inhibitor corticosterone (1 μM) resulted in a significant (p<0.01) decrease to 72% and 85% of the accumulation in control conditions, respectively. Experiments in pooled CHHS generally showed accumulation values that were comparable with the mean of the individual batches. A good correlation (R(2)=0.93) was observed between estrone-3-sulfate accumulation values and OATP1B3 mRNA levels, as determined in five batches of CHHS. Compared to substrate accumulation measured in sandwich-cultured human hepatocytes, accumulation values in CHHS were comparable (taurocholate and digoxin) to slightly higher (estrone-3-sulfate). Our data indicate that cryopreserved human hepatocyte suspensions are a reliable in vitro model to study transporter-mediated substrate uptake in the liver. Systematic characterization of multiple batches of CHHS for transporter activity supports rational selection of human hepatocytes for specific applications.

Mitochondria in peroxisome-deficient hepatocytes exhibit impaired respiration, depleted DNA, and PGC-1α independent proliferation

The tight interrelationship between peroxisomes and mitochondria is illustrated by their cooperation in lipid metabolism, antiviral innate immunity and shared use of proteins executing organellar fission. In addition, we previously reported that disruption of peroxisome biogenesis in hepatocytes severely impacts on mitochondrial integrity, primarily damaging the inner membrane. Here we investigated the molecular impairments of the dysfunctional mitochondria in hepatocyte selective Pex5 knockout mice. First, by using blue native electrophoresis and in-gel activity stainings we showed that the respiratory complexes were differentially affected with reduction of complexes I and III and incomplete assembly of complex V, whereas complexes II and IV were normally active. This resulted in impaired oxygen consumption in cultured Pex5(-/-) hepatocytes. Second, mitochondrial DNA was depleted causing an imbalance in the expression of mitochondrial- and nuclear-encoded subunits of the respiratory chain complexes. Third, mitochondrial membranes showed increased permeability and fluidity despite reduced content of the polyunsaturated fatty acid docosahexaenoic acid. Fourth, the affected mitochondria in peroxisome deficient hepatocytes displayed increased oxidative stress. Acute deletion of PEX5 in vivo using adeno-Cre virus phenocopied these effects, indicating that mitochondrial perturbations closely follow the loss of functional peroxisomes in time. Likely to compensate for the functional impairments, the volume of the mitochondrial compartment was increased several folds. This was not driven by PGC-1α but mediated by activation of PPARα, possibly through c-myc overexpression. In conclusion, loss of peroxisomal metabolism in hepatocytes perturbs the mitochondrial inner membrane, depletes mitochondrial DNA and causes mitochondrial biogenesis independent of PGC-1α.

Carbohydrate metabolism is perturbed in peroxisome-deficient hepatocytes due to mitochondrial dysfunction, AMP-activated protein kinase (AMPK) activation, and peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) suppression.

Background: It is not known whether peroxisomes influence hepatic carbohydrate metabolism. Results: Carbohydrate metabolism is perturbed in peroxisome-deficient mouse liver through mitochondrial deficits, AMPK, and PGC-1α. Conclusion: Dysfunctional peroxisome metabolism disrupts carbohydrate homeostasis by indirect mechanisms. Significance: The impact of peroxisome deficiency on liver metabolism is broader than expected. Hepatic peroxisomes are essential for lipid conversions that include the formation of mature conjugated bile acids, the degradation of branched chain fatty acids, and the synthesis of docosahexaenoic acid. Through unresolved mechanisms, deletion of functional peroxisomes from mouse hepatocytes (L-Pex5−/− mice) causes severe structural and functional abnormalities at the inner mitochondrial membrane. We now demonstrate that the peroxisomal and mitochondrial anomalies trigger energy deficits, as shown by increased AMP/ATP and decreased NAD+/NADH ratios. This causes suppression of gluconeogenesis and glycogen synthesis and up-regulation of glycolysis. As a consequence, L-Pex5−/− mice combust more carbohydrates resulting in lower body weights despite increased food intake. The perturbation of carbohydrate metabolism does not require a long term adaptation to the absence of functional peroxisomes as similar metabolic changes were also rapidly induced by acute elimination of Pex5 via adenoviral administration of Cre. Despite its marked activation, peroxisome proliferator-activated receptor α (PPARα) was not causally involved in these metabolic perturbations, because all abnormalities still manifested when peroxisomes were eliminated in a peroxisome proliferator-activated receptor α null background. Instead, AMP-activated kinase activation was responsible for the down-regulation of glycogen synthesis and induction of glycolysis. Remarkably, PGC-1α was suppressed despite AMP-activated kinase activation, a paradigm not previously reported, and they jointly contributed to impaired gluconeogenesis. In conclusion, lack of functional peroxisomes from hepatocytes results in marked disturbances of carbohydrate homeostasis, which are consistent with adaptations to an energy deficit. Because this is primarily due to impaired mitochondrial ATP production, these L-Pex5-deficient livers can also be considered as a model for secondary mitochondrial hepatopathies.

Role of PPARα in Hepatic Carbohydrate Metabolism

Tight control of storage and synthesis of glucose during nutritional transitions is essential to maintain blood glucose levels, a process in which the liver has a central role. PPARα is the master regulator of lipid metabolism during fasting, but evidence is emerging for a role of PPARα in balancing glucose homeostasis as well. By using PPARα ligands and PPARα −/− mice, several crucial genes were shown to be regulated by PPARα in a direct or indirect way. We here review recent evidence that PPARα contributes to the adaptation of hepatic carbohydrate metabolism during the fed-to-fasted or fasted-to-fed transition in rodents.

Lewis acid double metal cyanide catalysts for hydroamination of phenylacetylene

Double metal cyanides (DMCs) are highly active recyclable heterogeneous catalysts for hydroamination of phenylacetylene with 4-isopropylaniline. The best hydroamination yields are obtained with Zn-Co DMCs, especially if the particle size is decreased by a reverse emulsion synthesis technique.

Product recovery from ionic liquids by solvent-resistant nanofiltration: application to ozonation of acetals and methyl oleate

In this work we tackle the problematic separation of reaction products from ionic liquid media. Solvent-resistant nanofiltration proves to be an attractive technique for the separation of non-volatile polar products from ionic liquids. In view of the high compatibility between ozone and ionic liquids, two ozone-mediated model reactions were chosen: firstly the oxidation of acetals to esters in the presence of ozone and secondly the ozonation of methyl oleate to monomethyl azelate and pelargonic acid. The objective was to retain the ionic liquid phase by means of a solvent-resistant nanofiltration membrane, while the organic reaction products permeate through the polymeric membrane. First, the ozonations were studied in order to know the characteristic product compositions. Next, a screening of membranes was performed on synthetic product mixtures. The second generation polyimide-based DuraMem™ membranes showed the highest rejection, up to 96%, for the evaluated ionic liquids. These DuraMem™ membranes also proved suitable for the separation of the products on real reaction mixtures, even in a single filtration step.

Carbon dioxide as a reversible amine-protecting agent in selective Michael additions and acylations

Carbon dioxide can be used as a temporary protecting group for amines. A carbamic acid is formed reversibly when CO2 is bubbled through a solution of a sufficiently basic primary amine at room temperature and atmospheric pressure. This reaction is employed for the protection of the amine functionality in several reactions at room temperature where inter- or intramolecular selectivity is desired. The concept is demonstrated for the selective Michael additions to methyl acrylate of a normally less reactive sulfonamide in the presence of a strong amine nucleophile, or of a cyclic secondary amine in the presence of an aliphatic primary amine, or of a β-ketoester in the presence of amines. The selective acylation of an alcohol in the presence of an amine can be achieved under a CO2 atmosphere as well.

Oral Administration of the Broad-Spectrum Antibiofilm Compound Toremifene Inhibits Candida albicans and Staphylococcus aureus Biofilm Formation In Vivo

ABSTRACT We here report on the in vitro activity of toremifene to inhibit biofilm formation of different fungal and bacterial pathogens, including Candida albicans, Candida glabrata, Candida dubliniensis, Candida krusei, Pseudomonas aeruginosa, Staphylococcus aureus, and Staphylococcus epidermidis. We validated the in vivo efficacy of orally administered toremifene against C. albicans and S. aureus biofilm formation in a rat subcutaneous catheter model. Combined, our results demonstrate the potential of toremifene as a broad-spectrum oral antibiofilm compound.