Ergosterol reduction impairs mitochondrial DNA maintenance in S. cerevisiae

Sterols are essential lipids, involved in many biological processes. In Saccharomyces cerevisiae, the enzymes of the ergosterol biosynthetic pathway (Erg proteins) are localized in different cellular compartments. With the aim of studying organelle interactions, we discovered that Erg27p resides mainly in Lipid Droplets (LDs) in respiratory competent cells, while in absence of respiration, is found mostly in the ER. The results presented in this paper demonstrate an interplay between the mitochondrial respiration and ergosterol production: on the one hand, rho° cells show lower ergosterol content when compared with wild type respiratory competent cells, on the other hand, the ergosterol biosynthetic pathway influences the mitochondrial status, since treatment with ketoconazole, which blocks the ergosterol pathway, or the absence of the ERG27 gene, induced rho° production in S. cerevisiae. The loss of mitochondrial DNA in the ∆erg27 strain is fully suppressed by exogenous addition of ergosterol. These data suggest the notion that ergosterol is essential for maintaining the mitochondrial DNA attached to the inner mitochondrial membrane

Influence of dietary vitamin E supplementation on cholesterol oxidation and fresh colour in beef aged for 3 and 14 days

The effects of dietary vitamin E supplementation on the susceptibility to lipid oxidation and colour of the Longissimus thoracis (LT) muscle aged in vacuum packaged conditions for 3 or 14 days were studied. For this purpose, Charolais cattle were fed on a diet providing daily 60 mg (control) or 5500 mg of vitamin E per animal (supplemented) for 30 and 60 days before slaughter. Dietary vitamin E supplementation increased the liver vitamin E content, but not in the LT muscle of treated animals. The vitamin supplementation for 30 and 60 days has shown non-consistent effects in reducing cholesterol oxidation products of vacuum-packed aged meat. However, the vitamin E supplementation for 60 days was effective on Lightness stability in LT muscle during vacuum-packed ageing. Overall, from the practical standpoint, this study suggests that supranutritional supplementation up to 60 days may not increase the vitamin E content of Charolais LT muscle giving little, if any, benefits on meat colour and cholesterol oxidation. However, the present study suggests that it would be interesting to determine in which extent specific oxysterols are related to the meat colour and whether colour parameters can be useful for predicting the formation of cholesterol oxidation products along the industrial meat production chain.The effects of dietary vitamin E supplementation on the susceptibility to lipid oxidation and colour of the Longissimus thoracis (LT) muscle aged in vacuum packaged conditions for 3 or 14 days were studied. For this purpose, Charolais cattle were fed on a diet providing daily 60mg (control) or 5500mg of vitamin E per animal (supplemented) for 30 and 60 days before slaughter. Dietary vitamin E supplementation increased the liver vitamin E content, but not in the LT muscle of treated animals. The vitamin supplementation for 30 and 60 days has shown non-consistent effects in reducing cholesterol oxidation products of vacuum-packed aged meat. However, the vitamin E supplementation for 60 days was effective on Lightness stability in LT muscle during vacuum-packed ageing. Overall, from the practical standpoint, this study suggests that supranutritional supplementation up to 60 days may not increase the vitamin E content of Charolais LT muscle giving little, if any, benefits on meat colour and cholesterol oxidation. However, the present study suggests that it would be interesting to determine in which extent specific oxysterols are related to the meat colour and whether colour parameters can be useful for predicting the formation of cholesterol oxidation products along the industrial meat production chain

Linear cyclodextrin polymer prodrugs as novel yherapeutics for Niemann-Pick type C1 disorder

Niemann-Pick Type C1 disorder (NPC) is a rare lysosomal storage disease characterized by the accumulation of cholesterol in lysosomes. NPC has no FDA approved treatments yet, however 2-hydroxypropyl-β-cyclodextrin (HPβCD) has shown efficacy for treating the disease in both mouse and feline NPC models and is currently being investigated in late stage clinical trials. Despite promising results, therapeutic use of HPβCD is limited by the need for high doses, ototoxicity and intrathecal administration. These limitations can be attributed to its poor pharmacokinetic profile. In the attempt to overcome these limitations, we have designed a β-cyclodextrin (βCD) based polymer prodrugs (ORX-301) for an enhanced pharmacokinetic and biodistribution profile, which in turn can potentially provide an improved efficacy at lower doses. We demonstrated that subcutaneously injected ORX-301 extended the mean lifespan of NPC mice at a dosage 5-fold lower (800 mg/kg, body weight) the HPβCD dose proven efficacious (4000 mg/kg). We also show that ORX-301 penetrates the blood brain barrier and counteracts neurological impairment. These properties represent a substantial improvement and appear to overcome major limitations of presently available βCD-based therapy, demonstrating that this novel prodrug is a valuable alternative/complement for existing therapies

Modulation of STAT3 signaling, cell redox defenses and cell cycle checkpoints by β-caryophyllene in cholangiocarcinoma cells: possible mechanisms accounting for doxorubicin chemosensitization and chemoprevention

Cholangiocarcinoma (CCA) is an aggressive group of biliary tract cancers, characterized by late diagnosis, low effective chemotherapies, multidrug resistance, and poor outcomes. In the attempt to identify new therapeutic strategies for CCA, we studied the antiproliferative activity of a combination between doxorubicin and the natural sesquiterpene β-caryophyllene in cholangiocarcinoma Mz-ChA-1 cells and nonmalignant H69 cholangiocytes, under both long-term and metronomic schedules. The modulation of STAT3 signaling, oxidative stress, DNA damage response, cell cycle progression and apoptosis was investigated as possible mechanisms of action. β-caryophyllene was able to synergize the cytotoxicity of low dose doxorubicin in Mz-ChA-1 cells, while producing cytoprotective effects in H69 cholangiocytes, mainly after a long-term exposure of 24 h. The mechanistic analysis highlighted that the sesquiterpene induced a cell cycle arrest in G2/M phase along with the doxorubicin-induced accumulation in S phase, reduced the γH2AX and GSH levels without affecting GSSG. ROS amount was partly lowered by the combination in Mz-ChA-1 cells, while increased in H69 cells. A lowered expression of doxorubicin-induced STAT3 activation was found in the presence of β-caryophyllene in both cancer and normal cholangiocytes. These networking effects resulted in an increased apoptosis rate in Mz-ChA-1 cells, despite a lowering in H69 cholangiocytes. This evidence highlighted a possible role of STAT3 as a final effector of a complex network regulated by β-caryophyllene, which leads to an enhanced doxorubicin-sensitivity of cholangiocarcinoma cells and a lowered chemotherapy toxicity in nonmalignant cholangiocytes, thus strengthening the interest for this natural sesquiterpene as a dual-acting chemosensitizing and chemopreventive agent

Glucose Metabolism in the Progression of Prostate Cancer

Prostate cancer is one of the most common types of cancer in western country males but the mechanisms involved in the transformation processes have not been clearly elucidated. Alteration in cellular metabolism in cancer cells is recognized as a hallmark of malignant transformation, although it is becoming clear that the biological features of metabolic reprogramming not only differ in different cancers, but also among different cells in a type of cancer. Normal prostate epithelial cells have a peculiar and very inefficient energy metabolism as they use glucose to synthesize citrate that is secreted as part of the seminal liquid. During the transformation process, prostate cancer cells modify their energy metabolism from inefficient to highly efficient, often taking advantage of the interaction with other cell types in the tumor microenvironment that are corrupted to produce and secrete metabolic intermediates used by cancer cells in catabolic and anabolic processes. We recapitulate the metabolic transformations occurring in the prostate from the normal cell to the metastasis, highlighting the role of the microenvironment and summarizing what is known on the molecular mechanisms involved in the process

A pyrazolopyran derivative preferentially inhibits the activity of human cytosolic hydroxymethyltransferase and induces cell death in lung cancer cells

Serine hydroxymethyltransferase (SHMT) is a central enzyme in the metabolic reprogramming of cancer cells, providing activated one-carbon units in the serine-glycine one-carbon metabolism. Previous studies demonstrated that the cytoplasmic isoform of SHMT (SHMT1) plays a relevant role in lung cancer. SHMT1 is overexpressed in lung cancer patients and NSCLC cell lines. Moreover, SHMT1 is required to maintain DNA integrity. Depletion in lung cancer cell lines causes cell cycle arrest and uracil accumulation and ultimately leads to apoptosis. We found that a pyrazolopyran compound, namely 2.12, preferentially inhibits SHMT1 compared to the mitochondrial counterpart SHMT2. Computational and crystallographic approaches suggest binding at the active site of SHMT1 and a competitive inhibition mechanism. A radio isotopic activity assay shows that inhibition of SHMT by 2.12 also occurs in living cells. Moreover, administration of 2.12 in A549 and H1299 lung cancer cell lines causes apoptosis at LD50 34 μM and rescue experiments underlined selectivity towards SHMT1. These data not only further highlight the relevance of the cytoplasmic isoform SHMT1 in lung cancer but, more importantly, demonstrate that, at least in vitro, it is possible to find selective inhibitors against one specific isoform of SHMT, a key target in metabolic reprogramming of many cancer types

Immobilization of Lathyrus cicera Amine Oxidase on Magnetic Microparticles for Biocatalytic Applications

Amine oxidases are enzymes belonging to the class of oxidoreductases that are widespread, from bacteria to humans. The amine oxidase from Lathyrus cicera has recently appeared in the landscape of biocatalysis, showing good potential in the green synthesis of aldehydes. This enzyme catalyzes the oxidative deamination of a wide range of primary amines into the corresponding aldehydes but its use as a biocatalyst is challenging due to the possible inactivation that might occur at high product concentrations. Here, we show that the enzyme’s performance can be greatly improved by immobilization on solid supports. The best results are achieved using amino-functionalized magnetic microparticles: the immobilized enzyme retains its activity, greatly improves its thermostability (4 h at 75 ◦C), and can be recycled up to 8 times with a set of aromatic ethylamines. After the last reaction cycle, the overall conversion is about 90% for all tested substrates, with an aldehyde production ranging between 100 and 270 mg depending on the substrate used. As a proof concept, one of the aldehydes thus produced was successfully used for the biomimetic synthesis of a non-natural benzylisoquinoline alkaloid

Shmt2: a stat3 signaling new player in prostate cancer energy metabolism

Prostate cancer (PCa) is a multifactorial disease characterized by the aberrant activity of different regulatory pathways. STAT3 protein mediates some of these pathways and its activation is implicated in the modulation of several metabolic enzymes. A bioinformatic analysis indicated a STAT3 binding site in the upstream region of SHMT2 gene. We demonstrated that in LNCaP, PCa cells' SHMT2 expression is upregulated by the JAK2/STAT3 canonical pathway upon IL-6 stimulation. Activation of SHTM2 leads to a decrease in serine levels, pushing PKM2 towards the nuclear compartment where it can activate STAT3 in a non-canonical fashion that in turn promotes a transient shift toward anaerobic metabolism. These results were also confirmed on FFPE prostate tissue sections at different Gleason scores. STAT3/SHMT2/PKM2 loop in LNCaP cells can modulate a metabolic shift in response to inflammation at early stages of cancer progression, whereas a non-canonical STAT3 activation involving the STAT3/HIF-1α/PKM2 loop is responsible for the maintenance of Warburg effect distinctive of more aggressive PCa cells. Chronic inflammation might thus prime the transition of PCa cells towards more advanced stages, and SHMT2 could represent a missing factor to further understand the molecular mechanisms responsible for the transition of prostate cancer towards a more aggressive phenotyp

Dissecting the cytochrome P450 OleP substrate specificity: evidence for a preferential substrate

The cytochrome P450 OleP catalyzes the epoxidation of aliphatic carbons on both the aglycone 8.8a-deoxyoleandolide (DEO) and the monoglycosylated L-olivosyl-8.8a-deoxyoleandolide (L-O-DEO) intermediates of oleandomycin biosynthesis. We investigated the substrate versatility of the enzyme. X-ray and equilibrium binding data show that the aglycone DEO loosely fits the OleP active site, triggering the closure that prepares it for catalysis only on a minor population of enzyme. The open-to-closed state transition allows solvent molecules to accumulate in a cavity that forms upon closure, mediating protein–substrate interactions. In silico docking of the monoglycosylated L-O-DEO in the closed OleP–DEO structure shows that the L-olivosyl moiety can be hosted in the same cavity, replacing solvent molecules and directly contacting structural elements involved in the transition. X-ray structures of aglycone-bound OleP in the presence of L-rhamnose confirm the cavity as a potential site for sugar binding. All considered, we propose L-O-DEO as the optimal substrate of OleP, the L-olivosyl moiety possibly representing the molecular wedge that triggers a more efficient structural response upon substrate binding, favoring and stabilizing the enzyme closure before catalysis. OleP substrate versatility is supported by structural solvent molecules that compensate for the absence of a glycosyl unit when the aglycone is bound

Antioxidant Properties of Aminoethylcysteine Ketimine Decarboxylated Dimer: A Review

Aminoethylcysteine ketimine decarboxylated dimer is a natural sulfur-containing compound detected in human plasma and urine, in mammalian brain and in many common edible vegetables. Over the past decade many studies have been undertaken to identify its metabolic role. Attention has been focused on its antioxidant properties and on its reactivity against oxygen and nitrogen reactive species. These properties have been studied in different model systems starting from plasma lipoproteins to specific cellular lines. All these studies report that aminoethylcysteine ketimine decarboxylated dimer is able to interact both with reactive oxygen and nitrogen species (hydrogen peroxide, superoxide anion, hydroxyl radical, peroxynitrite and its derivatives). Its antioxidant activity is similar to that of Vitamin E while higher than other hydrophilic antioxidants, such as trolox and N-acetylcysteine