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Background: Tumorigenesis is associated with deregulation of nutritional requirements, intermediary metabolites production, and microenvironment interactions. Unlike their normal cell counterparts, tumor cells rely on aerobic glycolysis, through the Warburg effect. Summary: The pentose phosphate pathway (PPP) is a major glucose metabolic shunt that is upregulated in cancer cells. The PPP comprises an oxidative and a nonoxidative phase and is essential for nucleotide synthesis of rapidly dividing cells. The PPP also generates nicotinamide adenine dinucleotide phosphate, which is required for reductive metabolism and to counteract oxidative stress in tumor cells. This article reviews the regulation of the PPP and discusses inhibitors that target its main pathways. Key Message: Exploiting the metabolic vulnerability of the PPP offers potential novel therapeutic opportunities and improves patients’ response to cancer therapy.

Ichthyosis Follicularis, Atrichia, and Photophobia (IFAP) is a severe rare genetic disorder caused by mutations in the gene encoding the Membrane-Bound Transcription Factor Peptidase, Site 2 (MBTPS2). Olmsted syndrome is another rare genetic disease with overlapping clinical features caused by mutations in the gene encoding the Transient Receptor Potential Cation Channel, subfamily V (TRPV3). Mutations in MBTPS2 have been recently reported in Olmsted syndrome, underscoring the overlap and the confusion in separating Olmsted from IFAP syndrome. We studied a Lebanese family with IFAP syndrome both, clinically and molecularly, and investigated whether there is a cross relation between TRPV3 and MBTPS2. We identified a recurrent mutation designated p.F475S in MBTPS2 in the affected individuals. This mutation was not found in 100 control individuals from the same population. We determined that TRPV3 regulatory region is a target for MBTPS2. In addition, there was an increased cell death in the cells transfected with the mutant versus the wild-type MBTPS2. In conclusion, we identified a direct regulatory effect of MBTPS2 on TRPV3 which can partially contribute to the overlapping clinical features of IFAP and Olmsted syndromes under a common signaling pathway.

Colorectal cancer is the third most common neoplasia and the second cause of cancer-related deaths worldwide. Unlike normal cells, tumor cells reprogram metabolic pathways to meet their bioenergetics, redox, biosynthetic demands and rely mostly on aerobic glycolysis (Warburg effect). Colorectal cancer cells upregulate the pentose phosphate pathway (PPP), and p53 is a crucial regulator. The PPP is a significant route for glucose catabolism and is required for DNA synthesis of rapidly-proliferating cells. Its oxidative phase (oxPPP), catalyzed by the rate-limiting enzyme glucose-6-phosphate dehydrogenase (G6PD), provides the cell with nicotinamide adenine dinucleotide phosphate (NADPH) that is needed for biosynthetic and detoxifying processes. For the past six decades, 5-Fluorouracil (5-FU) has been the treatment of choice in colorectal cancer. However, 5-FU exhibits high toxicity and drug resistance in patients. Therefore, we hypothesized that targeting the PPP might offer novel therapeutic opportunities in colorectal cancer and improve the response to 5-FU at lower and safer doses. Accordingly, we aim to investigate and characterize the anti-tumor effect of the G6PD inhibitor, 6-aminonicotinamide (6-AN), alone, or in combination with 5-FU on colorectal cancer cells. We explored the effects of oxPPP inhibition on colorectal cancer cell growth, reactive oxygen species (ROS) production, cell cycle regulation, and senescence. We have shown that 6-AN arrested cell growth in HCT116 and HCT116 p53−/− colorectal cancer cells while spared normal-like colon cells (NCM460D). Moreover, 6-AN induced S-phase arrest and early senescence in HCT116 cells as demonstrated by cell cycle analysis using flow cytometry and senescence-associated beta-galactosidase assay, respectively. We also tested the effects of 6-AN/5-FU combination treatments on cellular viability using MTT assay and validated our results using SRB and trypan blue exclusion assays. Interestingly, combination treatments of 6-AN with 5-FU resulted in synergistic effects as estimated using Compusyn software. We used HCT116 colorectal cancer cells resistant to 5-FU (HCT116 5FU-R) or lacking p53, as models for tumor resistance. Importantly, the combination treatment sensitized both HCT116 p53−/− and HCT116 5FU-R colorectal cancer cells to 5-FU and was accompanied by a reduction in G6PD activity and increased ROS using NBT reduction assay. Our findings indicate that combining 6-AN with 5-FU may decrease resistance and further sensitize colorectal cancer cells to 5-FU treatment independently of p53 and drug resistance status. Exploiting this metabolic vulnerability may offer a novel clinical approach and improve patient's therapy. Citation Format: Noorhan Ghanem, Chirine El Baba, Lara Al Saleh, Berthe Hayar, Patrick Aouad, Marwa Al Hassan, Riyad El-Khoury, Julnar Usta, Nadine Darwiche. Therapeutic targeting of the pentose phosphate pathway in colorectal cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 236.

AIM To determine the phenotypes and predominant disease-causing mutations in Lebanese patients with Wilson’s disease, as compared to regional non-European data. METHODS The clinical profile of 36 patients diagnosed in Lebanon was studied and their mutations were determined by molecular testing. All patients underwent full physical exam, including ophthalmologic slit-lamp examination ultrasound imaging of the liver, as well as measurement of serum ceruloplasmin and 24-h urinary-Cu levels. In addition, genetic screening using PCR followed by sequencing to determine disease-causing mutations and polymorphisms in the ATP7B gene was carried on extracted DNA from patients and immediate family members. Our phenotypic-genotypic findings were then compared to reported mutations in Wilson’s disease patients from regional Arab and non-European countries. RESULTS Patients belonged to extended consanguineous families. The majority were homozygous for the disease-causing mutation, with no predominant mutation identified. The most common mutation, detected in 4 out of 13 families, involved the ATP hinge region and was present in patients from Lebanon, Egypt, Iran and Turkey. Otherwise, mutations in Lebanese patients and those of the region were scattered over 17 exons of ATP7B. While the homozygous exon 12 mutation Trp939Cys was only detected in patients from Lebanon but none from the regional countries, the worldwide common mutation H1069Q was not present in the Lebanese and was rare in the region. Pure hepatic phenotype was predominant in patients from both Lebanon and the region (25%-65%). Furthermore, the majority of patients, including those who were asymptomatic, had evidence of some hepatic dysfunction. Pure neurologic phenotype was rare. CONCLUSION Findings do not support presence of a founder effect. Clinical and genetic screening is recommended for family members with index patients and unexplained hepatic dysfunction.

Phytochemicals have been extensively researched for their potential anticancer effects. In previous study, direct exposure of rat liver mitochondria to eugenol main ingredient of clove, uncoupled mitochondria and increased F0F1ATPase activity. In the present study, we further investigated the effects of eugenol on MCF-7 cells in culture. Eugenol demonstrated: a dose-dependent decrease in viability (MTT assay), and proliferation (real time cell analysis) of MCF-7 cells, (EC50: 0.9 mM); an increase in reactive oxygen species; a decrease in ATP level and mitochondrial membrane potential (MitoPT JC-1 assay); and a release of cytochrome-c and lactate dehydrogenase (Cytotoxicity Detection Kit PLUS) into culture media at eugenol concentration >EC50. Pretreatment with the antioxidants Trolox and N-acetyl cysteine partially restored cell viability and decreased ROS, with Trolox being more potent. Expression levels of both anti- and pro-apoptotic markers (Bcl-2 and Bax, respectively) decreased with increasing eugenol concentration, with no variation in their relative ratios. Eugenol-treated MCF-7 cells overexpressing Bcl-2 exhibited results similar to those of MCF-7. Our findings indicate that eugenol toxicity is non-apoptotic Bcl-2 independent, affecting mitochondrial function and plasma membrane integrity with no effect on migration or invasion. We report here the chemo-sensitivity of MCF-7 cells to eugenol, a phytochemical with anticancer potential.

D-Penicillamine (PA), a copper chelator, and one of the recommended drugs for treatment of Wilson disease (WD) has been reported to worsen the symptoms of patients with neurologic presentations. However, the cause of this paradoxical response has not been fully elucidated and requires further investigations. Accordingly, we have studied the in vitro effect of Copper (Cu) and/or PA treatment on human glioblastoma U251 cells as an in vitro model of Cu cytotoxicity. Treatment of U251 cells with either Cu or PA exerted no significant effect on their morphology, viability or ROS level. In contrast, co-treatment with Cu-PA caused a decrease in viability, altered glutathione and ceruloplasmin expression coupled with marked increase in ROS; depolarization of mitochondrial membrane potential; and an increase in Sub G0 phase; along with alpha-Fodrin proteolysis. These findings along with the absence of LDH release in these assays, suggest that combined Cu-PA exposure induced apoptosis in U251 cells. In addition, pre-/or co-treatment with antioxidants showed a protective effect, with catalase being more effective than N-acetyl cysteine or trolox in restoring viability and reducing generated ROS levels. By comparison, a similar analysis using other cell lines showed that rat PC12 cells were resistant to Cu and/or PA treatment, while the neuroblastoma cell line SH-SY5Y was sensitive to either compound alone, resulting in decreased viability and increased ROS level. Taken together, this study shows that glioblastoma U251 cells provide a model for Cu-PA cytotoxicity mediated by H2O2. We postulate that PA oxidation in presence of Cu yields H2O2 which in turn permeates the plasma membrane and induced apoptosis. However, other cell lines exhibited different responses to these treatments, potentially providing a model for cell type- specific cytotoxic responses in the nervous system. The sensitivity of different neural and glial cell types to Cu-PA treatment may therefore underlie the neurologic worsening occurring in some PA-treated WD patients. Our results also raise the possibility that the side effects of PA treatment might be reduced or prevented by administering antioxidants.

Moringa oleifera (MO) is an important dietary component for many populations in West Africa and the Indian subcontinent. In addition to its highly nutritious value, almost all parts of this plant have been widely used in folk medicine in curing infectious, cardiovascular, gastrointestinal, hepatic, and other diseases. Evidence-based research supported its versatile medicinal properties; however, more rigorous research is required to establish it in cancer therapy. As such, in this study we aim to investigate the in vitro anticancerous effect of Moringa oleifera's aqueous leaf extract.Moringa extract was prepared by soaking pulverized leaves in hot water mimicking the people's mode of the leaf drink preparation. Several assays were used to study the effect of different percentage concentrations of the extract on viability of A549 cells; levels of adenosine triphosphate (ATP), reactive oxygen species (ROS), and glutathione (GSH) generated; as well as percentage of lactate dehydrogenase (LDH) released at different time points. In addition to mitochondrial membrane potential, apoptotic events were assessed using western blotting for apoptotic markers and immunoflourescent flourescent labeled inhibitor of caspases (FLICA) assay.MO extract treatment resulted in a significant decrease in mitochondrial membrane potential (1 hour) and ATP levels (3 hours), followed by an increase in (6 hours) ROS, caspase activation, proapoptotic proteins expression (p53, SMAC/Diablo, AIF), and PARP-1 cleavage. This eventually resulted in decreased GSH levels and a decrease in viability. The cytotoxic effect was prevented upon pretreatment with antioxidant N-acetyl-cysteine. MO decreased as well the viability of HepG2, CaCo2, Jurkat, and HEK293 cells.Our findings identify a plant extract with an anticancerous effect on cancer cell lines. MO extract exerts its cytotoxic effect in A549 cancer cells by affecting mitochondrial viability and inducing apoptosis in an ROS-dependent manner.

One proposed mechanism by which alum enhances an immune response is by its ability to induce an inflammatory response that results in the release of uric acid from necrotic cells. Uric acid is thought to be a mediator in enhancing the immune response. The aim of this study was to investigate the immunopotentiating effect of uric acid. Groups of BALB/c mice were injected intraperitoneally with ovalbumin, ovalbumin + alum, ovalbumin + uric acid, uric acid, alum, or allopurinol. Two other groups were pretreated with allopurinol and were given ovalbumin + alum, or ovalbumin + uric acid 24 hours later. An additional two groups served as controls. On days 4, 7 and 10 post-injection, the numbers of Interleukin 4(IL-4) and Interferon-γ (IFN-γ) secreting spleen cells were determined by the ELISPOT assay. Serum uric acid levels were determined using an autoanalyser and nitric oxide using the Greiss reagent. The groups that received alum + ovalbumin or uric acid + ovalbumin had the highest numbers of IL-4 and INF-γ secreting cells as compared to all the groups. Allopurinol administration one day prior to alum + ovalbumin or uric acid + ovalbumin resulted in a decrease in the number of IL-4 and INF-γ secreting cells when compared to alum+ ovalbumin or uric acid + ovalbumin allopurinol - untreated groups. Groups that received alum, alum + ovalbumin, uric acid, and uric acid + ovalbumin had high serum uric acid levels as compared to all the groups. All groups that received alum had the highest levels of nitric oxide when compared to the groups that were not given alum. In conclusion, it appears that uric acid might be a mediator in the adjuvant effect of alum.

We previously detected a membrane-bound, copper-containing oxidase that may be involved in iron efflux in BeWo cells, a human placental cell line. We have now identified a gene encoding a predicted multicopper ferroxidase (MCF) with a putative C-terminal membrane-spanning sequence and high sequence identity to hephaestin (Heph) and ceruloplasmin (Cp), the other known vertebrate MCF. Molecular modeling revealed conservation of all type I, II, and III copper-binding sites as well as a putative iron-binding site. Protein expression was observed in multiple diverse mouse tissues, including placenta and mammary gland, and the expression pattern was distinct from that of Cp and Heph. The protein possessed ferroxidase activity, and protein levels decreased in cellular copper deficiency. Knockdown with small interfering RNA in BeWo cells indicates that this gene represents the previously detected oxidase. We propose calling this new member of the MCF family “zyklopen.”

Objective To determine whether any correlation exists between the phenotype and genotype of 2 Lebanese families with members affected with Wilson disease (WD). Background WD is an autosomal-recessive disorder of copper transport with significant phenotypic diversity. Most patients are compound heterozygous making it difficult to establish a clear link between phenotype and genotype. Study We investigated 14 members from 2 Lebanese families (H and Z) with 5 members affected with WD. Mutation analysis of the ATP7B gene, and clinical assessments were carried out for both families. We also performed a literature search retrieving reported phenotypes of all patients homozygous to mutations in any of the 21 exons of the ATP7B. Results Patients of the H and Z-families were found homozygous for the respective Asn1270Ser and Pro1273Leu mutations in the adenosine triphosphate (ATP) hinge region of exon 18. Of the healthy members, 6 were heterozygous and 3 had normal sequences. Clinically, 4 patients had liver cirrhosis and 1 had asymptomatic transaminitis. One of the patients also had neurologic symptoms. Screening the literature for patients homozygous for mutations in the ATP hinge region identified 25 patients including ours. The overall prevalence of the hepatic phenotype among patients homozygous for mutation in exon 18 was 80% and was significantly higher than those in exons 7, 14, and 21. Conclusions We hereby report the association of liver disease with homozygous mutations in the conserved ATP hinge region of exon 18 of the ATP7B gene.

Coriander is used as an appetizer, a common food seasoning in Mediterranean dishes, and a remedy for many ailments. In this study we tested the biochemical effect of its essential oil components, in particular linalool, its main component. The oil extract was prepared by hydro-distillation of coriander seeds. The various components were identified by gas chromatography coupled to mass spectroscopy. The effect of the various oil components on the viability of different cell lines (HepG2, Caco2, NIH3t3, MCF7 and Hek293) was examined using MTT assay. Linalool was the most potent and HepG2 cells the most sensitive. A 50% and 100% decrease in the viability of HepG2 was obtained at 0.4 microM and 2 microM linalool, respectively. Whereas none of the other components exerted a significant effect at concentrations lower than 50 microM, myrcene and nerolidol, the structural analogues of linalool, were more potent at 100 microM than the other components decreasing HepG2 viability to 26%. The biochemical effect of linalool on mitochondria isolated from HepG2 showed a concentration-dependent inhibition in complexes I and II activities of the respiratory chain, and a time-dependent decrease in ATP level. In addition, a time-dependent decrease in glutathione (GSH) level and in the reduction of nitroblue tetrazolium was obtained, indicating increase in reactive oxygen species (ROS) generation. Pretreatment with the antioxidants: N-acetyl cysteine (2mM), Trolox (100 microM) and different flavonoids (50 microM) was partially protective against the linalool-induced cell death; the most effective response was that of rutin and apigenin which restored 91% of HepG2 viability. We hereby report a decrease in cell viability of HepG2 cells by linalool and identify the mitochondria as one possible target for its site of action, inhibiting complexes I and II and decreasing ATP. In addition linalool increased ROS generation and decreased GSH level.

Status epilepticus (SE) induces a number of events leading to programmed cell death (PCD). The aim of our work is to study the time sequence of activation of different factors in experimental SE (intraperitoneal kainic acid (KA) model). We studied ceramide, a known mediator of apoptosis in multiple models, sphingomyelinases (SMases), enzymes that break down sphingomyelin and increase ceramide thus leading to apoptosis in many models, Bcl(2), Bax, and caspase-3. SE induced a sustained ceramide increase starting 2h after kainic acid injection followed by an increase in Bax protein at 6 and 12h, and the appearance of caspase-3-activated fragment (caspase-3a) immunostaining and TUNEL positivity at 12h. Status epilepticus also induced an increase in acidic and neutral sphingomyelinases that preceded (acidic sphingomyelinase) and parallelled (acidic and neutral sphingomyelinase) the increases in ceramide. These data suggest that, in this model, Bax is activated early in the process and that its increase is sustained till 12h after kainic acid injection which is the time of first appearance of caspase-3 activation and TUNEL positivity, and that SMases contribute to increases in ceramide levels during and after status epilepticus.

A series of adenine-copper complexes (1-6) with various ligands (Cl(-), SCN(-), BF(4)(-) and acac [acetylacetonate ion]) have been synthesized and characterized by elemental analysis, infrared spectroscopy and thermal analysis. Among the six complexes only complex (1), Cu(2)(adenine)(4)Cl(4).2EtOH (abbreviated as Cu-Ad), demonstrated some toxic effect on different cell lines. In vitro investigations of the biological effect of Cu-Ad complex have shown that it: (1) binds genomic DNA; (2) decreases significantly, the viability of cells in culture in a concentration (15-125 microM)-dependant manner; an estimated IC(50) of: 45 microM with HepG2; 73 microM with C2C12; 103 microM with NIH3T3; and 108 microM with MCF7. Cu-Ad had no effect on A549 cells; (3) inhibits Taq polymerase-catalyzed reaction; (4) inhibits the binding of the transcription factor GATA-5 to labeled DNA probes; (5) inhibits mitochondrial NADH-UQ-reductase with an estimated IC(50) of 2.8 nmol, but had no effect on succinate dehydrogenase activity; (6) increases reactive oxygen species (60%) at 45 microM Cu-Ad; and (7) decreases ATP (80%) at 50 microM Cu-Ad. The new compound Cu(2)(adenine)(4)Cl(4).2EtOH (Cu-Ad), belongs to a class of copper-adenylate complexes that target many biochemical sites and with potential anti-cancer activity.

This study investigates the potential of essential oils from commonly used medical and culinary herbs in Lebanon as an environmentally safe measure to control the seaside mosquito, Ochlerotatus caspius. The composition of essential oils extracted from parsley seeds and leaves, alpine thyme inflorescences, anis seeds, and coriander fruits were analyzed by GC–MS, and the major components of these oils were found to be thymol, sabinene, carvacrol, anethole, and linalool, respectively. Mosquito larvicidal assays were conducted to evaluate the LC 50 and LC 90 after 24 and 48 h of the essential oils and their major constituents. All of the tested oils proved to have strong larvicidal activity (LC 50 : 15–156 ppm) against Oc. caspius fourth instars, with the most potent oil being thyme inflorescence extract, followed by parsley seed oil, aniseed oil, and then coriander fruit oil. Toxicity of each oil major constituent was also estimated and compared to a reported larvicidal compound, eugenol.

This project assesses the treatment role with insulin and (or) angiotensin II receptor subtype-1 (AT1-R) blocker (ARB) on insulin receptor and endothelin-1 receptor subtype (ETA-R and ETB-R) regulation in rat hearts suffering from insulin-dependent diabetes mellitus (IDDM). Animals were divided into 6 groups: groups 1, 3, and 5 were controls consisting of normal, diabetic (streptozotocin-treated, once at 0 time), and diabetic supplemented daily with insulin, respectively, whereas groups 2, 4, and 6 were the controls treated daily with losartan. One month after enrollment, rats were sacrificed and samples of cardiac tissue were snapped frozen for immunostaining and Western blotting. Insulin receptor density was observed to be upregulated in the cardiomyocytes of diabetic animals, but downregulated with insulin supplementation alone. Cotreatment with insulin and an ARB resulted in drastic increase in insulin-receptor density in the diabetic rats. In addition, expression of ETA-R in cardiomyocytes was upregulated and was consistently maintained within the various treatment modalities. However, ETB-R expression was significantly reduced in the diabetic group treated with both insulin and an ARB. The changes in the expression of the insulin, the ETA-Rs, and the ETB-Rs at the various sites of the myocardium and the effect of both insulin treatment and blockade of the AT1-R explain the new benefits related to the halting of myocardial remodeling in IDDM rats.

This study reports on the regulation and remodeling role of endothelin-1 (ET-1) and its receptor subtypes, ET(A)-Rs/ET(B)-Rs, at the coronary endothelium (CE) and cardiomyocyte (CM) sites. It is carried out in normal and normotensive rats with streptozotocin-induced diabetes mellitus receiving different treatment modalities. Normal rats were divided into two groups, namely a placebo (N) and a losartan-treated (NL), and diabetic rats into four groups receiving placebo (D), insulin-treated (DI), losartan-treated (DL), and insulin/losartan-treated (DIL) respectively. Binding kinetics of ET-1 to ET(A)-Rs/ET(B)-Rs on CE and CMs were assessed in the above groups to try to explain the effect of therapeutic doses of an angiotensin II receptor subtype-1 blocker on the dynamics of this ligand and its receptor in insulin supplemented diabetic animals. Each group was divided into two subgroups: CHAPS-untreated and CHAPS-treated rat hearts perfused with [125I]ET-1 to respectively estimate ET-1 binding affinity (tau = 1/k-n) to its receptor subtype(s) on CE and CMs using mathematical modeling describing a 1:1 reversible binding stoichiometry. Heart perfusion results revealed that insulin treatment significantly decreased tau on CE but not on CMs in diabetic rats. In diabetics treated with losartan, an increase in tau value on CE but not on CMs was noted. Cotreatment of diabetic rats with insulin and losartan normalized tau on CE but decreased it on CMs. Western blot, using snap-frozen heart tissues, revealed increase in ET(A)-R density in all diabetic groups. However, significant decrease in ET(B)-R density was observed in all groups compared to the normal, and was reconfirmed by immunohistochemical analysis. In conclusion, coadministration of insulin and losartan in nonhypertensive animals suffering from diabetes type 1 may offer new cardiac protection benefits by improving coronary blood flow and cardiomyocyte contractility through modulating ET-1 receptor subtypes density and affinity at CE and CM sites.

Anise (Pimpinella anisum) has been used as a traditional aromatic herb in many drinks and baked foods because of the presence of volatile oils in its fruits commonly known as seeds. Hot water extracts of the seeds have been used also in folk medicine for their diuretic and laxative effect, expectorant and anti-spasmodic action, and their ability to ease intestinal colic and flatulence. The aim of this work was to study the effect of aniseed oil on transport processes through intestinal and renal epithelia and determine its mechanism of action. The essential oils were extracted from the seeds by hydrodistillation and analyzed by gas chromatography. Aniseed oil enhanced significantly glucose absorption from the rat jejunum and increased the Na+-K+ ATPase activity in a jejunal homogenate in a dose dependent manner. The oil, however, exerted no effect on water absorption from the colon and did not alter the activity of the colonic Na+-K+ ATPase. When added to drinking water, it reduced the volume of urine produced in the rat and increased the activity of the renal Na+-K+ ATPase even at extremely low concentrations. It was concluded that aniseed oil increases glucose absorption by increasing the activity of the Na+-K+ ATPase and consequently the sodium gradient needed for the sugar transport. Its anti-diuretic effect is also mediated through a similar mechanism in the kidney whereby a stimulation of the Na+-K+ pump increases tubular sodium reabsorption and osmotic water movement. The colonic Na+-K+ ATPase was however, resistant to the oil.

The effect of water extracts of cinnamon and clove on rat mitochondrial F(0)F1ATPase was investigated. Both spices stimulated ATP hydrolysis. Gas chromatography analysis of the water extracts, confirmed the presence of eugenol and cinnamaldehyde as major components in clove and cinnamon, respectively. Both components (1) stimulated ATPase significantly at concentrations equal or greater then 0.3 mM; (2) reduced mitochondrial membrane potential: a 50% decrease in Deltapsi was obtained at 7.56 and 11.6 micromoles/mg protein for eugenol and cinnmaldehyde, respectively; (3) inhibited NADH oxidase or complex I of the respiratory chain with a 50% inhibition at 15 and 20 micromoles/mg protein for eugenol and cinnamaldehyde respectively; (4) had no effect on succinate dehydrogenase activity. The study proposes the mitochondria as a target for the action of the spices resulting in derangement of mitochondrial functions, particularly at proton transferring sites.

Treatment of A549 cells with C(6)-ceramide resulted in a significant increase in the endogenous long chain ceramide levels, which was inhibited by fumonisin B1 (FB1), and not by myriocin (MYR). The biochemical mechanisms of generation of endogenous ceramide were investigated using A549 cells treated with selectively labeled C(6)-ceramides, [sphingosine-3-(3)H]d-erythro-, and N-[N-hexanoyl-1-(14)C]d-erythro-C(6)-ceramide. The results demonstrated that (3)H label was incorporated into newly synthesized long chain ceramides, which was inhibited by FB1 and not by MYR. Interestingly, the (14)C label was not incorporated into long chain ceramides. Taken together, these results show that generation of endogenous ceramide in response to C(6)-ceramide is due to recycling of the sphingosine backbone of C(6)-ceramide via deacylation/reacylation and not due to the elongation of its fatty acid moiety. Moreover, the generation of endogenous long chain ceramide in response to C(6)-ceramide was completely blocked by brefeldin A, which causes Golgi disassembly, suggesting a role for the Golgi in the metabolism of ceramide. In addition, the generation of endogenous ceramide in response to short chain exogenous ceramide was induced by d-erythro- but not l-erythro-C(6)-ceramide, demonstrating the stereospecificity of this process. Interestingly, several key downstream biological activities of ceramide, such as growth inhibition, cell cycle arrest, and modulation of telomerase activity were induced by d-erythro-C(6)-ceramide, and not l-erythro-C(6)-ceramide (and inhibited by FB1) in A549 cells, suggesting a role for endogenous long chain ceramide in the regulation of these responses.

Objective: Cancer represents the second leading cause of death after stroke and heart diseases. Plant extracts have long been used in traditional medicine for the prevention and treatment of many illnesses, including some types of cancer. The aim of this study was to evaluate the antiproliferative effects of ethyl acetate fractions of two Lebanese herbs: Inula viscosa (I. vis) and Inula vulgaris (I. vul). Methods: Plants were extracted with ethanol followed by ethyl acetate, then dried and tested on three cell lines including CaCO2, HepG 2 , and MCF 7 , to check for their viability and antiproliferative activity, using trypan blue exclusion and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. Proton ( 1 H) and carbon ( 13 C) nuclear magnetic resonance spectrometry (NMR) were used to identify the compounds that have been isolated from both Inula species. Results : The current findings were consistent for both trypan blue and MTT assays. The results showed that the most potent effect for I. vul was HepG2 (IC 50 20 µg/ml, 27 µg/ml), and for I. vis on MCF7 (9 µg/ml, 15 µg/ml) and CaCO 2 (12 µg/ml, 22 µg/ml) in the two mentioned assays respectively. However, insignificant differences were observed among the studied plants for each of the evaluated cells indicating comparable potencies. Quercetin, quercetin glycoside, and epicatechin derivatives were isolated by fractionation on column chromatography and identified using NMR spectroscopy. Conclusion: The antiproliferative activities of the two plants could be related to their content that is significant for high levels of secondary metabolites. The identification of those compounds is necessary to establish a relationship between their chemical structures and their activities.

We demonstrated previously an inhibitory effect of luminal leptin on glucose absorption in differentiated Caco-2 cells. Since this process is dependent on the Na(+) gradient established by the Na(+)/K(+)ATPase this work was undertaken to investigate if the ATPase is one of the hormone's targets. Fully differentiated Caco-2 cells were incubated with 10nM luminal leptin and the activity of the Na(+)/K(+) ATPase was assayed by measuring the amount of inorganic phosphate liberated. To elucidate the signaling pathway involved, the suspected mediators, namely PKC, p38MAPK, ERK and PI3K, were inhibited with specific pharmacological inhibitors and their implication was confirmed by determining changes in the protein expression of their active phosphorylated forms by Western blot analysis. Leptin reduced significantly the activity of the Na(+)/K(+) ATPase, by activating p38MAPK via inhibition of PKC, an upstream inhibitor of the kinase. ERK and PI3K are modulators of the pump and are not along the pathway activated by leptin but cross talk with it at the level of p38MAPK.

This study was aimed at identifying the molecular mechanisms by which ceramide inhibits telomerase activity in the A549 human lung adenocarcinoma cell line. C(6)-ceramide (20 microm) caused a significant reduction of telomerase activity at 24 h as detected using the telomeric repeat amplification protocol, and this inhibition correlated with decreased telomerase reverse transcriptase (hTERT) protein. Semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and Northern blot analyses showed that C(6)-ceramide significantly decreased hTERT mRNA in a time-dependent manner. Electrophoretic mobility shift and supershift assays demonstrated that the binding activity of c-Myc transcription factor to the E-box sequence on the hTERT promoter was inhibited in response to C(6)-ceramide at 24 h. These results were also confirmed by transient transfections of A549 cells with pGL3-Basic plasmid constructs containing the functional hTERT promoter and its E-box deleted sequences cloned upstream of a luciferase reporter gene. Further analysis using RT-PCR and Western blotting showed that c-Myc protein but not its mRNA levels were decreased in response to C(6)-ceramide at 24 h. The effects of ceramide on the c-Myc protein were shown to be due to a reduction in half-life via increased ubiquitination. Similar results were obtained by increased endogenous ceramide levels in response to nontoxic concentrations of daunorubicin, resulting in the inhibition of telomerase and c-Myc activities. Furthermore, the elevation of endogenous ceramide by overexpression of bacterial sphingomyelinase after transient transfections also induced the inhibition of telomerase activity with concomitant decreased hTERT and c-Myc protein levels. Taken together, these results show for the first time that both exogenous and endogenous ceramides mediate the modulation of telomerase activity via decreased hTERT promoter activity caused by rapid proteolysis of the ubiquitin-conjugated c-Myc transcription factor.

Genotype phenotype correlations in Wilson disease (WD) are best established in homozygous patients or in compound heterozygous patients carrying the same set of mutations. We determined the clinical phenotype of patients with WD carrying the c.2298_2299insC in Exon 8 (c.2299insC) or the p. Ala1003Thr missense substitution in Exon 13 mutations in the homozygous or compound heterozygous state. We investigated 76 members of a single large Lebanese family. Their genotypes were determined, and clinical assessments were carried out for affected subjects. We also performed a literature search retrieving the phenotypes of patients carrying the same mutations of our patients in the homozygous or compound heterozygous state. There were 7 consanguineous marriages in this family and the prevalence of WD was 8.9% and of carriers of ATP7B mutation 44.7%. WD was confirmed in 9 out of 76 subjects. All 9 had the c.2299insC mutation, 5 homozygous and 4-compound heterozygous with p. Ala1003Thr. Six of our patients had hepatic, 2 had neurologic and 1 had asymptomatic phenotype. Based on our data and a literature review, clear phenotypes were reported for 38 patients worldwide carrying the c.2299insC mutation. About 53% of those have hepatic and 29% have neurologic phenotype. Furthermore, there were 10 compound heterozygous patients carrying the p. Ala1003Thr mutation. Among those, 80% having c.2299insC as the second mutation had hepatic phenotype, and all others had neurologic phenotype. We hereby report an association between the c.2299insC mutation and hepatic phenotype and between the p. Ala1003Thr mutation and neurologic phenotype.

Previous reports have suggested that 1,25-dihydroxychole-calciferol regulated cellular differentiation via its effects on protein kinase C activity. This study examined the in vivo effects of ergocalciferol on the activity of protein kinase C, and whether the differentiation of crypt intestinal cells is dependent on the activation of this enzyme. Ergocalciferol in saline was injected intramuscularly into rats and the animals sacrificed 24 hr after fasting. Protein kinase C specific activity was determined from the rate of incorporation of 32p-ATP into protamine. Injections of 60 micrograms ergocalciferol/200 g of body wt, raised protein kinase C specific activity to 59818 +/- 4010 (SEM, n = 5) cpm 32p-protamine/min/mg cell protein, compared with a control of 46173 +/- 4612 (P0.0005). Optimal specific activities were seen within 72 hr of injection. Administration of 120 micrograms ergocalciferol/200 g of body wt, raised the concentrations of serum calcium to 9.8 and 10.4 mg/dl following the intramuscular injection by 24 and 72 hr, respectively, compared with a control of 7.7 mg/dl. Actinomycin D (intramuscular, 100 micrograms/200 g of body wt) together with ergocalciferol (120 micrograms/200 g of body wt) reduced protein kinase C activity by 51% 24 hr after injection. Cycloheximide blocked the activation, but when injected alone stimulated endogenous protein kinase C activity by 34% 24 hr injection. The study shows activation of crypt protein kinase C by ergocalciferol. The inhibition of activation by actinomycin D and cycloheximide suggests the involvement of both transcriptional and translational processes in this activation.

Caritis SN, 2012, AM J OBSTET GYNECOL, V207; Caritis SN, 2012, AM J OBSTET GYNECOL, V207; Caritis SN, 2011, AM J OBSTET GYNECOL, V205, p[40, e1]

Phenylketonuria is an autosomal recessive inborn error of metabolism resulting from phenylalanine hydroxylase deficiency. Genetic basis of phenylalanine hydroxylase deficiency has been reported in various European and Asian countries with few reports available in Arab populations of the Mediterranean region. This is the first pilot study describing phenotype and genotype of 23 Lebanese patients with phenylketonuria. 48% of the patients presented mainly with neurological signs at a mean age of 2 years 9 months, as newborn screening is not yet a nationwide policy. 56.5% of the patients had classical phenylketonuria. Thirteen different mutations were identified: splice site 52%, frameshift 31%, and missense 17% with no nonsense mutations. IVS10-11G>A was found mainly in Christians at high relative frequency whereas Muslims carried the G352fs and R261Q mutations. A rare splice mutation IVS7+1G>T, not described before, was identified in the homozygous state in one family with moderate phenylketonuria phenotype. Genotype-phenotype correlation using Guldberg arbitrary value method showed high consistency between predicted and observed phenotypes. Calculated homozygosity rate was 0.07 indicating the genetic heterogeneity in our patients. Our findings underline the admixture of different ethnicities and religions in Lebanon that might help tracing back the PAH gene flux history across the Mediterranean region.

Context: Alum is thought to induce inflammation resulting in the release of danger signals such as uric acid (UA) which in turn enhances the immune response to an antigen. Hydrogen peroxide (H 2 O 2 ) is produced as a byproduct in the purine catabolic pathway that leads to the production of UA. In addition, serum nitric oxide (NO) levels are increased in inflammation. Objective: To further explore the mechanism of action of alum, this study was designed to determine the effects of catalase and 1400W on the number of interleukin-4 (IL-4) and interferon-γ (IFN-γ) secreting spleen cells in mice given ovalbumin (OVA) with alum. Materials and methods: Groups of BALB/c mice were injected intraperitoneally with alum + OVA, alum, OVA, catalase, or 1400W. Other groups were treated with catalase or 1400W and given alum + OVA. The number of IL-4 and IFN-γ secreting spleen cells were determined at days 4 and 7 postinjection by enzyme-linked immunosorbent spot (ELISPOT). Results: Catalase and 1400W caused a decrease in the number of IL-4 secreting spleen cells induced by alum + OVA. 1400W caused a decline in the IFN-γ secreting spleen cells induced by alum + OVA. Catalase caused an increase in IFN-γ secreting spleen cells. Discussion and conclusion: It appears that H 2 O 2 and NO are needed for alum-induced production of a T-helper 2 cytokine. NO also appears to be needed, whereas H 2 O 2 appeared to inhibit an alum-induced production of a T-helper 1 cytokine. These results might explain why alum is mainly a promoter of a T-helper 2 response.

We investigate in this study the biochemical effects on cells in culture of two commonly used fragrance chemicals: lyral and lilial. Whereas both chemicals exerted a significant effect on primary keratinocyte(s), HaCat cells, no effect was obtained with any of HepG2, Hek293, Caco2, NIH3T3, and MCF7 cells. Lyral and lilial: (a) decreased the viability of HaCat cells with a 50% cell death at 100 and 60 nM respectively; (b) decreased significantly in a dose dependant manner the intracellular ATP level following 12-h of treatment; (c) inhibited complexes I and II of electron transport chain in liver sub-mitochondrial particles; and (d) increased reactive oxygen species generation that was reversed by N-acetyl cysteine and trolox and the natural antioxidant lipoic acid, without influencing the level of free and/or oxidized glutathione. Lipoic acid protected HaCat cells against the decrease in viability induced by either compound. Dehydrogenation of lyral and lilial produce α,β-unsaturated aldehydes, that reacts with lipoic acid requiring proteins resulting in their inhibition. We propose lyral and lilial as toxic to mitochondria that have a direct effect on electron transport chain, increase ROS production, derange mitochondrial membrane potential, and decrease cellular ATP level, leading thus to cell death.

Most patients with Wilson's disease (WD) are compound heterozygote, which complicates establishing genotype-phenotype correlations. We identified five patients who presented with early and/or severe hepatic disease who are homozygous for W939C missense mutation on exon 12 of ATP7B. We therefore conducted a meta-analysis to determine the phenotype of patients homozygous for missense or nonsense mutations in all ATP7B exons.The meta-analysis showed that 69% and 31% of patients are homozygous for H1069Q and non-H1069Q mutations, respectively. Compared to patients with H1069Q, those with non-H1069Q mutations were significantly more likely to have a hepatic phenotype, severe liver disease, a mixed phenotype, and less likely to have a neurologic phenotype. Compared to patients with nonsense mutations, those with non-H1069Q ones were equally likely to present with a hepatic phenotype and to have severe liver disease. Mean age at symptom onset in the non-H1069Q versus the H1069Q group was 15.5 versus 20.5years (p0.001).Our data suggest that mutation W939C and other non-H1069Q missense mutations are associated with early disease onset, a hepatic phenotype, and a high risk of hepatic failure in homozygous patients. Early identification of such patients by genetic screening is important for timely initiation of treatment and prevention of complications.

Dibutyltin-3-hydroxyflavone bromide, Bu2Sn(of), is a fluorescent probe inhibitor of mitochondrial F1F0ATPase which reacts with and titrates a component of F0 with marked fluorescence enhancement and reacts similarly with chloroplast CF1CF0 and V-ATPases. Its use to monitor the interactions of other F0 inhibitors (venturicidin, oligomycin, DCCD) with F1F0ATPase, both membrane-bound and purified by solubilization is described. Trialkyltins (Bu3SnCl) back-titrate all Bu2Sn(of) interaction sites; whereas the macrolide inhibitor venturicidin backtitrates 60±5% and oligomycin only 30±3% of Bu2Sn(of) interaction sites. Bafilomycin, the macrolide inhibitor of V-ATPases, is inactive in this assay. DCCD acts in a different fashion from the other inhibitors. Current and potential applications of this fluorescent probe in mitochondrial bioenergetics and biogenesis are discussed.

Dibutyltin-3-hydroxyflavone bromide [Bu2SnBr-(of)] is a fluorescent inhibitor (excitation max, 395 nm; emission max., 450 nm) of mitochondrial F1F0–ATPase which does not inhibit F1–ATPase. Bu2SnBr(of) binding to mitochondria and submitochondrial particles results in a 10-fold fluorescence enhancement which correlates with the amount of F1F0–ATPase in the inner membrane. Enhancement is not affected by respiratory-chain substrates, ATP, uncoupling agents, ionophores or respiratory-chain inhibitors. It is reversed by tributyltin chloride (Bu3SnCl), indicating competition for a common triorganotin-binding site on the F0 segment of F1F0–ATPase. Enhancement is not reversed by dialkyltins, monoalkyltins, tributyl-lead acetate, efrapeptin or oligomycin. Bu2SnBr(of) is thus a new class of fluorescent probe of the F0 segment of F1F0–ATPase which titrates F0.

Dibutyltin-3-hydroxyflavone bromide, Bu2Sn(of), is a fluorescent probe inhibitor of mitochondrial F1F0ATPase which reacts with and titrates a component of F0 with marked fluorescence enhancement and reacts similarly with chloroplast CF1CF0 and V-ATPases. Its use to monitor the interactions of other F0 inhibitors (venturicidin, oligomycin, DCCD) with F1F0ATPase, both membrane-bound and purified by solubilization is described. Trialkyltins (Bu3SnCl) back-titrate all Bu2Sn(of) interaction sites; whereas the macrolide inhibitor venturicidin backtitrates 60±5% and oligomycin only 30±3% of Bu2Sn(of) interaction sites. Bafilomycin, the macrolide inhibitor of V-ATPases, is inactive in this assay. DCCD acts in a different fashion from the other inhibitors. Current and potential applications of this fluorescent probe in mitochondrial bioenergetics and biogenesis are discussed.

Fluorescent 5-coordinate organotin-flavone complexes of 3-hydroxyflavone (Hof) and 3,5,7,2′,4′,-pentahydroxyflavone (morin) are good inhibitors of mitochondrial F1F0ATPase but do not inhibit F1-ATPase and they have been examined as possible fluorescent probes of F1F0ATPase. R2SnX (morin) complexes exhibit low fluorescence enhancement on binding to mitochondrial membranes with no displacement by equimolar tributyltin. In contrast R2SnX (of) complexes exhibit high fluorescence enhancement whose extent is variable and is displacable by equimolar tributyltin. Fluorescence enhancement by R2SnX (of) complexes correlates with the ATPase I50 values. Dialkyltin-3-hydroxy flavone, R2SnX(of), complexes act as a new class of fluorescent probes which titrate the F0 segment of F1F0ATPase.

Ceramide is known to induce programmed cell death (PCD) in neural and non-neural tissues and to increase after kainic acid (KA) status epilepticus (SE). Ceramide increases have been shown to depend on NMDA receptor activation in the KA model, but these changes have not been studied in the lithium pilocarpine (LiPC) model. Thus, the purpose of this study was to determine if hippocampal ceramide levels increase after LiPC induced SE and if NMDA receptor blockade prevents PCD and any such ceramide increases. We found that LiPC induced SE resulted in ceramide increases and DNA fragmentation in the hippocampus of adult, P21, and P7 rats. The administration of MK-801, the NMDA receptor antagonist, in adults, 15 min prior to pilocarpine, prevented ceramide increases, and DNA fragmentation.

C18-sphingosine 1-phosphate (C18-SPP) is a sphingolipid with important functional and structural roles in cells. In this paper we report a new capillary electrophoresis technique that is coupled to a laser-induced fluorescence detection (CE-LIF) method to quantify the level of C18-SPP in biological samples. The method utilizes a commercial standard C17-sphingosine 1-phosphate (C17-SPP) derivatized with naphthalene-2,3-dicarboxaldehyde (NDA), a fluorogenic dye used to label primary amines. The detection limit for the C17-SPP NDA was 0.54 fmol. We quantified the C18-SPP in leukemic human cells before and after irradiation by gamma rays. We demonstrated that the amounts of this sphingolipid decreased after the irradiation. In a second part of this work, we used the technique to evaluate the ability of a novel transparent fused-silica capillary, which allows the use of fused-silica capillaries without burning a window. Solarization, homogeneity, and sensitivity were studied using this capillary. The results demonstrate that this new durable capillary can provide a sensitive and reproducible quantitation procedure for CE-LIF studies.

The tumor suppressor protein p53 and the putative lipid tumor suppressor ceramide play pivotal roles in inducing cell cycle arrest or in driving the cell towards apoptosis. Previously we had shown that, in a p53-dependent model of cell death, ceramide accumulated in a p53-dependent manner [Dbaibo GS, Pushkareva MY, Rachid RA, Alter N, Smyth MJ, Obeid LM, Hannun YA. J Clin Invest 1998;102:329–339]. In the current study, we investigated the biochemical pathways by which ceramide accumulated following p53 up-regulation. In both Molt-4 LXSN leukemia cells exposed to γ-irradiation and in EB-1 colon cancer cells treated with ZnCl 2 , p53 up-regulation led to de novo ceramide synthesis with predominance of N -palmitoylsphingosine (C16-ceramide) synthesis. The activation of the de novo pathway was not associated with increased activity of the key enzyme serine palmitoyltransferase (SPT) but rather with the increased activity of ceramide synthase. Furthermore, transcriptional up-regulation of the palmitoyl-specific Lass5 ceramide synthase gene was observed in Molt-4 but not in EB-1 cells. The SPT inhibitor ISP-1 or the ceramide synthase inhibitor fumonisin B1 led to substantial inhibition of ceramide accumulation in response to p53 up-regulation. Other biochemical pathways of ceramide generation such as sphingomyelinase activation were examined and found unlikely to contribute to p53-dependent ceramide formation. These studies indicate that p53 specifically drives de novo ceramide synthesis by activation of a ceramide synthase that favors the synthesis of N -palmitoylsphingosine.

A rapid, reproducible and accurate high-performance liquid chromatographic (HPLC) method for the quantitative determination of sphingomyelin in rat brain was developed and validated using normal-phase silica gel column, acetonitrile–methanol–water (65:18:17 (v/v)) at a flow rate of 1 ml/min, isocratic elution, UV detection at 207 nm and 1,2-dimyristoyl- sn -glycero-3-phosphocholine as an internal standard. Total run time was 10.0 min. The calibration curve was linear over the range of 0.025–0.4 mg/ml sphingomyelin ( R 2 > 0.99). The intra-day coefficient of variation ranged from 1.4% to 2.2%. The average inter-day coefficient of variation over a period of 4 days was 3.1%. The practical limit of detection was 0.005 mg/ml with a quantification limit of 0.01 mg/ml.

Sphingolipids (SLs) have a biomodulatory role in physiological as well as pathological cardiovascular conditions. This study aims to assess the variation of SL mediators and metabolizing enzymes in the growing and hypoxic rat heart. Sprague–Dawley rats were placed in a hypoxic environment at birth. Control animals remained in room air. In control animals, activities of acidic-sphingomyelinase (A-SMase), sphingomyelin synthase (SMS), glucosylceramide synthase (GCS), and ceramidase decreased with age in both ventricles whereas activity of neutral-sphingomyelinase (N-SMase) increased with age. Hypoxic RV mass was 171 and 229% that of controls, at 4 and 8 weeks, respectively. This was accompanied by an increase in RV myocardial ceramide synthesis, consumption and breakdown, with a net effect of suppression of ceramide accumulation and increase in diacylglycerol (DAG) concentration. In addition, significant increase in activities of: A-SMase by 26 and 29%, SMS by 108 and 40%, and ceramidase by 66 and 35%, in the hypoxic RV rats as compared to controls, was noted at 4 and 8 weeks of age, respectively. Sphingolipids and their regulating enzymes appear to play a role in adaptive responses to chronic hypoxia in the neonatal rat heart.

Objectives To assess the role of insulin or an angiotensin II receptor antagonist (losartan), or both, in preventing cardiomyocyte damage in rats suffering from insulin-dependent diabetes mellitus (IDDM), and to correlate it with insulin receptor modulation at the cardiomyocyte, coronary endothelium and skeletal muscle cell level. Design Animals were divided into groups of normal rats, diabetic rats, and diabetic rats given insulin, each subdivided into a control group and an experimental group treated with losartan. Methods The animals were killed 1 month after enrollment to the study. Perfusion of the heart with iodine-125-labelled insulin was carried out for all the groups and the binding kinetics of insulin to its receptors on the coronary endothelial cells and the cardiomyocytes were determined using a physical/mathematical model. In addition, tissue samples from the heart and intercostal skeletal muscle were snap frozen and used for histological, indirect immunofluorescence and western blot analysis. Results Cardiac muscle from diabetic animals exhibited diffuse cardiomyopathic changes consisting of widespread vacuolation, loss of striation and cellular hypertrophy, which were reduced and even prevented by treatment with insulin and losartan. In addition, losartan seemed to mediate the upregulation of insulin receptor density on cardiomyocytes and skeletal muscle, and increase insulin receptor affinity at the coronary endothelial site. Finally, treatment with losartan induced a significant decrease in glucose concentrations in the diabetic group compared with the appropriate controls. Conclusions Addition of losartan to the standard insulin treatment in non-hypertensive animals with IDDM offers new benefits concerning cardiac protection and prevention of damage. This may be attributed, in part, to insulin receptor density and sensitization.