Anti-Angiogenic and Cytotoxicity Effects of Selachyl Alcohol Analogues

BACKGROUND: The active ingredients in the shark liver oil (SLO) mixture were found to be a group of etherlinked glycerol known as alkylglycerols (AKGs). During the last century, initial clinical use of the SLO mixture was for treating leukemias and later preventing radiation sickness from cancer x-ray therapy. Selachyl alcohol is one of the most abundant AKGs in the SLO mixture and it displayed strong activity in reducing lung metastasis number on a model of grafted tumor in mice (Lewis lung carcinoma cells). OBJECTIVES: In this study, selachyl alcohol analogue containing methoxyl (7), gem-difluorinated (8), azide (9) and hydroxyl (10) group at the 12 position in the alkyl chain were synthesized and compared regarding their cytotoxicity and anti-migratory effects on Human Umbilical Vein Endothelial Cell line. METHODS: AKGs 7-10 were synthesized according to the literature procedure. The cytotoxicity of the studied AKGs was evaluated by the MTT test and Human Umbilical Vein Endothelial Cell line (HUVEC) was used as an in vitro model to evaluate their anti-migratory effects. RESULTS: The four AKGs have substantially the same toxicity threshold (≥ 12 µM), whereas they have an anti-migratory activity significantly different on endothelial cells. AKGs 9 and 10 significantly reduce the chemotactic migration induced by VEGF, but analogue (10) containing the hydroxyl group at the 12 position in the alkyl chain was the most potent anti-VEGF inhibitor. CONCLUSION: We presented here a series of four synthetic selachyl alcohol analogues, among which AKGs 9 and 10 showed the ability to inhibit endothelial cell migration. The relationship structures and anti-VEGF effects of these analogues were also evaluated and discussed. Unnatural synthesized AKGs could be explored as one new source of anticancer agents.

Disturbed expression of autophagy genes in blood of Parkinson's disease patients.

Parkinson's disease (PD) is a common neurodegenerative disorder which affects dopaminergic neurons leading to alteration of numerous cellular pathways. Several reports highlight that PD disturbs also other cells than CNS neurons including PBMCs, which could lead, among other things, to dysfunctions of immune functions. Because autophagy could be altered in PD, a monocentric pilot study was performed to quantify the transcripts levels of several autophagy genes in blood cells. MAP1LC3B, GABARAP, GABARAPL1, GABARAPL2 and P62/SQSTM1 were found to be overexpressed in patients. On the contrary, transcripts for HSPA8 and GAPDH were both decreased. Expression of MAP1LC3B and GABARAP was able to successfully segregate PD patients from healthy controls. The accuracy of this segregation was substantially increased when combined expressions of MAP1LC3B and GAPDH or GABARAP and GAPDH were used as categorical variables. This pilot study suggests that autophagy genes expression is dysregulated in PD patients and may open new perspectives for the characterisation of prediction markers.

Dysregulation of apoptosis and autophagy gene expression in peripheral blood mononuclear cells of efficiently treated HIV-infected patients.

OBJECTIVE: We measure the transcript levels of the proapoptotic GALIG, antiapoptotic MCL1 genes and those of the autophagy genes BECN1, MAP1LC3B, ATG9a, P62/SQSTM1, GABARAP, GABARAPL1 and GABARAPL2 to define if mRNA alteration can characterize HIV-infected patients effectively treated with combined antiretroviral therapy (cART). DESIGN: Monocentric pilot study conducted on peripheral blood mononuclear cell (PBMC) of 40 uninfected donors and 27 HIV-positive patients effectively treated by cART for at least 8.4 years. METHODS: Transcripts of the various genes were quantified by reverse transcription (RT)-quantitative PCR (qPCR) and RT-droplet digital PCR and compared using the standard statistical Mann-Whitney U test and machine learning algorithms. RESULTS: A concomitant overexpression of GALIG and MCL1 is detected in PBMC of effectively cART-treated patients. Overexpression of MAP1LC3B and GABARAPL1 is also measured, whereas BECN1 is underexpressed. Finally, accurate classification (94.5%) of our PBMC samples as HIV-negative donors or HIV-positive cART-treated is obtained in three separate machine-learning algorithms with GABARAPL1 and ATG9a as input variables. CONCLUSION: cART-treated HIV patients display altered transcript levels for three genes of basal autophagy. Some of these alterations may appear contradictory: BECN1 and ATG9a, both key actors in the formation of mammalian autophagosome, exhibit decreased amount of transcripts, whereas mRNA from the ATG8 family increase. Given the known role of impaired basal autophagy in immune senescence and chronic inflammation, the functional significance of our findings should be explored in larger studies.

Recombinant yeast and human cells as screening tools to search for antibacterial agents targeting the transcription termination factor Rho.

The alarming issue of antibiotic resistance expansion requires a continuous search for new and efficient antibacterial agents. Here we describe the design of new tools to screen for target-specific inhibitors of the bacterial Rho factor directly inside eukaryotic cells. Rho factor is a global regulator of gene expression which is essential to most bacteria, especially Gram-negative. Since Rho has no functional or structural homolog in eukaryotes, it constitutes a valuable and well known bacterial target as evidenced by its inhibition by the natural antibiotic, Bicyclomycin. Our screening tools are based on perturbation of mRNA processing and packaging reactions in the nucleus of eukaryotic cells by the RNA-dependent helicase/translocase activity of bacterial Rho factor leading to a growth defect phenotype. In this approach, any compound that impedes Rho activity should restore growth to yeast or human cells expressing Rho protein, providing valuable means to screen for target-specific antibacterial agents within the environment of a eukaryotic cell. The yeast tool expressing E. coli Rho factor was validated using Bicyclomycin as the control antibacterial agent. The validation of the screening tool was further extended with a stable human cell line expressing Rho factor conditionally. Finally, we show that Rho factors from different bacterial pathogens can also be designed as yeast-based screening tools which can reveal subtle variations in the functional features of the proteins.

Interaction of Alpha-synuclein with Cytogaligin, a protein encoded by the proapoptotic gene GALIG.

GALIG, an internal gene to the human galectin-3 gene, encodes two distinct proteins, Mitogaligin and Cytogaligin through translation of a unique mRNA in two overlapping alternative reading frames. When overexpressed GALIG induces apoptosis. In cultured cells, Mitogaligin destabilizes mitochondria membranes through interaction with cardiolipin. Little is known regarding the role of Cytogaligin. This protein displays multiple subcellular localizations; cytosol, nucleus, and mitochondria. We illustrate here that Cytogaligin is also secreted in the extracellular medium. Cytogaligin is shown to interact with α-Synuclein, the major component of Lewy bodies in Parkinson's disease. Overexpression of Cytogaligin reduces α-Synuclein dimerization raising a possible role in the evolution of α-Synuclein aggregation, a key molecular event underlying the pathogenesis of Parkinson's disease.

Opposing Mcl-1, the GALIG proapoptotic gene is upregulated as neutrophils die and underexpressed in Acute Myeloid Leukemia cells.

GALIG gene expression induces apoptosis in cultured cells through a pathway still under investigation. It is highly expressed in leukocytes but weakly detectable in bone marrow, suggesting a role in the myeloid lineage homeostasis. We show here that GALIG-induced cell death is counteracted by the overexpression of MCL-1, a pro-survival member of the Bcl2 family. Moreover, during spontaneous neutrophil apoptosis, a substantial increase in GALIG gene expression is observed: GALIG still opposes MCL-1. Finally, in bone marrow and peripheral blood cells from patients with Acute Myeloid Leukemia type 2, the level of GALIG transcripts is massively down-regulated when compared to their normal counterparts, while MCL-1 is expressed to the same extent. These data suggest that GALIG could be a key player in the cell death pathway involved in leukocytes homeostasis and myeloid malignancies.

The addressing fragment of mitogaligin: first insights into functional and structural properties.

Mitogaligin is a mitochondrion-targeting protein involved in cell death. The sequence of the protein is unrelated to that of any known pro- or antiapoptotic protein. Mitochondrial targeting is controlled by an internal sequence from residues 31 to 53, and although this sequence is essential and sufficient to provoke cell death, the precise mechanism of action at the mitochondrial membrane remains to be elucidated. Here, by focusing on the [31-53] fragment, we first assessed and confirmed its cell cytotoxicity by microinjection. Subsequently, with the aid of membrane models, we evaluated the impact of the membrane environment on the 3D structure of the peptide and on how the peptide is embedded and oriented within membranes. The fragment is well organized, even though it does not contain a canonical secondary structure, and adopts an interfacial location. Structural comparison with other membrane-interacting Trp-rich peptides demonstrated similarities with the antimicrobial peptide tritrpcidin.

Mitochondrial complex I deficiency of nuclear origin II. Non-structural genes.

Complex I deficiency is the most frequent cause of respiratory chain diseases. This large multiprotein complex is composed in human of 45 structural subunits, of which 7 are mitochondrial-encoded and 38 are nuclear-encoded. Most of the pathological mutations responsible for complex I deficiencies have been identified to date in complex I structural subunits. Numerous studies from last decade gave some insight into the biogenesis of this huge multi subunit complex of double genetic origin. A sequential incorporation of the structural subunits as well as ten complex I assembly factors has been described. Here, we present a short overview of the human complex I biogenesis and we review the pathological mutations identified to date in eight of the ten known complex I assembly factors.

Mitochondrial complex I deficiency of nuclear origin I. Structural genes.

Complex I (or NADH-ubiquinone oxidoreductase), is by far the largest respiratory chain complex with 38 subunits nuclearly encoded and 7 subunits encoded by the mitochondrial genome. Its deficiency is the most frequently encountered in mitochondrial disorders. Here, we summarize recent data obtained on architecture of complex I, and review the pathogenic mutations identified to date in nuclear structural complex I genes. The structural NDUFS1, NDUFS2, NDUFV1, and NDUFS4 genes are mutational hot spot genes for isolated complex I deficiency. The majority of the pathogenic mutations are private and the genotype-phenotype correlation is inconsistent in the rare recurrent mutations.

Ex vivo and in vivo diffusion of ropivacaine through spinal meninges: influence of absorption enhancers.

Following epidural administration, cerebrospinal fluid bioavailability of local anesthetics is low, one major limiting factor being diffusion across the arachnoid mater barrier. The aim of this study was to evaluate the influence of absorption enhancers on the meningeal permeability of epidurally administered ropivacaine. Five enhancers known for their ability to increase drug permeability via transcellular and/or paracellular pathways, i.e. palmitoyl carnitine, ethylenediaminetetraacetic acid, sodium caprate, dodecylphosphocholine and pentylglycerol, were tested ex vivo on fresh specimen of meninges removed from cervical to lumbar level of rabbit spine following laminectomy and placed in diffusion chambers. Among them, sodium caprate lead to the best permeability improvement for both marker and drug (440% and 112% for mannitol and ropivacaine, respectively) and was therefore selected for in vivo study in a sheep model using microdialysis technique to evaluate epidural and intrathecal ropivacaine concentrations following epidural administration. Resulting dialysate and plasma concentrations were used to calculate pharmacokinetic parameters. Following sodium caprate pre-treatment, ropivacaine intrathecal maximal concentration (Cmax) was 1.6 times higher (78 ± 16 µg ml(-1) vs 129 ± 26 µg ml(-1), p<0.05) but the influence of the absorption enhancer was only effective the first 30 min following ropivacaine injection, as seen with the significantly increase of intrathecal AUC(0-30 min) (1629 ± 437 µg min ml(-1) vs 2477 ± 559 µg min ml(-1), p<0.05) resulting in a bioavailable fraction 130% higher 30 min after ropivavaine administration. Co-administration of local anesthetics with sodium caprate seems to allow a transient and reversible improvement of transmeningeal passage into intrathecal space.

Which alkylglycerols from shark liver oil have anti-tumour activities?

Alkylglycerols (alkyl-Gro) are ether lipids abundant in shark liver oil (SLO), and oral SLO or alkyl-Gro mix from this source have several in vivo biological activities including stimulation of haematopoiesis an immunological defences, or anti-tumour and anti-metastasis activities in vivo. Composition of natural alkyl-Gro mix contains several alkyl-Gro varying by chain length and unsaturation, and individual anti-tumour activity of each molecule present in natural mix remained unknown. We synthesized six prominent constituents of natural alkyl-Gro mix, namely 12:0, 14:0 16:0, 18:0, 16:1 n-7, and 18:1 n-9 alkyl-Gro. Using an in vivo model of grafted tumour in mice (3LL cells), we studied and compared the oral anti-tumour and anti-metastasis activities of each of these 6 alkyl-Gro. 16:1 and 18:1 alkyl-Gro showed strong activity in reducing lung metastasis number, while saturated alkyl-Gro had weaker (16:0) or no (12:0, 14:0, 18:0) effect. Spleen weights at day 20 after graft were also measured and showed tremendous variations depending on the treatment. Tumour graft resulted in a raise in spleen weight in control group, this raise was nearly abolished in 16:1 and 18:1 alkyl-Gro-treated mice, and was reduced in 14:0 and 16:0 alkyl-Gro-treated mice. Conversely, 18:0 alkyl-Gro-treated mice showed spleen weigh raise as compared with untreated grafted mice. These new data demonstrate a prominent role of unsaturation in the anti-tumour activities of alkyl-Gro.

MSM law in francophone Africa and the fight against AIDS: the hypocrisy of certain countries.

In addition to being the targets of frequent discrimination and violence,African men who have sex with men (MSM) are being hit hard by the HIV/AIDS epidemic. Although there is still insufficient research regarding the methods of HIV transmission in sub-Saharan Africa, several studies show that the prevalence of HIV infection among MSM is more than ten times higher than among the general population.

Multiple beneficial health effects of natural alkylglycerols from shark liver oil.

Alkylglycerols (alkyl-Gro) are ether lipids abundant in the liver of some elasmobranch fish species such as ratfishes and some sharks. Shark liver oil from Centrophorus squamosus (SLO), or alkyl-Gro mix from this source, have several in vivo biological activities including stimulation of hematopoiesis and immunological defences, sperm quality improvement, or anti-tumor and anti-metastasis activities. Several mechanisms are suggested for these multiple activities, resulting from incorporation of alkyl-Gro into membrane phospholipids, and lipid signaling interactions. Natural alkyl-Gro mix from SLO contains several alkyl-Gro, varying by chain length and unsaturation. Six prominent constituents of natural alkyl-Gro mix, namely 12:0, 14:0, 16:0, 18:0, 16:1 n-7, and 18:1 n-9 alkyl-Gro, were synthesized and tested for anti-tumor and anti-metastatic activities on a model of grafted tumor in mice (3LL cells). 16:1 and 18:1 alkyl-Gro showed strong activity in reducing lung metastasis number, while saturated alkyl- Gro had weaker (16:0) or no (12:0, 14:0, 18:0) effect. Multiple compounds and mechanisms are probably involved in the multiple activities of natural alkyl-Gro.

The mitogaligin protein is addressed to the nucleus via a non-classical localization signal.

Mitogaligin, a protein encoded by galig, an internal cytotoxic gene of the galectin-3 locus, is mostly a mitochondrial protein. Mitochondrial targeting is due to an already identified mitochondrial localization signal. Interaction of mitogaligin with mitochondria leads to cytochrome c cytosolic leakage and ultimately to cell death. We have previously pointed out that mitogaligin can also be directed to the nucleus when the mitochondrial addressing signal is inactivated, indicating a possible dual intracellular localization of the protein. When expressed in the nucleus, mitogaligin exhibits also apoptotic properties leading to cell death. In this report, we show that nuclear addressing of mitogaligin depends on a sequence differing from classical signals containing basic, lysine or proline-tyrosine rich residues. The signal consists of a long sequence of amino acids residues based on a series of a short repetitive degenerated sequence.