Comparative analysis of nonaspanin protein sequences and expression studies in zebrafish.

Nonaspanins constitute a family of proteins, also called TM9SF, characterized by a large non-cytoplasmic domain and nine putative transmembrane domains. This family is highly conserved through evolution and comprises three members in Saccharomyces cerevisiae, Dictyostelium discoideum, and Drosophila melanogaster, and four members are reported in mammals (TM9SF1-TM9SF4). Genetic studies in Dictyostelium and Drosophila have shown that TM9SF members are required for adhesion and phagocytosis in innate immune response, furthermore, human TM9SF1 plays a role in the regulation of autophagy and human TM9SF4 in tumor cannibalism. Here we report that the zebrafish genome encodes five members of this family, TM9SF1-TM9SF5, which show high level of sequence conservation with the previously reported members. Expression analysis in zebrafish showed that all members are maternally expressed and continue to be present throughout embryogenesis to adults. Gene expression could not be regulated by pathogen-associated molecular patterns such as LPS, CpG, or Poly I:C. By bioinformatic analyses of 80 TM9SF protein sequences from yeast, plants, and animals, we confirmed a very conserved protein structure. An evolutionary conserved immunoreceptor tyrosine-based inhibition motif has been detected in the cytoplasmic domain between transmembrane domain (TM) 7 and TM8 in TM9SF1, TM9SF2, TM9SF4 and TM9SF5, and at the extreme C-terminal end of TM9SF4. Finally, a conserved TRAF2 binding domain could also be predicted in the cytoplasmic regions of TM9SF2, TM9SF3, TM9SF4, and TM9SF5. This confirms the hypothesis that TM9SF proteins may play a regulatory role in a specific and ancient cellular mechanism that is involved in innate immunity.

Senescence and Cancer: Role of Nitric Oxide (NO) in SASP.

Cellular senescence is a cell state involved in both physiological and pathological processes such as age-related diseases and cancer. While the mechanism of senescence is now well known, its role in tumorigenesis still remains very controversial. The positive and negative effects of senescence on tumorigenesis depend largely on the diversity of the senescent phenotypes and, more precisely, on the senescence-associated secretory phenotype (SASP). In this review, we discuss the modulatory effect of nitric oxide (NO) in SASP and the possible benefits of the use of NO donors or iNOS inducers in combination with senotherapy in cancer treatment.

Glyceryl trinitrate­induced cytotoxicity of docetaxel­resistant prostatic cancer cells is associated with differential regulation of clusterin.

Metastatic castration resistant prostate cancer (mCRPC) relapse due to acquired resistance to chemotherapy, such as docetaxel, remains a major threat to patient survival. Resistance of mCRPC to docetaxel can be associated with elevated levels of soluble clusterin (sCLU) and growth differentiation factor­15 (GDF­15). Any strategies aiming to modulate sCLU and/or GDF­15 in docetaxel­resistant prostate cancer cells present a therapeutic interest. The present study reports the cytotoxic effect of a nitric oxide donor, glyceryl trinitrate (GTN), on docetaxel­resistant mCRPC human cell lines and demonstrates that GTN displays greater inhibition of cell viability toward docetaxel­resistant mCRPC cells than on mCRPC cells. It is also demonstrated that GTN modulates the level of expression of clusterin (CLU) which is dependent of GDF­15, two markers associated with docetaxel resistance in prostate cancer. The results indicate that GTN represses the level of expression of the cytoprotective isoform of CLU (sCLU) and can increase the level of expression of the cytotoxic isoform (nuclear CLU) in docetaxel resistant cells. Furthermore, it was observed that GTN differentially regulates the level of the precursor form of GDF­15 between resistant and parental cells, and that recombinant GDF­15 can modulate the expression of CLU isoforms and counteract GTN­induced cytotoxicity in resistant cells. A link was established between GDF­15 and the expression of CLU isoforms. The present study thus revealed GTN as a potential therapeutic strategy to overcome docetaxel­resistant mCRPC.

Imaging of nitric oxide in a living vertebrate using a diamino-fluorescein probe.

Numerous approaches have been described to identify nitric oxide (NO), a free radical involved in various physiological and pathophysiological processes. One of these approaches is based on the use of chemical probes whose transformation by NO generates highly fluorescent derivatives, permitting detection of NO down to nanomolar concentrations. Here, we show that the cell-permeant diamino-fluorophore 4-amino-5-methylamino-2'-7'-difluoro-fluorescein diacetate (DAF-FM-DA) can be used to detect NO production sites in a living vertebrate, the zebrafish Danio rerio. The staining pattern obtained in larvae includes the bulbus arteriosus, forming bones, the notochord, and the caudal fin. The specificity of the signal was confirmed by its decrease in animals exposed to a NO scavenger or a NO synthase inhibitor and its increase in the presence of a NO donor. Using this method, NO production was observed to change along development in the notochord and the caudal fin whereas it remained stable in the bulbus arteriosus. Local changes in NO production in response to stressful conditions were also detected by this method. Altogether, labeling with DAF-FM DA is an efficient method to monitor changes in NO production in live zebrafish under physiological as well as pathophysiological conditions, suggesting applications to drug screening and molecular pharmacology.

[NO and cancer: itinerary of a double agent]. / Itinéraire d'un agent double - NO, S-nitrosylation et cancer.

Protein S-nitrosylation is now recognized as a ubiquitous regulatory mechanism. Like any post-translational modifications, S-nitrosylation is critical for the control of numerous cellular processes. It is now clear that S-nitrosylation is playing a double game, enhancing or inhibiting the tumor growth or the induction of cell death. Thanks to research aimed at demonstrating NO cytotoxic effects, new therapeutic strategies based on NO donor drugs have emerged. Although therapeutic NO donors can target a large number of proteins, the cellular mechanism is still not fully understood. This review reflects the current state of knowledge on S-nitrosylated proteins that take part of the oncogenic and apoptotic signaling, putting forward proteins with potential interest in cancer therapy.

S-Nitrosylation in TNF superfamily signaling pathway: Implication in cancer.

One of the key features of tumor cells is the acquisition of resistance to apoptosis. Thus, novel therapeutic strategies that circumvent apoptotic resistance and result in tumor elimination are needed. One strategy to induce apoptosis is to activate death receptor signaling pathways. In the tumor microenvironment, stimulation of Fas, Death receptor 4 (DR4) and tumor necrosis factor receptor 1 (TNFR1) can initiate multiple signaling pathways driving either tumor promotion or elimination. Nitric oxide (NO) is an important signaling molecule now understood to play a dual role in cancer biology. More and more attention is directed toward the role displayed by S-nitrosylation, the incorporation of an NO moiety to a cysteine thiol group, in promoting cell death in tumor cells. Protein post-translation modification by S-nitrosylation has decisive roles in regulating signal-transduction pathways. In this review, we summarize several examples of protein modification by S-nitrosylation that regulate signaling pathways engaged by members of the TNF superfamily (Fas ligand (FasL), Tumor-necrosis-factor-related apoptosis inducing ligand (TRAIL) and TNFalpha (TNFα)) and the way it influences cell fate decisions.

The impact of tumor nitric oxide production on VEGFA expression and tumor growth in a zebrafish rat glioma xenograft model.

To investigate the effect of nitric oxide on tumor development, we established a rat tumor xenograft model in zebrafish embryos. The injected tumor cells formed masses in which nitric oxide production could be detected by the use of the cell-permeant DAF-FM-DA (diaminofluorophore 4-amino-5-methylamino-2'-7'-difluorofluorescein diacetate) and DAR-4M-AM (diaminorhodamine-4M). This method revealed that nitric oxide production could be co-localized with the tumor xenograft in 46% of the embryos. In 85% of these embryos, tumors were vascularized and blood vessels were observed on day 4 post injection. Furthermore, we demonstrated by qRT-PCR that the transplanted glioma cells highly expressed Nos2, Vegfa and Cyclin D1 mRNA. In the xenografted embryos we also found increased zebrafish vegfa expression. Glioma and zebrafish derived Vegfa and tumor Cyclin D1 expression could be down regulated by the nitric oxide scavenger 2-(4-Carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide or CPTIO. We conclude that even if there is a heterogeneous nitric oxide production by the xenografted glioma cells that impacts Vegfa and Cyclin D1 expression levels, our results suggest that reduction of nitric oxide levels by nitric oxide scavenging could be an efficient approach to treat glioma.

Anticancer Agents: Does a Phosphonium Behave Like a Gold(I) Phosphine Complex? Let a "Smart" Probe Answer!

Gold phosphine complexes, such as auranofin, have been recognized for decades as antirheumatic agents. Clinical trials are now underway to validate their use in anticancer or anti-HIV treatments. However, their mechanisms of action remain unclear. A challenging question is whether the gold phosphine complex is a prodrug that is administered in an inactive precursor form or rather that the gold atom remains attached to the phosphine ligand during treatment. In this study, we present two novel gold complexes, which we compared to auranofin and to their phosphonium analogue. The chosen ligand is a phosphine-based smart probe, whose strong fluorescence depends on the presence of the gold atom. The in vitro biological action of the gold complexes and the phosphonium derivative were investigated, and a preliminary in vivo study in healthy zebrafish larvae allowed us to evaluate gold complex biodistribution and toxicity. The different analyses carried out showed that these gold complexes were stable and behaved differently from phosphonium and auranofin, both in vitro and in vivo. Two-photon microscopy experiments demonstrated that the cellular targets of these gold complexes are not the same as those of the phosphonium analogue. Moreover, despite similar IC50 values in some cancer cell lines, gold complexes displayed a low toxicity in vivo, in contrast to the phosphonium salt. They are therefore suitable for future in vivo investigations.

Comparative analysis of zebrafish nos2a and nos2b genes.

Nitric oxide synthase (NOS) produces nitric oxide (NO) from arginine. Three NOS isoforms have been identified in mammals, namely a neuronal (NOS1), an inducible (NOS2) and an endothelial (NOS3) enzyme. In zebrafish genome, one nos1 gene and two nos2 genes (nos2a and nos2b) were observed. We cloned zebrafish nos2a cDNA and compared nos2a and nos2b sequences, expression and inducibility. When analyzed by reverse transcription-PCR, the expression of nos2a remained very low during initial development, then increased at 96 hpf, while nos2b was expressed from 6 hpf and subsequently remained stable. Expression of nos2a is detected in the head, eye and gut regions by WISH experiments performed at 48, 72 and 96 hpf larvae. In adults, nos2a expression varies from one tissue to another whereas nos2b is expressed in all studied tissues. Both nos2 isoforms can be induced by pro-inflammatory or mechanical stresses (tissue injury). In vitro as in vivo stimulations with Poly I:C and lipopolysaccharides (LPS) enhanced more dramatically nos2a than nos2b expression. After tail transection in 4 dpf larvae a strong increase of nos2a and nos2b expression was evidenced in the regeneration site, skin cells and for the nos2b gene in neuromasts. Phylogenetic and syntenic analyses show that nos2b gene was associated with syntenic genes identified for nos2 genes in vertebrate. This is not the case for the nos2a gene, despite zebrafish nos2a presenting the inducible property of a classical vertebrate nos2 isoform. A myristoylation consensus site was detected at the N-terminal extremity of zebrafish Nos2b, a property shared with mammal NOS3 isoforms. Thus, the evolution of nos2 genes in zebrafish provides a typical example of gene divergence after duplication.

Induction of apoptosis in a leukemia cell line by triterpene saponins from Albizia adianthifolia.

Triterpenoid saponins, which are present in plants and some marine animals, exert various important pharmacological effects. The present study examines the effects of adianthifoliosides A, B, and D (AdA, AdB, and AdD) together with two prosapogenins (Pro1 and Pro2) obtained from Albizia adianthifolia (Mimosaceae) on human leukemia T-cells (Jurkat cells) and on splenocytes. AdA, AdB, and AdD were found to exhibit a cytotoxic effect on Jurkat cells, whereas the prosapogenins were found to exert a lymphoproliferative effect on this cell type. Furthermore, all tested compounds were found to exert a synergistic lymphoproliferative activity with concanavalin A (ConA) on splenocytes. The concentrations where the saponins were found to be cytotoxic on Jurkat cells are far below the concentration of hemolysis. These results indicate that another mechanism than membrane permeabilization formation is responsible of the cell cytotoxicity. Thus, we demonstrated that at 5 microM for AdA and at 1 microM for AdD, these compounds induce apoptosis in Jurkat cells. Early apoptotic events were detected by flow cytometry analysis by using a double annexin-V-FITC and propidium iodide staining. In addition, a disrupted mitochondrial membrane potential was observed in cells treated with AdA, AdB, and AdD. Furthermore, a DNA ladder was observed when Jurkat cells were incubated with 1 microM AdD for 24h. By comparison between the biological activities of the native compounds with the prosapogenins, we show in this work the important role of the acylation and esterification by different moieties at C-21 and C-28 of the aglycone (acacic acid) in the apoptosis-inducing capacity. Particularly, the monoterpene-quinovosyl moiety is shown to be important for the induction of apoptosis.

New acylated triterpene saponins from Silene fortunei that modulate lymphocyte proliferation.

Three new acylated triterpene saponins 1-3, with a quillaic acid as aglycon, were isolated from the roots of Silene fortunei together with a known phytoecdysteroid (20-hydroxyecdysone). The compounds were characterized mainly by a combination of 2D NMR techniques, mass spectrometry, and chemical methods. Saponins 1-3, jenisseensosides C and D (4, 5), and 6 (deacylated form of 2/3 and 4/5) were found to stimulate the proliferation of the Jurkat tumor cell lines at low concentration. At high concentration, 2/3 and 4/5 inhibited the proliferation of the cells and suggested the induction of apoptosis.

Two new biologically active triterpenoidal saponins acylated with salicylic acid from Albizia adianthifolia.

Two new oleanane-type triterpene saponins, adianthifoliosides A (1) and B (2), were isolated from a 95% ethanolic extract of roots of Albizia adianthifolia. Their structures were elucidated mainly by using a combination of 600 MHz 1D and 2D NMR techniques (COSY, NOESY, TOCSY, HSQC, and HMBC) and by FABMS and HRESIMS. Compounds 1 and 2 were characterized as glycosides of acacic acid acylated by an o-hydroxybenzoyl unit. The crude saponin mixture (CSM), compounds 1 and 2 together with 3 and 4 (prosapogenins obtained from the mild alkaline hydrolysate of the CSM), were evaluated for immunomodulatory activity on the Jurkat T cell line and for hemolytic property against sheep erythrocytes. Compound 2 and, to a lesser extent, 1 and 3 were found to exhibit a dose-dependent immunomodulatory effect in the concentration range 10(-2)-10 microM, whereas 4 showed a lymphoproliferative activity in the same concentration range. Among the compounds tested, only 1 and 2 were found to be hemolytic.