Increased acid sphingomyelinase levels in pediatric patients with obesity.

The level of secretory acid sphingomyelinase (S-ASM), a key enzyme in the sphingolipid metabolism, is elevated in a variety of human diseases, including in the serum of obese adults. Alterations in S-ASM were also found to induce morphological changes in erythrocytes. Consequently, the inhibition of S-ASM by functional Inhibitors of ASM (FIASMA) may have broad clinical implications. The purpose of this study was to assess S-ASM activity in pediatric patients with obesity and healthy matched controls, as well as to investigate the erythrocyte morphology using transmission electron microscopy. We recruited 46 obese patients (mean age 11 ± 2.9 years) and 44 controls (mean age 10.8 ± 2.9 years). S-ASM activity was significantly higher (Wilcoxon signed-rank test p-value: 0.004) in obese patients (mean 396.4 ± 49.7 pmol/ml/h) than in controls (mean 373.7 ± 23.1 pmol/ml/h). No evidence of morphological differences in erythrocytes was found between the two populations. We then carried out a case-control study based on the spontaneous reporting system database to compare FIASMAs with NON-FIASMAs in terms of weight gain risk. Children who received FIASMA had a significantly lower frequency of weight gain reports than patients who took NON-FIASMA agents (p < 0.001). Our findings suggest there is an intriguing possibility that S-ASM may play a role in pediatric obesity. This pilot study could serve as the basis for future studies in this interesting field of research.

Acid Sphingomyelinase Controls Early Phases of Skeletal Muscle Regeneration by Shaping the Macrophage Phenotype.

Skeletal muscle regeneration is a complex process involving crosstalk between immune cells and myogenic precursor cells, i.e., satellite cells. In this scenario, macrophage recruitment in damaged muscles is a mandatory step for tissue repair since pro-inflammatory M1 macrophages promote the activation of satellite cells, stimulating their proliferation and then, after switching into anti-inflammatory M2 macrophages, they prompt satellite cells' differentiation into myotubes and resolve inflammation. Here, we show that acid sphingomyelinase (ASMase), a key enzyme in sphingolipid metabolism, is activated after skeletal muscle injury induced in vivo by the injection of cardiotoxin. ASMase ablation shortens the early phases of skeletal muscle regeneration without affecting satellite cell behavior. Of interest, ASMase regulates the balance between M1 and M2 macrophages in the injured muscles so that the absence of the enzyme reduces inflammation. The analysis of macrophage populations indicates that these events depend on the altered polarization of M1 macrophages towards an M2 phenotype. Our results unravel a novel role of ASMase in regulating immune response during muscle regeneration/repair and suggest ASMase as a supplemental therapeutic target in conditions of redundant inflammation that impairs muscle recovery.

Defects of full-length dystrophin trigger retinal neuron damage and synapse alterations by disrupting functional autophagy.

Dystrophin (dys) mutations predispose Duchenne muscular disease (DMD) patients to brain and retinal complications. Although different dys variants, including long dys products, are expressed in the retina, their function is largely unknown. We investigated the putative role of full-length dystrophin in the homeostasis of neuro-retina and its impact on synapsis stabilization and cell fate. Retinas of mdx mice, the most used DMD model which does not express the 427-KDa dys protein (Dp427), showed overlapped cell death and impaired autophagy. Apoptotic neurons in the outer plexiform/inner nuclear layer and the ganglion cell layer had an impaired autophagy with accumulated autophagosomes. The autophagy dysfunction localized at photoreceptor axonal terminals and bipolar, amacrine, and ganglion cells. The absence of Dp427 does not cause a severe phenotype but alters the neuronal architecture, compromising mainly the pre-synaptic photoreceptor terminals and their post-synaptic sites. The analysis of two dystrophic mutants of the fruit fly Drosophila melanogaster, the homozygous DysE17 and DysEP3397, lacking functional large-isoforms of dystrophin-like protein, revealed rhabdomere degeneration. Structural damages were evident in the internal network of retina/lamina where photoreceptors make the first synapse. Both accumulated autophagosomes and apoptotic features were detected and the visual system was functionally impaired. The reactivation of the autophagosome turnover by rapamycin prevented neuronal cell death and structural changes of mutant flies and, of interest, sustained autophagy ameliorated their response to light. Overall, these findings indicate that functional full-length dystrophin is required for synapsis stabilization and neuronal survival of the retina, allowing also proper autophagy as a prerequisite for physiological cell fate and visual properties.

Acid Sphingomyelinase Downregulation Enhances Mitochondrial Fusion and Promotes Oxidative Metabolism in a Mouse Model of Melanoma.

Melanoma is the most severe type of skin cancer. Its unique and heterogeneous metabolism, relying on both glycolysis and oxidative phosphorylation, allows it to adapt to disparate conditions. Mitochondrial function is strictly interconnected with mitochondrial dynamics and both are fundamental in tumour progression and metastasis. The malignant phenotype of melanoma is also regulated by the expression levels of the enzyme acid sphingomyelinase (A-SMase). By modulating at transcriptional level A-SMase in the melanoma cell line B16-F1 cells, we assessed the effect of enzyme downregulation on mitochondrial dynamics and function. Our results demonstrate that A-SMase influences mitochondrial morphology by affecting the expression of mitofusin 1 and OPA1. The enhanced expression of the two mitochondrial fusion proteins, observed when A-SMase is expressed at low levels, correlates with the increase of mitochondrial function via the stimulation of the genes PGC-1alpha and TFAM, two genes that preside over mitochondrial biogenesis. Thus, the reduction of A-SMase expression, observed in malignant melanomas, may determine their metastatic behaviour through the stimulation of mitochondrial fusion, activity and biogenesis, conferring a metabolic advantage to melanoma cells.

Drp1 overexpression induces desmin disassembling and drives kinesin-1 activation promoting mitochondrial trafficking in skeletal muscle.

Mitochondria change distribution across cells following a variety of pathophysiological stimuli. The mechanisms presiding over this redistribution are yet undefined. In a murine model overexpressing Drp1 specifically in skeletal muscle, we find marked mitochondria repositioning in muscle fibres and we demonstrate that Drp1 is involved in this process. Drp1 binds KLC1 and enhances microtubule-dependent transport of mitochondria. Drp1-KLC1 coupling triggers the displacement of KIF5B from kinesin-1 complex increasing its binding to microtubule tracks and mitochondrial transport. High levels of Drp1 exacerbate this mechanism leading to the repositioning of mitochondria closer to nuclei. The reduction of Drp1 levels decreases kinesin-1 activation and induces the partial recovery of mitochondrial distribution. Drp1 overexpression is also associated with higher cyclin-dependent kinase-1 (Cdk-1) activation that promotes the persistent phosphorylation of desmin at Ser-31 and its disassembling. Fission inhibition has a positive effect on desmin Ser-31 phosphorylation, regardless of Cdk-1 activation, suggesting that induction of both fission and Cdk-1 are required for desmin collapse. This altered desmin architecture impairs mechanotransduction and compromises mitochondrial network stability priming mitochondria transport through microtubule-dependent trafficking with a mechanism that involves the Drp1-dependent regulation of kinesin-1 complex.

XIAP as a Target of New Small Organic Natural Molecules Inducing Human Cancer Cell Death.

X-linked inhibitor of apoptosis protein (XIAP) is an emerging crucial therapeutic target in cancer. We report on the discovery and characterisation of small organic molecules from Piper genus plants exhibiting XIAP antagonism, namely erioquinol, a quinol substituted in the 4-position with an alkenyl group and the alkenylphenols eriopodols A-C. Another isolated compound was originally identified as gibbilimbol B. Erioquinol was the most potent inhibitor of human cancer cell viability when compared with gibbilimbol B and eriopodol A was listed as intermediate. Gibbilimbol B and eriopodol A induced apoptosis through mitochondrial permeabilisation and caspase activation while erioquinol acted on cell fate via caspase-independent/non-apoptotic mechanisms, likely involving mitochondrial dysfunctions and aberrant generation of reactive oxygen species. In silico modelling and molecular approaches suggested that all molecules inhibit XIAP by binding to XIAP-baculoviral IAP repeat domain. This demonstrates a novel aspect of XIAP as a key determinant of tumour control, at the molecular crossroad of caspase-dependent/independent cell death pathway and indicates molecular aspects to develop tumour-effective XIAP antagonists.

The Natural Compound Climacostol as a Prodrug Strategy Based on pH Activation for Efficient Delivery of Cytotoxic Small Agents.

We synthesized and characterized MOMO as a new small molecule analog of the cytotoxic natural product climacostol efficiently activated in mild extracellular acidosis. The synthesis of MOMO had a key step in the Wittig olefination for the construction of the carbon-carbon double bond in the alkenyl moiety of climacostol. The possibility of obtaining the target (Z)-alkenyl MOMO derivative in very good yield and without presence of the less active (E)-diastereomer was favored from the methoxymethyl ether (MOM)-protecting group of hydroxyl functions in aromatic ring of climacostol aldehyde intermediate. Of interest, the easy removal of MOM-protecting group in a weakly acidic environment allowed us to obtain a great quantity of climacostol in biologically active (Z)-configuration. Results obtained in free-living ciliates that share the same micro-environment of the climacostol natural producer Climacostomum virens demonstrated that MOMO is well-tolerated in a physiological environment, while its cytotoxicity is rapidly and efficiently triggered at pH 6.3. In addition, the cytostatic vs. cytotoxic effects of acidified-MOMO can be modulated in a dose-dependent manner. In mouse melanoma cells, MOMO displayed a marked pH-sensitivity since its cytotoxic and apoptotic effects become evident only in mild extracellular acidosis. Data also suggested MOMO being preferentially activated in the unique extra-acidic microenvironment that characterizes tumoural cells. Finally, the use of the model organism Drosophila melanogaster fed with an acidic diet supported the efficient activity and oral delivery of MOMO molecule in vivo. MOMO affected oviposition of mating adults and larvae eclosion. Reduced survival of flies was due to lethality during the larval stages while emerging larvae retained their ability to develop into adults. Interestingly, the gut of eclosed larvae exhibited an extended damage (cell death by apoptosis) and the brain tissue was also affected (reduced mitosis), demonstrating that orally activated MOMO efficiently targets different tissues of the developing fly. These results provided a proof-of-concept study on the pH-dependence of MOMO effects. In this respect, MOM-protection emerges as a potential prodrug strategy which deserves to be further investigated for the generation of efficient pH-sensitive small organic molecules as pharmacologically active cytotoxic compounds.

Dysfunctional autophagy induced by the pro-apoptotic natural compound climacostol in tumour cells.

Autophagy occurs at a basal level in all eukaryotic cells and may support cell survival or activate death pathways. Due to its pathophysiologic significance, the autophagic machinery is a promising target for the development of multiple approaches for anti-neoplastic agents. We have recently described the cytotoxic and pro-apoptotic mechanisms, targeting the tumour suppressor p53, of climacostol, a natural product of the ciliated protozoan Climacostomum virens. We report here on how climacostol regulates autophagy and the involvement of p53-dependent mechanisms. Using both in vitro and in vivo techniques, we show that climacostol potently and selectively impairs autophagy in multiple tumour cells that are committed to die by apoptosis. In particular, in B16-F10 mouse melanomas climacostol exerts a marked and sustained accumulation of autophagosomes as the result of dysfunctional autophagic degradation. We also provide mechanistic insights showing that climacostol affects autophagosome turnover via p53-AMPK axis, although the mTOR pathway unrelated to p53 levels plays a role. In particular, climacostol activated p53 inducing the upregulation of p53 protein levels in the nuclei through effects on p53 stability at translational level, as for instance the phosphorylation at Ser15 site. Noteworthy, AMPKα activation was the major responsible of climacostol-induced autophagy disruption in the absence of a key role regulating cell death, thus indicating that climacostol effects on autophagy and apoptosis are two separate events, which may act independently on life/death decisions of the cell. Since the activation of p53 system is at the molecular crossroad regulating both the anti-autophagic action of climacostol and its role in the apoptosis induction, it might be important to explore the dual targeting of autophagy and apoptosis with agents acting on p53 for the selective killing of tumours. These findings also suggest the efficacy of ciliate bioactive molecules to identify novel lead compounds in drug discovery and development.

Nitric Oxide Generated by Tumor-Associated Macrophages Is Responsible for Cancer Resistance to Cisplatin and Correlated With Syntaxin 4 and Acid Sphingomyelinase Inhibition.

Tumor microenvironment is fundamental for cancer progression and chemoresistance. Among stromal cells tumor-associated macrophages (TAMs) represent the largest population of infiltrating inflammatory cells in malignant tumors, promoting their growth, invasion, and immune evasion. M2-polarized TAMs are endowed with the nitric oxide (NO)-generating enzyme inducible nitric oxide synthase (iNOS). NO has divergent effects on tumors, since it can either stimulate tumor cells growth or promote their death depending on the source of it; likewise the role of iNOS in cancer differs depending on the cell type. The role of NO generated by TAMs has not been investigated. Using different tumor models in vitro and in vivo we found that NO generated by iNOS of M2-polarized TAMs is able to protect tumor cells from apoptosis induced by the chemotherapeutic agent cisplatin (CDDP). Here, we demonstrate that the protective effect of NO depends on the inhibition of acid sphingomyelinase (A-SMase), which is activated by CDDP in a pathway involving the death receptor CD95. Mechanistic insights indicate that NO actions occur via generation of cyclic GMP and activation of protein kinase G (PKG), inducing phosphorylation of syntaxin 4 (synt4), a SNARE protein responsible for A-SMase trafficking and activation. Noteworthy, phosphorylation of synt4 at serine 78 by PKG is responsible for the proteasome-dependent degradation of synt4, which limits the CDDP-induced exposure of A-SMase to the plasma membrane of tumor cells. This inhibits the cytotoxic mechanism of CDDP reducing A-SMase-triggered apoptosis. This is the first demonstration that endogenous NO system is a key mechanism through which TAMs protect tumor cells from chemotherapeutic drug-induced apoptosis. The identification of the pathway responsible for A-SMase activity downregulation in tumors leading to chemoresistance warrants further investigations as a means to identify new anti-cancer molecules capable of specifically inhibiting synt4 degradation.

The Numb/p53 circuitry couples replicative self-renewal and tumor suppression in mammary epithelial cells.

The cell fate determinant Numb orchestrates tissue morphogenesis and patterning in developmental systems. In the human mammary gland, Numb is a tumor suppressor and regulates p53 levels. However, whether this function is linked to its role in fate determination remains unclear. Here, by exploiting an ex vivo system, we show that at mitosis of purified mammary stem cells (SCs), Numb ensures the asymmetric outcome of self-renewing divisions by partitioning into the progeny that retains the SC identity, where it sustains high p53 activity. Numb also controls progenitor maturation. At this level, Numb loss associates with the epithelial-to-mesenchymal transition and results in differentiation defects and reacquisition of stemness features. The mammary gland of Numb-knockout mice displays an expansion of the SC compartment, associated with morphological alterations and tumorigenicity in orthotopic transplants. This is because of low p53 levels and can be inhibited by restoration of Numb levels or p53 activity, which results in successful SC-targeted treatment.