Ilaria Carnevale

SIRT5 regulation of ammonia-induced autophagy and mitophagy

In liver the mitochondrial sirtuin, SIRT5, controls ammonia detoxification by regulating CPS1, the first enzyme of the urea cycle. However, while SIRT5 is ubiquitously expressed, urea cycle and CPS1 are only present in the liver and, to a minor extent, in the kidney. To address the possibility that SIRT5 is involved in ammonia production also in nonliver cells, clones of human breast cancer cell lines MDA-MB-231 and mouse myoblast C2C12, overexpressing or silenced for SIRT5 were produced. Our results show that ammonia production increased in SIRT5-silenced and decreased in SIRT5-overexpressing cells. We also obtained the same ammonia increase when using a new specific inhibitor of SIRT5 called MC3482. SIRT5 regulates ammonia production by controlling glutamine metabolism. In fact, in the mitochondria, glutamine is transformed in glutamate by the enzyme glutaminase, a reaction producing ammonia. We found that SIRT5 and glutaminase coimmunoprecipitated and that SIRT5 inhibition resulted in an increased succinylation of glutaminase. We next determined that autophagy and mitophagy were increased by ammonia by measuring autophagic proteolysis of long-lived proteins, increase of autophagy markers MAP1LC3B, GABARAP, and GABARAPL2, mitophagy markers BNIP3 and the PINK1-PARK2 system as well as mitochondrial morphology and dynamics. We observed that autophagy and mitophagy increased in SIRT5-silenced cells and in WT cells treated with MC3482 and decreased in SIRT5-overexpressing cells. Moreover, glutaminase inhibition or glutamine withdrawal completely prevented autophagy. In conclusion we propose that the role of SIRT5 in nonliver cells is to regulate ammonia production and ammonia-induced autophagy by regulating glutamine metabolism.

Bridging hypoxia, inflammation and estrogen receptors in thyroid cancer progression

Thyroid cancer is a common endocrine-related cancer with a higher incidence in women than in men. Thyroid tumors are classified on the basis of their histopathology as papillary, follicular, medullary, and undifferentiated or anaplastic. Epidemiological and in vitro or in vivo studies have suggested a correlation between incidence of thyroid malignancies and hormones. In particular, growing evidence indicates a role of estrogens and estrogen receptors (ERs) in thyroid tumorigenesis, reprogramming and progression. In this scenario, estrogens are hypothesized to contribute to the observed female predominance of thyroid cancer in reproductive years. However, the precise contribution of estrogens in thyroid proliferative disease initiation and progression is not well understood. HIF-1α and NF-κB are two transcription factors very frequently activated in tumors and involved in tumor growth, progression and resistance to chemotherapy. In fact, HIF-1α and NF-κB together regulate transcription of over a thousand genes that, in turn, control vital cellular processes such as adaptation to the hypoxia, metabolic and differentiation reprogramming, inflammatory-reparative response, extracellular matrix digestion, migration and invasion, adhesion, etc. Because of this wide involvement, they could control in an integrated manner the origin of the malignant phenotype. Interestingly, hypoxia and inflammation have been sequentially bridged in tumors by the discovery that alarmin receptors genes such as RAGE, P2X7 and some TLRs are activated by HIF-1α; and that, in turn, alarmin receptors strongly activate NF-κB and proinflammatory gene expression, evidencing all the hallmarks of the malignant phenotype. Recently, a large number of drugs have been identified that inhibit one or both transcription factors with promising results in terms of controlling tumor progression. In addition, many of these inhibitors are natural compounds or off-label drugs already used to cure other pathologies. Some of them are undergoing clinical trials and soon they will be used alone or in combination with standard anti-tumoral agents to achieve a better treatment of tumors to achieve a reduction of metastasis formation and, more importantly, a net increase in survival. This review highlights the central role of HIF-1α activated in hypoxic regions of the tumor, of NF-κB activation and proinflammatory gene expression in transformed thyroid cells to understand their progression toward malignancy. The role of ER-α will be underlined, considering also its role in reprogramming cancer cells.

1,4-Dihydropyridines Active on the SIRT1/AMPK Pathway Ameliorate Skin Repair and Mitochondrial Function and Exhibit Inhibition of Proliferation in Cancer Cells

Modulators of sirtuins are considered promising therapeutic targets for the treatment of cancer, cardiovascular, metabolic, inflammatory, and neurodegenerative diseases. Here we prepared new 1,4-dihydropyridines (DHPs) bearing changes at the C2/C6, C3/C5, C4, or N1 position. Tested with the SIRTainty procedure, some of them displayed increased SIRT1 activation with respect to the prototype 3a, high NO release in HaCat cells, and ameliorated skin repair in a mouse model of wound healing. In C2C12 myoblasts, two of them improved mitochondrial density and functions. All the effects were reverted by coadministration of compound C (9), an AMPK inhibitor, or of EX-527 (10), a SIRT1 inhibitor, highlighting the involvement of the SIRT1/AMPK pathway in the action of DHPs. Finally, tested in a panel of cancer cells, the water-soluble form of 3a, compound 8, displayed antiproliferative effects in the range of 8-35 μM and increased H4K16 deacetylation, suggesting a possible role for SIRT1 activators in cancer therapy.

Sirtuins and resveratrol-derived compounds: a model for understanding the beneficial effects of the Mediterranean diet.

The beneficial effects of the Mediterranean diet (MD) had been first observed about 50 years ago. Consumption of fresh vegetables and fruits, cereals, red wine, nuts, legumes, etc. has been regarded as the primary factor for protection from many human pathologies by the Mediterranean diet. Subsequently, this was attributed to the presence of polyphenols and their derivatives that, by exerting an anti-inflammatory and anti-oxidative effect, can be involved in the prevention of many diseases. Clinical trials, observational studies and meta-analysis have demonstrated an antiageing effect of MD accompanied by a reduced risk of age-related pathologies, such as cardiovascular, metabolic and neurodegenerative diseases, as well as cancer. The scientific explanation of such beneficial effects was limited to the reduction of the oxidative stress by compounds present in the MD. However, recently, this view is changing thanks to new studies aimed to uncover the molecular mechanism(s) activated by components of this diet. In particular, a new class of proteins called sirtuins have gained the attention of the scientific community because of their antiageing effects, their ability to protect from cardiovascular, metabolic, neurodegenerative diseases, cancer and to extend lifespan in lower organisms as well as in mammals. Interestingly, resveratrol a polyphenol present in grapes, nuts and berries has been shown to activate sirtuins and such activation is able to explain most of the beneficial effects of the MD. In this review, we will highlight the importance of MD with particular attention to the possible molecular pathways that have been shown to be influenced by it. We will describe the state of the art leading to demonstrate the important role of sirtuins as principal intracellular mediators of the beneficial effects of the MD. Finally, we will also introduce how Mediterranean diet may influence microbioma composition and stem cells function.

SIRT1‐SIRT3 Axis Regulates Cellular Response to Oxidative Stress and Etoposide

Sirtuins are conserved NAD+‐dependent deacylases. SIRT1 is a nuclear and cytoplasmic sirtuin involved in the control of histones a transcription factors function. SIRT3 is a mitochondrial protein, which regulates mitochondrial function. Although, both SIRT1 and SIRT3 have been implicated in resistance to cellular stress, the link between these two sirtuins has not been studied so far. Here we aimed to unravel: i) the role of SIRT1‐SIRT3 axis for cellular response to oxidative stress and DNA damage; ii) how mammalian cells modulate such SIRT1‐SIRT3 axis and which mechanisms are involved. Therefore, we analyzed the response to different stress stimuli in WT or SIRT1‐silenced cell lines. Our results demonstrate that SIRT1‐silenced cells are more resistant to H2O2 and etoposide treatment showing decreased ROS accumulation, γ‐H2AX phosphorylation, caspase‐3 activation and PARP cleavage. Interestingly, we observed that SIRT1‐silenced cells show an increased SIRT3 expression. To explore such a connection, we carried out luciferase assays on SIRT3 promoter demonstrating that SIRT1‐silencing increases SIRT3 promoter activity and that such an effect depends on the presence of SP1 and ZF5 recognition sequences on SIRT3 promoter. Afterwards, we performed co‐immunoprecipitation assays demonstrating that SIRT1 binds and deacetylates the transcription inhibitor ZF5 and that there is a decreased interaction between SP1 and ZF5 in SIRT1‐silenced cells. Therefore, we speculate that acetylated ZF5 cannot bind and sequester SP1 that is free, then, to increase SIRT3 transcription. In conclusion, we demonstrate that cells with low SIRT1 levels can maintain their resistance and survival by increasing SIRT3 expression. J. Cell. Physiol. 232: 1835–1844, 2017.

Pyrazole-based inhibitors of enhancer of zeste homologue 2 induce apoptosis and autophagy in cancer cells

Novel pyrazole-based EZH2 inhibitors have been prepared through a molecular pruning approach from known inhibitors bearing a bicyclic moiety as a central scaffold. The hit compound 1o (N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-5-methyl-1-phenyl-1H-pyrazole-4-carboxamide) showed low micromolar EZH2/PRC2 inhibition and high selectivity towards a panel of other methyltransferases. Moreover, 1o displayed cell growth arrest in breast MDA-MB231, leukaemia K562, and neuroblastoma SK-N-BE cancer cells joined to reduction of H3K27me3 levels and induction of apoptosis and autophagy. This article is part of a discussion meeting issue ‘Frontiers in epigenetic chemical biology’.

Correction to ‘Pyrazole-based inhibitors of enhancer of zeste homologue 2 induce apoptosis and autophagy in cancer cells’

Phil. Trans. R. Soc. B 373, 20170150. (Published 23 April 2018) (doi:10.1098/rstb.2017.0150) An affiliation was missing for Elisabetta Ferretti. The correct author list and affiliations is as follows: Paolo Mellini1, Biagina Marrocco1, Diana Borovika2, Lucia Polletta …

Infertility in Fabry’s disease: role of hypoxia and inflammation in determining testicular damage

Fabry’s disease (FD) is a genetic X-linked systemic and progressive rare disease, which is characterized by the accumulation of glycolipid bodies (GB) into the lys- osomes of almost all cell types and consequently by a multiform clinical picture. Here we studied testicular biopsies of a 42 ys old FD patient, presenting infertility with reduced number of spermatozoa and preserved sexual activity. Testicular biopsies have been analyzed by optical microscopy (OM) and transmis- sion electron microscopy (TEM). OM, showed a severe involvement of testis inter- stitium blood vessels with reduced or closed lumen, an increased of connective tis- sue and a substantial thicketing of peritubular region. TEM, showed that GB were abundant in vessel wall cells and in myofibroblast of peritubular region. In contrast with literature reports, Leydig cells were constantly unaffected by GB accumulation showing well preserved ultrastructural organization. On the contrary, tubular cells, although not affected by GB accumulation, appeared severely damaged. These data led us to hypothesize that diffusion of oxygen and nutrients from blood to tubules could be impaired. To test this hypothesis we explored, by immunofluorescence (IF) and molecular biology (MB) coupled to laser capture micro-dissection (LCMD), the activation of HIF/NFkB pathway. IF showed increased signal for HIF1a in all stromal components, while it appeared almost absent in seminipherous tubules. On the contrary, NFkB fluores- cence was evident in tubules. mRNA of tubular and interstitial tissue fractions, separately extracted by LCMD, confirms that HIF1a and hypoxic-related genes such as alarmin recepters (RAGE, TLR4) were overexpressed in the interstitial cells. At the same time, NFkB and a number of proinflammatory genes such as HMOX1, PTGES, SAA1-SAA2 were up- regulated in the tubule microenviroment. Taken together, these results suggest that the GB accumulation in interstitium, reducing vessel lumen and increasing the distance between vessel and tubular cells, leads to chronic progressive hypoxia. Hypoxia has two effects: 1)Necrosis of cells more distant from vessels, especially germinative epithelium and Sertoli cells, releas- ing alarmins; 2)Adaptation to low levels of O2, with activation of HIF1a. In both cas- es a strong activation of NFkB occurs that trigger a inflammatory response (IR). We suggest a role for the IR activation in determining intratubular cells damage and con- sequently, infertility in FD.