• Cancers (Basel). 2020 Dec 25;12(1). pii: E68. doi: 10.3390/cancers12010068.

TCA Cycle Rewiring as Emerging Metabolic Signature of Hepatocellular Carcinoma.

• Int J Mol Sci. 2019 Apr 16;20(8). pii: E1888. doi: 10.3390/ijms20081888.

Transcriptional Regulation Factors of the Human Mitochondrial Aspartate/Glutamate Carrier Gene, Isoform 2 (SLC25A13): USF1 as Basal Factor and FOXA2 as Activator in Liver Cells.

• 
  Biochim Biophys Acta Mol Basis Dis. 2019 Jan;1865(1):38-47. doi: 10.1016/j.bbadis.2018.10.018. 

  Epigenetic upregulation and functional role of the mitochondrial aspartate/glutamate carrier isoform 1 in hepatocellular carcinoma.

  Infantino V, Dituri F, Convertini P, Santarsiero A, Palmieri F, Todisco S, Mancarella S, Giannelli G, Iacobazzi V.

   

  Metabolic reprogramming is a common hallmark of cancer cells. Although some biochemical features have been clarified, there is still much to learn about cancer cell metabolism and its regulation. Aspartate-glutamate carrier isoform 1 (AGC1), encoded by SLC25A12 gene, catalyzes an exchange between intramitochondrial aspartate and cytosolic glutamate plus a proton across the mitochondrial membrane, so supplying aspartate to the cytosol. SLC25A12, expressed in brain, heart, and skeletal muscle, is silenced in normal liver. Here, we demonstrate that SLC25A12 gene is reactivated in hepatocellular carcinoma (HCC) HepG2 cell line through histone acetylation and CREB recruitment. Furthermore, SLC25A12 knockdown by small interfering RNA, impairs HepG2 cell proliferation by inducing cell cycle arrest. AGC1 sustains HCC cell growth by supplying cytosolic aspartate for nucleotide biosynthesis. In addition, SLC25A12-silenced HCC cells show a strong reduction of cell migration. Overall, we have provided evidence for molecular mechanisms controlling SLC25A12 gene expression in liver and pointing to an important role for AGC1 in HCC.

  KEYWORDS: Aspartate-glutamate carrier isoform 1 (AGC1); Cancer metabolism; Epigenetics; Gene expression; Hepatocellular carcinoma (HCC); SLC25A12

  PMID: 30321589

• 
  Curr Med Chem. 2018 May 10. doi: 10.2174/0929867325666180510124558. [Epub ahead of print]

  Metabolic routes in inflammation: the citrate pathway and its potential as therapeutic target.

  Infantino V, Pierri CL, Iacobazzi V.

  Significant metabolic changes occur in inflammation to respond to the new energetic needs of cells. Mitochondria are addressed not only to produce ATP, but also to supply substrates, such citrate, to produce pro-inflammatory molecules. In this context most of citrate is diverted from Krebs cycle and channeled into the "citrate pathway" consisting in the increase of the export of citrate into cytosol by the mitochondrial citrate carrier followed by its cleavage into acetyl-CoA and oxaloacetate by ATP citrate lyase. Acetyl-CoA is used to produce PGE2 and oxaloacetate to make NADPH needed for NO and ROS. In addition cytosolic citrate provides also for itaconate synthesis. Citrate-derived itaconate acts as a negative regulator of inflammation by modulating the synthesis of the inflammatory mediators. Inhibition of CIC or ACLY by different synthetic and natural molecules results in reduction of NO, ROS and PGE2 levels suggesting that the citrate pathway can be a new target to be addressed in inflammation. Beneficial effects can be obtained also in the oxidative stress and inflammatory conditions observed in Down syndrome.

  KEYWORDS:  ATP citrate lyase; Citrate pathway; NO; PGE2.; ROS; inflammation; inhibition; mitochondrial citrate carrier

  PMID:29745322

   

• Mediators Inflamm. 2018 Jun 28;2018:1419352. doi: 10.1155/2018/1419352. eCollection 2018.

  New Insights into Behçet's Syndrome Metabolic Reprogramming: Citrate Pathway Dysregulation.

  Santarsiero A, Leccese P, Convertini P, Padula A, Abriola P, D'Angelo S, Bisaccia F, Infantino V. 

  To date, a major research effort on Behçet's syndrome (BS) has been concentrated on immunological aspects. Little is known about the metabolic reprogramming in BS. Citrate is an intermediary metabolite synthesized in mitochondria, and when transported into the cytosol by the mitochondrial citrate carrier-SLC25A1-encoded protein-it is cleaved into acetyl-CoA and oxaloacetate by ATP citrate lyase (ACLY). In induced macrophages, mitochondrial citrate is necessary for the production of inflammatory mediators. The aim of our study was to evaluate SLC25A1 and ACLY expression levels in BS patients. Following a power analysis undertaken on few random samples, the number of enrolled patients was set. Thirty-nine consecutive BS patients fulfilling ISG criteria, and 21 healthy controls suitable for age and sex were recruited. BS patients were divided into two groups according to the presence (active) or absence (inactive) of clinical manifestations. Real-time PCR experiments were performed on PBMCs to quantify SLC25A1 and ACLY mRNA levels. Data processing through the Kruskal-Wallis test and Dunn's multiple comparison test as post hoc showed higher SLC25A1 and ACLY mRNA levels in BS patients compared to those in healthy controls. Therefore, SLC25A1 and ACLY upregulation suggests that metabolic reprogramming in BS involves the citrate pathway dysregulation.

  PMID: 30050389

   

    
•  

  Eur J Med Chem. 2017 Feb 15;127:379-397. doi: 10.1016/j.ejmech.2016.12.047. Epub 2016 Dec 24.

  New diphenylmethane derivatives as peroxisome proliferator-activated receptor alpha/gamma dual agonists endowed with anti-proliferative effects and mitochondrial activity.

  Piemontese L, Cerchia C, Laghezza A, Ziccardi P, Sblano S, Tortorella P, Iacobazzi V, Infantino V, Convertini P, Dal Piaz F, Lupo A, Colantuoni V, Lavecchia A, Loiodice F.

  We screened a short series of new chiral diphenylmethane derivatives and identified potent dual PPARα/γ partial agonists. As both enantiomers of the most active compound 1 displayed an unexpected similar transactivation activity, we performed docking experiments to provide a molecular understanding of their similar partial agonism. We also evaluated the ability of both enantiomers of 1 and racemic 2 to inhibit colorectal cancer cells proliferation: (S)-1 displayed a more robust activity due, at least in part, to a partial inhibition of the Wnt/β-catenin signalling pathway that is upregulated in the majority of colorectal cancers. Finally, we investigated the effects of (R)-1, (S)-1 and (R,S)-2 on mitochondrial function and demonstrated that they activate the carnitine shuttle system through upregulation of carnitine/acylcarnitine carrier (CAC) and carnitine-palmitoyl-transferase 1 (CPT1) genes. Consistent with the notion that these are PPARα target genes, we tested and found that PPARα itself is regulated by a positive loop. Moreover, these compounds induced a significant mitochondrial biogenesis. In conclusion, we identified a new series of dual PPARα/γ agonists endowed with novel anti-proliferative properties associated with a strong activation of mitochondrial functions and biogenesis, a potential therapeutic target of the treatment of insulin resistance.

  KEYWORDS: Anti-proliferative effects; Docking experiments; Gene expression analysis; Mitochondrial biogenesis; PPAR

  PMID:28076827

   

• FEBS J. 2017 Mar;284(6):967-984. doi: 10.1111/febs.14028. Epub 2017 Feb 21.

  SLC25A26 overexpression impairs cell function via mtDNA hypermethylation and rewiring of methyl metabolism.

  Menga A, Palmieri EM, Cianciulli A, Infantino V, Mazzone M, Scilimati A5, Palmieri F, Castegna A1, Iacobazzi V.

  Cancer cells down-regulate different genes to give them a selective advantage in invasiveness and/or metastasis. The SLC25A26 gene encodes the mitochondrial carrier that catalyzes the import of S-adenosylmethionine (SAM) into the mitochondrial matrix, required for mitochondrial methylation processes, and is down-regulated in cervical cancer cells. In this study we show that SLC25A26 is down-regulated due to gene promoter hypermethylation, as a mechanism to promote cell survival and proliferation. Furthermore, overexpression of SLC25A26 in CaSki cells increases mitochondrial SAM availability and promotes hypermethylation of mitochondrial DNA, leading to decreased expression of key respiratory complex subunits, reduction of mitochondrial ATP and release of cytochrome c. In addition, increased SAM transport into mitochondria leads to impairment of the methionine cycle with accumulation of homocysteine at the expense of glutathione, which is strongly reduced. All these events concur to arrest the cell cycle in the S phase, induce apoptosis and enhance chemosensitivity of SAM carrier-overexpressing CaSki cells to cisplatin.

  KEYWORDS:S-adenosylmethionine; SLC25A26 mitochondrial carrier; epigenetic mechanisms; methyl cycle; mtDNA methylation

  PMID:28118529

   

• Biol Chem. 2016 Oct 11. pii: /j/bchm.just-accepted/hsz-2016-0260/hsz-2016-0260.xml. doi: 10.1515/hsz-2016-0260. [Epub ahead of print]

Mitochondrial carriers in inflammation induced by bacterial endotoxin and cytokines.

Iacobazzi V, Infantino V, Castegna A, Menga A, Palmieri EM, Convertini P, Palmieri F.

Significant metabolic changes occur in the shift from resting to activated cellular status in inflammation. Thus, changes in expression of a large number of genes and extensive metabolic reprogramming gives rise to acquisition of new functions (e.g. production of cytokines, intermediates for biosynthesis, lipid mediators, PGE, ROS and NO). In this context, mitochondrial carriers, which catalyze the transport of solute across mitochondrial membrane, change their expression to transport mitochondrially produced molecules, among which citrate and succinate, to be used as intracellular signalling molecules in inflammation. This review summarizes the mitochondrial carriers studied so far that are, directly or indirectly, involved in inflammation. 

PMID: 27727142

• Immunology. 2016 Aug 9. doi: 10.1111/imm.12659. [Epub ahead of print]

The contribution of the citrate pathway to oxidative stress in Down syndrome.

Convertini P, Menga A, Andria G, Scala I, Santarsiero A, Castiglione Morelli MA, Iacobazzi V, Infantino V.

Inflammatory conditions and oxidative stress have a crucial role in Down syndrome (DS). Emerging studies have also reported an altered lipid profile in the early stages of DS. Our previous works demonstrate that citrate pathway activation is required for oxygen radical production during inflammation. Here, we find up-regulation of the citrate pathway and down-regulation of carnitine/acylcarnitine carrier and carnitine palmitoyl-transferase 1 genes in cells from children with DS. Interestingly, when the citrate pathway is inhibited, we observe a reduction in oxygen radicals as well as in lipid peroxidation levels. Our preliminary findings provide evidence for a citrate pathway dysregulation, which could be related to some phenotypic traits of people with DS.

© 2016 John Wiley & Sons Ltd. PMID: 27502741

• Cell Discov. 2016 Feb 16;2:15046. doi: 10.1038/celldisc.2015.46. eCollection 2016.

 Involvement of PARP1 in the regulation of alternative splicing.

Matveeva E, Maiorano J, Zhang Q, Eteleeb AM, Convertini P, Chen J, Infantino V, Stamm S, Wang J, Rouchka EC, Fondufe-Mittendorf YN.

Specialized chromatin structures such as nucleosomes with specific histone modifications decorate exons in eukaryotic genomes, suggesting a functional connection between chromatin organization and the regulation of pre-mRNA splicing. Through profiling the functional location of Poly (ADP) ribose polymerase, we observed that it is associated with the nucleosomes at exon/intron boundaries of specific genes, suggestive of a role for this enzyme in alternative splicing. Poly (ADP) ribose polymerase has previously been implicated in the PARylation of splicing factors as well as regulation of the histone modification H3K4me3, a mark critical for co-transcriptional splicing. In light of these studies, we hypothesized that interaction of the chromatin-modifying factor, Poly (ADP) ribose polymerase with nucleosomal structures at exon-intron boundaries, might regulate pre-mRNA splicing. Using genome-wide approaches validated by gene-specific assays, we show that depletion of PARP1 or inhibition of its PARylation activity results in changes in alternative splicing of a specific subset of genes. Furthermore, we observed that PARP1 bound to RNA, splicing factors and chromatin, suggesting that Poly (ADP) ribose polymerase serves as a gene regulatory hub to facilitate co-transcriptional splicing. These studies add another function to the multi-functional protein, Poly (ADP) ribose polymerase, and provide a platform for further investigation of this protein's function in organizing chromatin during gene regulatory processes.

PMID: 27462443

• Physiol Genomics. 2015 Aug;47(8):299-307. doi:10.1152/physiolgenomics.00096.2014. Epub 2015 Jun 2.

The mitochondrial side of epigenetics.

Castegna A, Iacobazzi V, Infantino V.

The bidirectional cross talk between nuclear and mitochondrial DNA is essential for cellular homeostasis and proper functioning. Mitochondria depend on nuclear contribution for much of their functionality, but their activities have been recently recognized to control nuclear gene expression as well as cell function in many different ways. Epigenetic mechanisms, which tune gene expression in response to environmental stimuli, are key regulatory events at the interplay between mitochondrial and nuclear interactions. Emerging findings indicate that epigenetic factors can be targets or instruments of mitochondrial-nuclear cross talk. Additionally, the growing interest into mtDNA epigenetic modifications opens new avenues into the interaction mechanisms between mitochondria and nucleus. In this review we summarize the points of mitochondrial and nuclear reciprocal control involving epigenetic factors, focusing on the role of mitochondrial genome and metabolism in shaping epigenetic modulation of gene expression. The relevance of the new findings on the methylation of mtDNA is also highlighted as a new frontier in the complex scenario of mitochondrial-nuclear communication.

Copyright © 2015 the American Physiological Society. PMID: 26038395 [PubMed - in process]

• Chem Biol Interact. 2015 Jul 25;237:1-8. doi: 10.1016/j.cbi.2015.05.005. Epub 2015 May 14.

Permethylated Anigopreissin A inhibits human hepatoma cell proliferation by mitochondria-induced apoptosis.

Convertini P, Tramutola F, Iacobazzi V, Lupattelli P, Chiummiento L, Infantino V.

Anigopreissin A belongs to stilbene di- and oligomeric forms containing a benzofuran ring system whose biological activity is unknown. Recently, a completely protected Anigopreissin A - Permethylated Anigopreissin A - has been synthesized. We use MTT bioassay to assess Permethylated Anigopreissin A cytotoxicity in different human cell lines. Furthermore, fluorescence microscopy, caspase activity, real-time PCR and Western-blot methods are employed to evaluate apoptotic cell death pathway in liver cancer cells. Permethylated Anigopreissin A kills different types of human cancer cells but does not affect non-tumorigenic cells. The Permethylated Anigopreissin A concentration that causes 50% inhibition of liver tumor cells is 0.24μM. Hepatoma cells treated with Permethylated Anigopreissin A arrest their cell cycle in G1 phase. We also demonstrate that Permethylated Anigopreissin A-triggered cell death occurs by apoptosis. Decrease of the BCL2 expression levels, loss of the mitochondrial membrane potential, release of cytochrome c and increase of caspase 9 activity highlight a key role for mitochondria in Permethylated Anigopreissin A-induced apoptosis. Our study shows that Permethylated Anigopreissin A kills liver cancer cells through intrinsic apoptotic pathway.

Copyright © 2015 Elsevier Ireland Ltd. All rights reserved. PMID: 25980588 [PubMed - in process]

• Biochim Biophys Acta. 2015 Aug;1847(8):729-38. doi: 10.1016/j.bbabio.2015.04.009. Epub 2015 Apr 24.

Acetylation of human mitochondrial citrate carrier modulates mitochondrial citrate/malate exchange activity to sustain NADPH production during macrophage activation.

Palmieri EM, Spera I, Menga A, Infantino V, Porcelli V, Iacobazzi V, Pierri CL, Hooper DC, Palmieri F, Castegna A.

The mitochondrial citrate-malate exchanger (CIC), a known target of acetylation, is up-regulated in activated immune cells and plays a key role in the production of inflammatory mediators. However, the role of acetylation in CIC activity is elusive. We show that CIC is acetylated in activated primary human macrophages and U937 cells and the level of acetylation is higher in glucose-deprived compared to normal glucose medium. Acetylation enhances CIC transport activity, leading to a higher citrate efflux from mitochondria in exchange with malate. Cytosolic citrate levels do not increase upon activation of cells grown in deprived compared to normal glucose media, indicating that citrate, transported from mitochondria at higher rates from acetylated CIC, is consumed at higher rates. Malate levels in the cytosol are lower in activated cells grown in glucose-deprived compared to normal glucose medium, indicating that this TCA intermediate is rapidly recycled back into the cytosol where it is used by the malic enzyme. Additionally, in activated cells CIC inhibition increases the NADP+/NADPH ratio in glucose-deprived cells; this ratio is unchanged in glucose-rich grown cells due to the activity of the pentose phosphate pathway. Consistently, the NADPH-producing isocitrate dehydrogenase level is higher in activated glucose-deprived as compared to glucose rich cells. These results demonstrate that, in the absence of glucose, activated macrophages increase CIC acetylation to enhance citrate efflux from mitochondria not only to produce inflammatory mediators but also to meet the NADPH demand through the actions of isocitrate dehydrogenase and malic enzyme.

Copyright © 2015 Elsevier B.V. All rights reserved. PMID: 25917893 [PubMed - indexed for MEDLINE]

• Eur J Pharmacol. 2015 Jul 5;758:16-23. doi: 10.1016/j.ejphar.2015.03.067. Epub 2015 Apr 3.

Elements in support of the 'non-identity' of the PGRMC1 protein with the σ2 receptor.

Abate C, Niso M, Infantino V, Menga A, Berardi F.

σ2 Receptor subtype is overexpressed in a variety of human tumors, with σ2 agonists showing antiproliferative effects towards tumor cells through multiple pathways that depend both on the tumor cell type and on the molecule type. Therefore, σ2 receptor is an intriguing target for tumor diagnosis and treatment despite the fact that that it has not yet been cloned. One of the last attempts to characterize σ2 receptors led to identify it as the progesterone receptor membrane component 1 (PGRMC1). Although still controversial, such identity appears to have been accepted. We the aim of contributing to solve this controversy, in this work we stably silenced or overexpressed PGRMC1 protein in human MCF7 adenocarcinoma cells. Western blotting analyses were performed to quantify the presence of PGRMC1 protein on each of the three MCF7 cell lines variants, while scatchard analyses with radioligand were performed in order to determine the expression of the σ2 receptors. In order to correlate the antiproliferative effect of σ2 receptor agonist with PGRMC1 density, some σ2 ligands were administered to each of the three MCF7 cells variants. The results suggested that PGRMC1 and σ2 receptors are two different molecular entities.

Copyright © 2015 Elsevier B.V. All rights reserved. PMID: 25843410 [PubMed - in process]

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