Domenico Iacopetta

A fourth ADP/ATP carrier isoform in man: identification, bacterial expression, functional characterization and tissue distribution.

The mitochondrial ADP/ATP carriers (AACs) catalyze the exchange of cytosolic ADP for matrix ATP. We have identified and characterized a novel member of the AAC subfamily of mitochondrial metabolite transport proteins, termed AAC4. The AAC4 gene maps to human chromosome 4q28.1, and its product AAC4 is 66–68% identical to human AAC 1–3 and is localized to mitochondria. AAC4 transcripts are exclusively present in liver, testis and brain unlike those of AAC 1–3. Consistent with its belonging to the AAC subfamily, upon heterologous expression and reconstitution into liposomes AAC4 exchanges ADP for ATP by an electrogenic antiport mechanism with high specificity and high sensitivity to carboxyatractyloside and bongkrekic acid.

Oleuropein and hydroxytyrosol inhibit MCF-7 breast cancer cell proliferation interfering with ERK1/2 activation.

The growth of many breast tumors is stimulated by estradiol (E2), which activates a classic mechanism of regulation of gene expression and signal transduction pathways inducing cell proliferation. Polyphenols of natural origin with chemical similarity to estrogen have been shown to interfere with tumor cell proliferation. The aim of this study was to investigate whether hydroxytyrosol (HT) and oleuropein (OL), two polyphenols contained in extra-virgin olive oil, can affect breast cancer cell proliferation interfering with E2-induced molecular mechanisms. Both HT and OL inhibited proliferation of MCF-7 breast cancer cells. Luciferase gene reporter experiments, using a construct containing estrogen responsive elements able to bind estrogen receptor alpha (ERalpha) and the study of the effects of HT or OL on ERalpha expression, demonstrated that HT and OL are not involved in ERalpha-mediated regulation of gene expression. However, further experiments pointed out that both OL and HT determined a clear inhibition of E2-dependent activation of extracellular regulated kinase1/2 belonging to the mitogen activating protein kinase family. Our study demonstrated that HT and OL can have a chemo-preventive role in breast cancer cell proliferation through the inhibition of estrogen-dependent rapid signals involved in uncontrolled tumor cell growth.

G protein-coupled estrogen receptor mediates the up-regulation of fatty acid synthase induced by 17β-estradiol in cancer cells and cancer-associated fibroblasts.

Background: Fatty acid synthase (FASN) is a key lipogenic enzyme regulated by various factors, including estrogens. Results: GPER mediates FASN expression and activity induced by estrogens in cancer cells. Conclusion: Fatty acid biogenesis is regulated by estrogens through GPER. Significance: GPER may be included among the transduction mediators involved by estrogens in regulating FASN expression and activity. Activation of lipid metabolism is an early event in carcinogenesis and a central hallmark of many tumors. Fatty acid synthase (FASN) is a key lipogenic enzyme catalyzing the terminal steps in the de novo biogenesis of fatty acids. In cancer cells, FASN may act as a metabolic oncogene, given that it confers growth and survival advantages to these cells, whereas its inhibition effectively and selectively kills tumor cells. Hormones such as estrogens and growth factors contribute to the transcriptional regulation of FASN expression also through the activation of downstream signaling and a cross-talk among diverse transduction pathways. In this study, we demonstrate for the first time that 17β-estradiol (E2) and the selective GPER ligand G-1 regulate FASN expression and activity through the GPER-mediated signaling, which involved the EGF receptor/ERK/c-Fos/AP1 transduction pathway, as ascertained by using specific pharmacological inhibitors, performing gene-silencing experiments and ChIP assays in breast SkBr3, colorectal LoVo, hepatocarcinoma HepG2 cancer cells, and breast cancer-associated fibroblasts. In addition, the proliferative effects induced by E2 and G-1 in these cells involved FASN as the inhibitor of its activity, named cerulenin, abolished the growth response to both ligands. Our data suggest that GPER may be included among the transduction mediators involved by estrogens in regulating FASN expression and activity in cancer cells and cancer-associated fibroblasts that strongly contribute to cancer progression.

The biochemical properties of the mitochondrial thiamine pyrophosphate carrier from Drosophila melanogaster.

The mitochondrial carriers are a family of transport proteins that shuttle metabolites, nucleotides and cofactors across the inner mitochondrial membrane. The genome of Drosophila melanogaster encodes at least 46 members of this family. Only five of these have been characterized, whereas the transport functions of the remainder cannot be assessed with certainty. In the present study, we report the functional identification of two D. melanogaster genes distantly related to the human and yeast thiamine pyrophosphate carrier (TPC) genes as well as the corresponding expression pattern throughout development. Furthermore, the functional characterization of the D. melanogaster mitochondrial thiamine pyrophosphate carrier protein (DmTpc1p) is described. DmTpc1p was over‐expressed in bacteria, the purified protein was reconstituted into liposomes, and its transport properties and kinetic parameters were characterized. Reconstituted DmTpc1p transports thiamine pyrophosphate and, to a lesser extent, pyrophosphate, ADP, ATP and other nucleotides. The expression of DmTpc1p in Saccharomyces cerevisiaeTPC1 null mutant abolishes the growth defect on fermentable carbon sources. The main role of DmTpc1p is to import thiamine pyrophosphate into mitochondria by exchange with intramitochondrial ATP and/or ADP.

SLC37A1 Gene expression is up-regulated by epidermal growth factor in breast cancer cells

Phospholipid biosynthesis exerts an important role in the proliferation of tumor cells; however, the regulation of the proteins involved in this context still remains to be fully evaluated. SLC37A1 protein belongs to a small family of sugar-phosphate/phosphate exchangers. The sequence homology with the bacterial glycerol-3-phosphate transporter (30%) suggests that SLC37A1 might be able to catalyze an exchange of glycerol-3-phosphate against phosphate. Glycerol-3-phosphate, found in different cellular compartments, is a fundamental substrate in phospholipid biosynthesis. In the present study, we demonstrate for the first time that epidermal growth factor (EGF) transactivates SLC37A1 promoter sequence and induces SLC37A1 mRNA, and protein expression through the EGFR/MAPK/Fos transduction pathway in ER-negative SkBr3 breast cancer cells. These findings were corroborated by comparable results obtained in ER-positive endometrial Ishikawa tumor cells. Interestingly, we also show that SLC37A1 protein localizes in the endoplasmic reticulum, hence supporting its possible involvement in phospholipid biosynthesis. On the basis of our data, the up-regulation of SLC37A1 gene expression should be included among the well-known stimulatory action exerted by EGF in breast cancer cells. In addition, further studies are required to provide evidence concerning the potential role of EGF-mediated SLC37A1 induction in breast tumor cells.

Identification of the Drosophila melanogaster Mitochondrial Citrate Carrier: Bacterial Expression, Reconstitution, Functional Characterization and Developmental Distribution

The mitochondrial carriers are a family of transport proteins that shuttle metabolites, nucleotides and cofactors across the inner mitochondrial membrane. The genome of Drosophila melanogaster encodes at least 46 members of this family. Only four of them have been characterized: the two isoforms of the ADP/ATP translocase, the brain uncoupling protein and the carnitine/acylcarnitine carriers. The transport functions of the remainders cannot be assessed with certainty. One of them, the product of the gene CG6782, shows a fairly close sequence homology to the known sequence of the rat mitochondrial citrate carrier. In this article the fruit fly protein coding by the CG6782 gene has been functionally characterized by over-expression in Escherichia coli and reconstitution into liposomes. It shows to have similar transport properties of the eukaryotic mitochondrial citrate carriers previously biochemically characterized. This indicates that in addition to the protein sequence conservation, insect and mammalian citrate carriers are also significantly related at the functional level suggesting that Drosophila may be used as model organism for the study of mitochondrial solute transporter. The DmCIC expression pattern throughout development was also investigated; the transcripts were detected at equal levels in all stages analysed.

Carbazole derivatives: a promising scenario for breast cancer treatment

Chemotherapeutics used in cancer treatment may elicit pleiotropic effects interfering, for instance, directly on DNA metabolism or on endoplasmic organelles functions. Recently there has been a trend towards the use of molecular-targeted therapies as alternative treatments of cancer, arising from the need to overcome the onset of undesired side effects or drug-resistance. Thus, a major challenge is the design and synthesis of new agents able to interact with specific cellular components, often over-expressed or altered in cancerous cells, such as telomerase and topoisomerase or protein kinases, with reduced toxicity at effective doses. The main molecular targets for the development of new anticancer drugs include: cell surface receptors, signal transduction pathways, enzymes, gene transcription, ubiquitin-proteasome/heat shock proteins, and anti-angiogenic agents. Several natural or synthetic polycyclic molecules with carbazolic nucleus, which show attractive drug-like properties, were identified with the aim to increase their biological activities and their specificity, obtaining cytotoxic agents effective in a panel of cancer cell lines. The cytotoxic profile of these compounds has been assessed using several in vitro assays as, for instance, MTT, colony formation, and flow cytometry assays and some of these compounds showed an interesting profile at sub-micromolar concentrations. The usefulness of some carbazole derivatives has been demonstrated, as well, in preclinical studies.

N-Alkyl Carbazole Derivatives as New Tools for Alzheimer’s Disease: Preliminary Studies

Alzheimer’s disease (AD) is a progressive and age-related neurodegenerative disorder affecting brain cells and is the most common form of “dementia”, because of the cognitive detriment which takes place. Neuronal disruption represents its major feature, due to the cytosolic accumulation of amyloid β-peptide (Aβ) which leads to senile plaques formation and intracellular neurofibrillary tangles. Many studies have focused on the design and therapeutic use of new molecules able to inhibit Aβ aggregation. In this context, we evaluated the ability of two recently synthesized series of N-alkyl carbazole derivatives to increase the Aβ soluble forms, through molecular docking simulations and in vitro experiments. Our data evidenced that two carbazole derivatives, the most active, adopt distinct binding modes involving key residues for Aβ fibrillization. They exhibit a good interfering activity on Aβ aggregation in mouse (N2a) cells, stably expressing wild-type human amyloid precursor protein (APP) 695. These preliminary results are promising and we are confident that the N-alkyl carbazole derivatives may encourage next future studies needed for enlarging the knowledge about the AD disease approach.

Bergamot (Citrus bergamia Risso) Flavonoids and Their Potential Benefits in Human Hyperlipidemia and Atherosclerosis: an Overview.

Elevated serum cholesterol, triglycerides and LDL levels are often associated with an increased incidence of atherosclerosis and coronary artery disease. The most effective therapeutic strategy against these diseases is based on statins administration, nevertheless some patients, especially those with metabolic syndrome fail to achieve their recommended LDL targets with statin therapy, moreover, it may induce many serious side effects. Several scientific studies have highlighted a strong correlation between diets rich in flavonoids and cardiovascular risk reduction. In particular, Citrus bergamia Risso, also known as bergamot, has shown a significant degree of hypocholesterolemic and antioxidant/radical scavenging activities. In addition, this fruit has attracted considerable attention due to its peculiar flavonoid composition, since it contains some flavanones that can act as natural statins. Hence, the study of bergamot flavonoids as metabolic regulators offers a great opportunity for screening and discovery of new therapeutic agents. Cholesterol metabolism, flavonoid composition and potential therapeutic use of C. bergamia Risso will be discussed in the following review.

3-(Dipropylamino)-5-hydroxybenzofuro[2,3-f]quinazolin-1(2H)-one (DPA-HBFQ-1) plays an inhibitory role on breast cancer cell growth and progression.

A series of unknown 3-(alkyl(dialkyl)amino)benzofuro[2,3-f]quinazolin-1(2H)-ones 4-17 has been synthesized as new ellipticine analogs, in which the carbazole moiety and the pyridine ring were replaced by a dibenzofuran residue and a pyrimidine ring, respectively. The synthesis of these benzofuroquinazolinones 4-17 was performed in a simple one-pot reaction using 3-aminodibenzofuran or its 2-methoxy derivative, as starting materials. From 3-(dipropylamino)-5-methoxybenzofuro[2,3-f] quinazolin-1(2H)-one (13), we prepared 3-(dipropylamino)-5-hydroxybenzofuro[2,3-f]quinazolin-1(2H)-one (18), referred to as DPA-HBFQ-1. The cytotoxic activities of all the synthesized compounds, tested in different human breast cancer cell lines, revealed that DPA-HBFQ-1 was the most active compound. In particular, the latter was able to inhibit anchorage-dependent and -independent cell growth and to induce apoptosis in estrogen receptor alpha (ERα)-positive and -negative breast cancer cells. It did not affect proliferation and apoptotic responses in MCF-10A normal breast epithelial cells. The observed effects have been ascribed to an enhanced p21(Cip1/WAF1) expression in a p53-dependent manner of tumor suppressor and to a selective inhibition of human topoisomerase II. In addition, DPA-HBFQ-1 exerted growth inhibitory effects also in other cancer cell lines, even though with a lower cytotoxic activity. Our results indicate DPA-HBFQ-1 as a good candidate to be useful as cancer therapeutic agent, particularly for breast cancer.