Vincenza Dolce

G-Protein-Coupled Receptor 30 and Estrogen Receptor-[Alpha] Are Involved in the Proliferative Effects Induced by Atrazine in Ovarian Cancer Cells

Background Atrazine, one of the most common pesticide contaminants, has been shown to up-regulate aromatase activity in certain estrogen-sensitive tumors without binding or activating the estrogen receptor (ER). Recent investigations have demonstrated that the orphan G-protein–coupled receptor 30 (GPR30), which is structurally unrelated to the ER, mediates rapid actions of 17β-estradiol and environmental estrogens. Objectives Given the ability of atrazine to exert estrogen-like activity in cancer cells, we evaluated the potential of atrazine to signal through GPR30 in stimulating biological responses in cancer cells. Methods and results Atrazine did not transactivate the endogenous ERα in different cancer cell contexts or chimeric proteins encoding the ERα and ERβ hormone-binding domain in gene reporter assays. Moreover, atrazine neither regulated the expression of ERα nor stimulated aromatase activity. Interestingly, atrazine induced extracellular signal-regulated kinase (ERK) phosphorylation and the expression of estrogen target genes. Using specific signaling inhibitors and gene silencing, we demonstrated that atrazine stimulated the proliferation of ovarian cancer cells through the GPR30–epidermal growth factor receptor transduction pathway and the involvement of ERα. Conclusions Our results indicate a novel mechanism through which atrazine may exert relevant biological effects in cancer cells. On the basis of the present data, atrazine should be included among the environmental contaminants potentially able to signal via GPR30 in eliciting estrogenic action.

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.

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.

Copper activates HIF-1α/GPER/VEGF signalling in cancer cells

Copper promotes tumor angiogenesis, nevertheless the mechanisms involved remain to be fully understood. We have recently demonstrated that the G-protein estrogen receptor (GPER) cooperates with hypoxia inducible factor-1α (HIF-1α) toward the regulation of the pro-angiogenic factor VEGF. Here, we show that copper sulfate (CuSO4) induces the expression of HIF-1α as well as GPER and VEGF in breast and hepatic cancer cells through the activation of the EGFR/ERK/c-fos transduction pathway. Worthy, the copper chelating agent TEPA and the ROS scavenger NAC prevented the aforementioned stimulatory effects. We also ascertained that HIF-1α and GPER are required for the transcriptional activation of VEGF induced by CuSO4. In addition, in human endothelial cells, the conditioned medium from breast cancer cells treated with CuSO4 promoted cell migration and tube formation through HIF-1α and GPER. The present results provide novel insights into the molecular mechanisms involved by copper in triggering angiogenesis and tumor progression. Our data broaden the therapeutic potential of copper chelating agents against tumor angiogenesis and progression.

Effects of atrazine on estrogen receptor α- and G protein-coupled receptor 30-mediated signaling and proliferation in cancer cells and cancer-associated fibroblasts.

Background: The pesticide atrazine does not bind to or activate the classical estrogen receptor (ER), but it up-regulates the aromatase activity in estrogen-sensitive tumor cells. The G protein estrogen receptor (GPR30/GPER) has been reported to be involved in certain biological responses to endogenous estrogens and environmental compounds exerting estrogen-like activity. Objectives: We aimed to evaluate the potential of atrazine to trigger GPER-mediated signaling in cancer cells and cancer-associated fibroblasts (CAFs). Methods and Results: Using gene reporter assays in diverse types of cancer cells, we found that atrazine did not transactivate endogenous ERα or chimeric proteins that encode the ERα and ERβ hormone binding domains. Conversely, atrazine was able to bind to GPER to induce ERK activation and the expression of estrogen target genes, which, interestingly, appeared to rely on both GPER and ERα expression. As a biological counterpart, atrazine stimulated the proliferation of ovarian cancer cells that depend on GPER and ERα, as evidenced by gene silencing experiments and the use of specific signaling inhibitors. Of note, through GPER, atrazine elicited ERK phosphorylation, gene expression, and migration in CAFs, thus extending its stimulatory role to these main players of the tumor microenvironment. Conclusions: Our results suggest a novel mechanism through which atrazine may exert relevant biological effects in cancer cells and CAFs. On the basis of our data, atrazine should be included among the environmental contaminants that may elicit estrogenic activity through GPER-mediated signaling. Citation: Albanito L, Lappano R, Madeo A, Chimento A, Prossnitz ER, Capello AR, Dolce V, Abonante S, Pezzi V, Maggiolini M. 2015. Effects of atrazine on estrogen receptor α– and G protein–coupled receptor 30–mediated signaling and proliferation in cancer cells and cancer-associated fibroblasts. Environ Health Perspect 123:493–499; http://dx.doi.org/10.1289/ehp.1408586

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.

The Mitochondrial Citrate Carrier (CIC) Is Present and Regulates Insulin Secretion by Human Male Gamete

The mechanisms through which sperm manage their energy metabolism are poorly understood. The present study provides biochemical and morphological evidence that mitochondrial citrate carrier (CIC) is present in ejaculated human sperm and is restricted to the midpiece. The inhibition of CIC with the specific substrate analog 1,2,3-benzenetricarboxylate resulted in the reduction of cholesterol efflux, protein tyrosine phosphorylation, phospho-AKT, phospho-p60src, hyperactivated motility and acrosome reaction, suggesting a role for this mitochondrial carrier in sperm physiology. Furthermore, inhibition of CIC by 1,2,3-benzenetricarboxylate resulted in a reduction of glucose-stimulated insulin secretion and autocrine insulin secretion by sperm. Remarkably, blocking CIC also reduced glucose-6-phosphate dehydrogenase activity, probably in accordance with its regulation on insulin secretion. Capacitation and glucose metabolism were stimulated by glucose as well as citrate, the specific substrate of CIC, implying a similar action because glucose and citrate both induced insulin secretion by sperm. In the present finding, we discovered a new site of action for CIC in the regulation of metabolism, and it may be assumed that CIC works with other factors in the regulation of sperm energy metabolism to sustain capacitation process and acrosome reaction.

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.

Substrate-induced conformational changes of the mitochondrial oxoglutarate carrier : a spectroscopic and molecular modelling study

The structural and dynamic properties of the oxoglutarate carrier were investigated by introducing a single tryptophan in the Trp-devoid carrier in position 184, 190 or 199 and by monitoring the fluorescence spectra in the presence and absence of the substrate oxoglutarate. In the absence of substrate, the emission maxima of Arg190Trp, Cys184Trp and Leu199Trp are centered at 342, 345 and 348 nm, respectively, indicating that these residues have an increasing degree of solvent exposure. The emission intensity of the Arg190Trp and Cys184Trp mutants is higher than that of Leu199Trp. Addition of substrate increases the emission intensity of Leu199Trp, but not that of Cys184Trp and Arg190Trp. A 3D model of the oxoglutarate carrier was built using the structure of the ADP/ATP carrier as a template and was validated with the experimental results available in the literature. The model identifies Lys122 as the most likely candidate for the quenching of Trp199. Consistently, the double mutant Lys122Ala-Leu199Trp exhibits a higher emission intensity than Leu199Trp and does not display further fluorescence enhancement in response to substrate addition. Substitution of Lys122 with Cys and evaluation of its reactivity with a sulphydryl reagent in the presence and absence of substrate confirms that residue 122 is masked by the substrate, likely through a substrate-induced conformational change.