Impact of STAT-signaling pathway on cancer-associated fibroblasts in colorectal cancer and its role in immunosuppression.

Colorectal cancer (CRC) remains one of the most commonly diagnosed and deadliest types of cancer worldwide. CRC displays a desmoplastic reaction (DR) that has been inversely associated with poor prognosis; less DR is associated with a better prognosis. This reaction generates excessive connective tissue, in which cancer-associated fibroblasts (CAFs) are critical cells that form a part of the tumor microenvironment. CAFs are directly involved in tumorigenesis through different mechanisms. However, their role in immunosuppression in CRC is not well understood, and the precise role of signal transducers and activators of transcription (STATs) in mediating CAF activity in CRC remains unclear. Among the myriad chemical and biological factors that affect CAFs, different cytokines mediate their function by activating STAT signaling pathways. Thus, the harmful effects of CAFs in favoring tumor growth and invasion may be modulated using STAT inhibitors. Here, we analyze the impact of different STATs on CAF activity and their immunoregulatory role.

Identification of flap structure-specific endonuclease 1 as a factor involved in long-term memory formation of aversive learning.

We previously proposed that DNA recombination/repair processes play a role in memory formation. Here, we examined the possible role of the fen-1 gene, encoding a flap structure-specific endonuclease, in memory consolidation of conditioned taste aversion (CTA). Quantitative real-time PCR showed that amygdalar fen-1 mRNA induction was associated to the central processing of the illness experience related to CTA and to CTA itself, but not to the central processing resulting from the presentation of a novel flavor. CTA also increased expression of the Fen-1 protein in the amygdala, but not the insular cortex. In addition, double immunofluorescence analyses showed that amygdalar Fen-1 expression is mostly localized within neurons. Importantly, functional studies demonstrated that amygdalar antisense knockdown of fen-1 expression impaired consolidation, but not short-term memory, of CTA. Overall, these studies define the fen-1 endonuclease as a new DNA recombination/repair factor involved in the formation of long-term memories.

P2Y2 receptor desensitization on single endothelial cells.

Receptor desensitization, or decreased responsiveness of a receptor to agonist stimulation, represents a regulatory process with the potential to have a significant impact on cell behavior. P2Y(2), a G-protein-coupled receptor activated by extracellular nucleotides, undergoes desensitization at many tissues, including the vascular endothelium. Endothelial cells from a variety of vascular beds are normally exposed to extracellular nucleotides released from damaged cells and activated platelets. The purpose of the present study was to compare P2Y(2) receptor desensitization observed in endothelial cells derived from bovine retina, a model of microvascular endothelium, and human umbilical vein endothelial cells (HUVECs), a model of a large blood vessel endothelium. P2Y(2) receptor desensitization was monitored by following changes in UTP-stimulated intracellular free Ca(2 +) in single cells using fura-2 microfluorometry. Both endothelial cell models exhibited desensitization of the P2Y(2) receptor after stimulation with UTP. However, the cells differed in the rate, dependence on agonist concentration, and percentage of maximal desensitization. These results suggest differential mechanisms of P2Y(2) receptor desensitization and favors heterogeneity in extracellular nucleotide activity in endothelial cells according to its vascular bed origin.

P2Y2 nucleotide receptors inhibit trauma-induced death of astrocytic cells.

Nucleotides as well as other neurotransmitters are known to be released to the extracellular space upon injury. To determine whether nucleotides acting on P2Y(2) nucleotide receptors promote protective or degenerative events after trauma in astrocytic cells, a well-established model of in vitro brain trauma was applied to 1321N1 cells expressing recombinant P2Y(2) nucleotide receptors (P2Y(2)R-1321N1). Cellular death was examined by measuring DNA fragmentation and caspase activation. Fragmented DNA was observed 48 h post-injury in 1321N1 cells, while P2Y(2) nucleotide receptor expressing cells did not show DNA fragmentation. A laddering pattern of fragmented DNA following injury was observed upon inhibition of P2Y(2) nucleotide receptors with suramin. Time-dependent increases of cleaved caspase-9, a mitochondrial-associated caspase, correlated with injury-induced cellular death. A decreased bax/bcl-2 gene expression ratio was observed in P2Y(2)R-1321N1 cells after traumatic injury, while untransfected 1321N1 cells showed a significant time-dependent increase of the bax/bcl-2 gene expression ratio. Activation of protein kinases was assessed to determine the signaling pathways involved in cell death and survival responses following traumatic injury. In P2Y(2)R-1321N1 and 1321N1 cells p38 phosphorylation was stimulated in a time-dependent manner but the phosphatidylinositol 3-kinase-dependent activation of extracellular signal-regulated kinase 1/2 and protein kinase B (PKB)/Akt was only observed in P2Y(2)R-1321N1 cells after injury. The stress-activated protein kinase/c-Jun NH2-terminal kinase (SAPK/JNK) signaling pathway was not activated by traumatic injury in either astrocytic cell line. Inhibition of p38 kinase signaling pathway by treatment with PD1693, a MKK3/6 inhibitor, abolished the expression of cleaved caspase-9, the increase in the bax/bcl-2 gene expression ratio, as well as the fragmentation of DNA that followed injury of 1321N1 cells. Taken together, our results demonstrate a novel role for P2Y(2) nucleotide receptors and extracellular nucleotides in mediating survival responses to glial cells undergoing cellular death induced by trauma.

Immobilized P2X2 purinergic receptor stationary phase for chromatographic determination of pharmacological properties and drug screening.

The purinergic receptor signaling system plays an important role in communication between cells in the nervous system and opens new opportunities for screening of potential drugs. Our objective was to explore the pharmacological properties and establish a new methodology for ligand screening for the P2X2 receptor, which has been developed by the combinatorial library approach Systematic Evolution of Ligands by Exponential enrichment (SELEX). To this end, membranes of 1321N1 cells stably transfected with rat P2X2 receptors were resuspended in 2% cholate detergent and subsequently coupled onto an immobilized artificial membrane (IAM). The IAM-cholate-P2X2 mixture was then dialyzed, centrifuged and packed into a FPLC column. Equilibrium binding to the receptor and competition between ATP and the purinergic antagonists suramin and 2'3'-O-(2,4,6-trinitrophenyl) adenosine 5'-triphosphate (TNP-ATP) were analyzed by a chromatographic assay using 32P alpha ATP as a radioligand. Our data indicate that suramin does not compete with ATP for the ligand binding site and TNP-ATP is a competitive antagonist, confirming previous studies [C.A. Trujillo, A.A. Nery, A.H. Martins, P. Majumder, F.A. Gonzalez, H. Ulrich, Biochemistry 45 (2006) 224-233]. In addition, we demonstrate that this assay can be used in in vitro selection procedures for RNA aptamers binding to P2X2 receptors. The results demonstrate that the receptor can be immobilized in a stable format and reused over an extended period of time, facilitating the exploration of ligand-receptor interactions and screening of combinatorial pools for possible ligands.

Postmenopause is associated with recurrence of differentiated papillary thyroid carcinoma.

UNLABELLED: Differentiated papillary thyroid carcinoma (D-PTC) is the most common malignancy arising in the thyroid gland. There are gender differences in the incidence of PTC being mainly observed in females. Low-risk groups consisted of men younger than 40-year-old and women younger than 50-year-old, whereas the high-risk group are older patients. We believe that age is not enough to explain the clinical course of this neoplasm and hypothesize that aggressive behavior of D-PTC may be correlated with hormonal status. Studies that support this idea showed that the follicular neoplastic cells had higher estrogen receptor-alpha in premenopausal (28.1+/-4.5) than in postmenopausal women (14.2+/-2.9). According to author's prior observations, there are evidences correlating recurrence of D-PTC with postmenopause in women. Postmenopause status is characterized by estrogen decrease and FSH increases both associated with EGFR activation. Previous observations identified EGFR over-expression in D-PTC of postmenopause when compared with premenopausal ladies. CONCLUSIONS: Postmenopause is an adverse factor for tumor evolution in women with D-PTC and is associated with EGFR expression. It's introduction in thyroid tumor stratification could be a fine tuning in predicting papillary thyroid carcinoma behavior.

Inhibition mechanism of the recombinant rat P2X(2) receptor in glial cells by suramin and TNP-ATP.

P2X receptors play an important role in communication between cells in the nervous system. Therefore, understanding the mechanisms of inhibition of these receptors is important for the development of new tools for drug discovery. Our objective has been to determine the pharmacological activity of the antagonist suramin, the most important antagonist of purinergic receptor function, as well as to demonstrate its noncompetitive inhibition and confirm a competitive mechanism between ATP and TNP-ATP in 1321N1 glial cells stably transfected with the recombinant rat P2X(2) receptor. A radioligand binding assay was employed to determine whether suramin, TNP-ATP, and ATP compete for the same binding site on the receptor. TNP-ATP displaced [alpha-32P]ATP, whereas suramin did not interfere with [alpha-32P]ATP-receptor binding. To determine the inhibition mechanism relevant for channel opening, currents obtained in fast kinetic whole-cell recording experiments, following stimulation of cells by ATP in the presence of suramin, were compared to those obtained by ATP in the presence of TNP-ATP. Supported by a mathematical model for receptor kinetics [Breitinger, H. G., Geetha, N., and Hess, G. P. (2001) Biochemistry 40, 8419-8429], the inhibition factors were plotted as functions of inhibitor or agonist concentrations. Analysis of the data indicated a competitive inhibition mechanism for TNP-ATP and a noncompetitive inhibition for suramin. Taken together, both data support a noncompetitive inhibition mechanism of the rat recombinant P2X(2) receptor by suramin, confirm the competitive inhibition by TNP-ATP, and allow the prediction of a model for P2X(2) receptor inhibition.

Mechanisms for inhibition of P2 receptors signaling in neural cells.

Trophic factors are required to ensure neuronal viability and regeneration after neural injury. Although abundant information is available on the factors that cause the activation of astrocytes, little is known about the molecular mechanisms underlying the regulation of this process. Nucleotides released into the extracellular space from injured or dying neural cells can activate astrocytes via P2 nucleotide receptors. After a brief historical review and update of novel P2 receptor antagonists, this article focuses on recent advancements toward understanding molecular mechanisms that regulate G protein-coupled P2Y receptor signaling. Among P2Y receptor subtypes, the heptahelical P2Y2 nucleotide receptor interacts with vitronectin receptors via an RGD sequence in the first extracellular loop, and this interaction is required for effective signal transduction to activate mitogen-activated protein kinases ERK1/2, to mobilize intracellular calcium stores via activation of phospholipase C, protein kinase C isoforms, and to activate focal adhesion kinase and other signaling events. Ligation of vitronectin receptors with specific antibodies caused an inhibition of P2Y2 receptor-induced ERK1/2 and p38 phosphorylation and P2Y2 receptor-induced cytoskeleton rearrangement and DNA synthesis. Structure-function studies have identified agonist-induced phosphorylation of the C-terminus of the P2Y2 receptor, an important mechanism for receptor desensitization. Understanding selective mechanisms for regulating P2Y2 receptor signaling could provide novel targets for therapeutic strategies in the management of brain injury, synaptogenesis, and neurological disorders.

P2Y receptors activate neuroprotective mechanisms in astrocytic cells.

Mechanical or ischemic trauma to the CNS causes the release of nucleotides and other neurotransmitters into the extracellular space. Nucleotides can activate nucleotide receptors that modulate the expression of genes implicated in cellular adaptive responses. In this investigation, we used human 1321N1 astrocytoma cells expressing a recombinant P2Y2 receptor to assess the role of this receptor in the regulation of anti-apoptotic (bcl-2 and bcl-xl) and pro-apoptotic (bax) gene expression. Acute treatment with the P2Y2 receptor agonist UTP up-regulated bcl-2 and bcl-xl, and down-regulated bax, gene expression. Activation of P2Y2 receptors was also coupled to the phosphorylation of cyclic AMP responsive element binding protein that positively regulates bcl-2 and bcl-xl gene expression. Cyclic AMP responsive element decoy oligonucleotides markedly attenuated the UTP-induced increase in bcl-2 and bcl-xl mRNA levels. Activation of P2Y2 receptors induced the phosphorylation of the pro-apoptotic factor Bad and caused a reduction in bax/bcl-2 mRNA expression ratio. All these signaling pathways are known to be involved in cell survival mechanisms. Using cDNA microarray analysis and RT-PCR, P2Y2 receptors were found to up-regulate the expression of genes for neurotrophins, neuropeptides and growth factors including nerve growth factor 2; neurotrophin 3; glia-derived neurite-promoting factor, as well as extracellular matrix proteins CD44 and fibronectin precursor--genes known to regulate neuroprotection. Consistent with this observation, conditioned media from UTP-treated 1321N1 cells expressing P2Y2 receptors stimulated the outgrowth of neurites in PC-12 cells. Taken together, our results suggest an important novel role for the P2Y2 receptor in survival and neuroprotective mechanisms under pathological conditions.

P2X7 receptors stimulate AKT phosphorylation in astrocytes.

1. Emerging evidence indicates that nucleotide receptors are widely expressed in the nervous system. Here, we present evidence that P2Y and P2X receptors, particularly the P2X(7) subtype, are coupled to the phosphoinositide 3-kinase (PI3K)/Akt pathway in astrocytes. 2. P2Y and P2X receptor agonists ATP, uridine 5'-triphosphate (UTP) and 2',3'-O-(4-benzoyl)-benzoyl ATP (BzATP) stimulated Akt phosphorylation in primary cultures of rat cortical astrocytes. BzATP induced Akt phosphorylation in a concentration- and time-dependent manner, similar to the effect of BzATP on Akt phosphorylation in 1321N1 astrocytoma cells stably transfected with the rat P2X(7) receptor. Activation was maximal at 5 - 10 min and was sustained for 60 min; the EC(50) for BzATP was approximately 50 microM. In rat cortical astrocytes, the positive effect of BzATP on Akt phosphorylation was independent of glutamate release. 3. The effect of BzATP on Akt phosphorylation in rat cortical astrocytes was significantly reduced by the P2X(7) receptor antagonist Brilliant Blue G and the P2X receptor antagonist iso-pyridoxal-5'-phosphate-6-azophenyl-2',4'-disulfonic acid, but was unaffected by trinitrophenyl-ATP, oxidized ATP, suramin and reactive blue 2. 4. Results with specific inhibitors of signal transduction pathways suggest that extracellular and intracellular calcium, PI3K and a Src family kinase are involved in the BzATP-induced Akt phosphorylation pathway. 5. In conclusion, our data indicate that stimulation of astrocytic P2X(7) receptors, as well as other P2 receptors, leads to Akt activation. Thus, signaling by nucleotide receptors in astrocytes may be important in several cellular downstream effects related to the Akt pathway, such as cell cycle and apoptosis regulation, protein synthesis, differentiation and glucose metabolism.

The first total synthesis of the marine fatty acid (+/-)-2-methoxy-13-methyltetradecanoic acid: a cytotoxic fatty acid to leukemia cells.

The recently discovered marine fatty acid (+/-)-2-methoxy-13-methyltetradecanoic acid was synthesized for the first time in six steps (26% overall yield) starting from commercially available methyl 12-methyltridecanoate. The synthetic approach provided enough material to corroborate the structure of the acid, which was recently identified in the sponge Amphimedon complanata from Aguadilla, Puerto Rico, and to test its cytotoxicity to three leukemia cell lines. The key step in the synthesis was the addition of trimethylsilyl cyanide to 12-methyltridecanal under triethylamine catalysis. Nuclear magnetic resonance data are provided for the first time for this methoxylated fatty acid and the synthetic approach utilized is of general applicability since it can be used in the synthesis of other methyl-branched 2-methoxylated fatty acids. We also report that the acid (+/-)-2-methoxy-13-methyltetradecanoic acid is cytotoxic to human chronic myelogenous leukemia K-562 (EC50=238 microM), histiocytic lymphoma U-937 (EC50=250 microM), and promielocytic leukemia HL-60 (EC50=476 microM) in RPMI 1640 medium.