Inhibition of transcellular tumor cell migration and metastasis by novel carba-derivatives of cyclic phosphatidic acid

Cyclic phosphatidic acid (1-acyl-sn-glycerol-2,3-cyclic phosphate; cPA) is a naturally occurring analog of lysophosphatidic acid (LPA) with a variety of distinctly different biological activities from those of LPA. In contrast to LPA, a potent inducer of tumor cell invasion, palmitoyl-cPA inhibits FBS- and LPA-induced transcellular migration and metastasis. To prevent the conversion of cPA to LPA we synthesized cPA derivatives by stabilizing the cyclic phosphate ring; to prevent the cleavage of the fatty acid we generated alkyl ether analogs of cPA. Both sets of compounds were tested for inhibitory activity on transcellular tumor cell migration. Carba derivatives, in which the phosphate oxygen was replaced with a methylene group at either the sn-2 or the sn-3 position, showed much more potent inhibitory effects on MM1 tumor cell transcellular migration and the pulmonary metastasis of B16-F0 melanoma than the natural pal-cPA. The antimetastatic effect of carba-cPA was accompanied by the inhibition of RhoA activation and was not due to inhibition of the activation of LPA receptors.     

alpha- and beta-substituted phosphonate analogs of LPA as autotaxin inhibitors

Autotaxin (ATX) is an attractive pharmacological target due to its lysophospholipase D activity which leads to the production of lysophosphatidic acid (LPA). Blockage of ATX produced LPA by small molecules could be a potential anticancer chemotherapy. In our previous study, we have identified the two beta-hydroxy phosphonate analogs of LPA (compounds f17 and f18) as ATX inhibitors. With this work, we investigated alpha- and beta-substituted phosphonate analogs of LPA and evaluated them for ATX inhibitory activity. The stereochemistry of beta-hydroxy phosphonates was also studied.     

Lysophosphatidic acids, cyclic phosphatidic acids and autotaxin as promising targets in therapies of cancer and other diseases

Lysophospholipids have long been recognized as membrane phospholipid metabolites, but only recently lysophosphatidic acids (LPA) have been demonstrated to act on specific G protein-coupled receptors. The widespread expression of LPA receptors and coupling to several classes of G proteins allow LPA-dependent regulation of numerous processes, such as vascular development, neurogenesis, wound healing, immunity, and cancerogenesis. Lysophosphatidic acids have been found to induce many of the hallmarks of cancer including cellular processes such as proliferation, survival, migration, invasion, and neovascularization. Furthermore, autotaxin (ATX), the main enzyme converting lysophosphatidylcholine into LPA was identified as a tumor cell autocrine motility factor. On the other hand, cyclic phosphatidic acids (naturally occurring analogs of LPA generated by ATX) have anti-proliferative activity and inhibit tumor cell invasion and metastasis. Research achievements of the past decade suggest implementation of preclinical and clinical evaluation of LPA and its analogs, LPA receptors, as well as autotaxin as potential therapeutic targets.     

Myristic acid analogs are inhibitors of Junin virus replication

The effects of two myristic acid analogs on Junin virus (JV) replication were investigated. The compounds chosen for the study were DL-2-hydroxymyristic acid (2OHM), an inhibitor of N-myristoyltransferase (NMT), which binds the enzyme and blocks protein myristoylation, and 13-oxamyristic acid (13OM), a competitive inhibitor of NMT which incorporates into the protein instead of myristic acid. Both types of analogs achieved dose-dependent inhibition of viral multiplication at concentrations not affecting cell viability. The 50% inhibitory concentration values determined by a virus-yield inhibition assay for different strains of JV, including a human pathogenic strain, and for the related arenavirus, Tacaribe, were in the range 1.6 to 20.1 microM, with 13OM as the most active compound. From time of addition and removal experiments, it can be concluded that both analogs inhibit a late stage in the JV replicative cycle, and their effect was partially reversible. The cytoplasmic and surface expression of JV glycoproteins was not affected in the presence of the compounds, as revealed by immunofluorescence staining, suggesting that JV glycoprotein myristoylation would not be essential for the intracellular transport of the envelope proteins, but it may have an important role in their interaction with the plasma membrane during virus budding.     

Design and pharmacophore modeling of biaryl methyl eugenol analogs as breast cancer invasion inhibitors

Cell invasion and migration are required for the parent solid tumor cells to metastasize to distant organs. Microtubules form a polarized network, enabling organelle and protein movement throughout the cell. Cytoskeletal elements coordinately regulate cell’s motility, adhesion, migration, exocytosis, endocytosis, and division. Thus, microtubule disruption can be a useful target to control cancer cell invasion and metastasis. The phenolic ether methyl eugenol (1), the major component of the essential oil of the leaves of Melaleuca ericifolia Sm. (Myrtaceae), was used as a starting scaffold to design eleven new and three known anti-tubulin agents 2–15 using carbon–carbon coupling reactions. A computer-assisted approach was used to design these new biaryl derivatives using colchicine-binding site of tubulin as the molecular target and colchicine as an active ligand. Several derivatives showed potent inhibitory activity against MDA-MB-231 cell migration at the 1–4 μM dose range. The Z isomers, 4 and 15 were more active as invasion inhibitors compared to their structurally related E isomers, 2 and 14. The cytotoxic activities of compounds 2–15 against two breast cancer cell lines MDA-MB-231 and MCF-7 were evaluated. Anti-invasive activity of the semisynthetic derivatives is not due to a direct cytotoxic effect on MDA-MB-231. Analogs 2–15 may promote their anti-invasive activity through the induction of changes in cell morphology. A pharmacophore model was generated involving seven essential features for activity, which was consistent with a previously generated colchicine site inhibitors model.     

Phosphatase-resistant analogues of lysophosphatidic acid: agonists promote healing, antagonists and autotaxin inhibitors treat cancer

Isoform-selective agonists and antagonists of the lysophosphatidic acid (LPA) G protein-coupled receptors (GPCRs) have important potential applications in cell biology and therapy. LPA GPCRs regulate cancer cell proliferation, invasion, angiogenesis, and also biochemical resistance to chemotherapy- and radiotherapy-induced apoptosis. LPA and its analogues also are feedback inhibitors of the enzyme lysophospholipase D (lysoPLD, a.k.a., autotaxin, ATX), a central regulator of invasion and metastasis. For cancer therapy, the optimal therapeutic profile would be a metabolically-stabilized, pan-LPA receptor antagonist that also inhibited lysoPLD. For protection of gastrointestinal mucosa and lymphocytes, LPA agonists would be desirable to minimize or reverse radiation or chemical-induced injury. Analogues of lysophosphatidic acid (LPA) that are chemically modified to be less susceptible to phospholipases and phosphatases show activity as long-lived receptor-specific agonists and antagonists for LPA receptors, as well as inhibitors for the lysoPLD activity of ATX.     

Pharmacological evaluation of a novel cyclic phosphatidic acid derivative 3-S-cyclic phosphatidic acid (3-S-cPA)

Cyclic phosphatidic acid (cPA) is a naturally occurring phospholipid mediator possessing cyclic phosphate ring, which is necessary for its specific biological activities. To stabilize cyclic phosphate ring of cPA, we synthesized a series of cPA derivatives. We have shown that racemic 3-S-cPA, with a phosphate oxygen atom replaced with a sulfur atom at the sn-3, was a more effective autotaxin (ATX) inhibitor than cPA. In this study, we showed that racemic 3-S-cPA also had potent biological activities such as inhibition of cancer cell migration, suppression of the nociceptive reflex, and attenuation of ischemia-induced delayed neuronal cell death in the hippocampal CA1. Moreover, we synthesized both enantiomers of palmitoleoyl derivative of 3-S-cPA, and found that the chirality of 3-S-cPA is not involved in ATX inhibition. Based on these findings, racemic 3-S-cPA is suggested as an effective therapeutic compound like cPA.     

MFAP3L activation promotes colorectal cancer cell invasion and metastasis

An abundance of microfibril-associated glycoprotein 3-like (MFAP3L) significantly correlates with distant metastasis in colorectal cancer (CRC), although the mechanism has yet to be explained. In this study, we observed that MFAP3L knock-down resulted in reduced CRC cell invasion and hepatic metastasis. We evaluated the cellular location and biochemical functions of MFAP3L and found that this protein was primarily localized in the nucleus of CRC cells and acted as a protein kinase. When EGFR translocated into the nucleus upon stimulation with EGF, MFAP3L was phosphorylated at Tyr287 within its SH2 motif, and the activated form of MFAP3L phosphorylated ERK2 at Thr185 and Tyr187. Moreover, the metastatic behavior of CRC cells in vitro and in vivo could be partially explained by activation of the nuclear ERK pathway through MFAP3L phosphorylation. Hence, we experimentally demonstrated for the first time that MFAP3L likely participates in the nuclear signaling of EGFR and ERK2 and acts as a novel nuclear kinase that impacts CRC metastasis.     

MicroRNA-221 promotes colorectal cancer cell invasion and metastasis by targeting RECK

MicroRNAs (miRNAs) have recently emerged as regulators of metastasis. We provide insight into the behavior of miR-221 in colorectal cancer (CRC) metastasis by showing that miR-221 is significantly upregulated in metastatic CRC cell lines and tissues. miR-221 overexpression enhances, whereas miR-221 depletion reduces CRC cell migration and invasion in vitro and metastasis in vivo. We identify RECK as a direct target of miR-221, reveal its expression to be inversely correlated with miR-221 in CRC samples and show that its re-introduction reverses miR-221-induced CRC invasiveness. Collectively, miR-221 is an oncogenic miRNA which may regulate CRC migration and invasion through targeting RECK.     

Directed cell invasion and migration during metastasis

Metastasis requires tumor cell dissemination to different organs from the primary tumor. Dissemination is a complex cell motility phenomenon that requires the molecular coordination of the protrusion, chemotaxis, invasion and contractility activities of tumor cells to achieve directed cell migration. Recent studies of the spatial and temporal activities of the small GTPases have begun to elucidate how this coordination is achieved. The direct visualization of the pathways involved in actin polymerization, invasion and directed migration in dissemination competent tumor cells will help identify the molecular basis of dissemination and allow the design and testing of more specific and selective drugs to block metastasis.     

Cannabidiol inhibits lung cancer cell invasion and metastasis via intercellular adhesion molecule-1

Cannabinoids inhibit cancer cell invasion via increasing tissue inhibitor of matrix metalloproteinases-1 (TIMP-1). This study investigates the role of intercellular adhesion molecule-1 (ICAM-1) within this action. In the lung cancer cell lines A549, H358, and H460, cannabidiol (CBD; 0.001-3 μM) elicited concentration-dependent ICAM-1 up-regulation compared to vehicle via cannabinoid receptors, transient receptor potential vanilloid 1, and p42/44 mitogen-activated protein kinase. Up-regulation of ICAM-1 mRNA by CBD in A549 was 4-fold at 3 μM, with significant effects already evident at 0.01 μM. ICAM-1 induction became significant after 2 h, whereas significant TIMP-1 mRNA increases were observed only after 48 h. Inhibition of ICAM-1 by antibody or siRNA approaches reversed the anti-invasive and TIMP-1-upregulating action of CBD and the likewise ICAM-1-inducing cannabinoids Δ(9)-tetrahydrocannabinol and R(+)-methanandamide when compared to isotype or nonsilencing siRNA controls. ICAM-1-dependent anti-invasive cannabinoid effects were confirmed in primary tumor cells from a lung cancer patient. In athymic nude mice, CBD elicited a 2.6- and 3.0-fold increase of ICAM-1 and TIMP-1 protein in A549 xenografts, as compared to vehicle-treated animals, and an antimetastatic effect that was fully reversed by a neutralizing antibody against ICAM-1 [% metastatic lung nodules vs. isotype control (100%): 47.7% for CBD + isotype antibody and 106.6% for CBD + ICAM-1 antibody]. Overall, our data indicate that cannabinoids induce ICAM-1, thereby conferring TIMP-1 induction and subsequent decreased cancer cell invasiveness.     

2-Carba cyclic phosphatidic acid inhibits lipopolysaccharide-induced prostaglandin E2 production in a human macrophage cell line

Cyclic phosphatidic acid (cPA) is a naturally occurring phospholipid mediator that contains a unique cyclic phosphate ring at the sn-2 and sn-3 positions of its glycerol backbone. Using mouse models for multiple sclerosis (cuprizone-induced demyelination and experimental autoimmune encephalomyelitis) and traumatic brain injury, we revealed that cPA and its metabolically stabilized cPA derivative, 2-carba-cPA (2ccPA), have potential to protect against neuroinflammation. In this study, we investigated whether 2ccPA has anti-inflammatory effect on peripheral immune function or not using inflammation-induced macrophages-like cell line, THP-1 monocytes differentiated by phorbol 12-myristate 13-acetate (PMA). Lipopolysaccharide (LPS)-stimulated THP-1 cells were found to have higher expression of the mRNAs of several inflammation-related cytokines and of the enzyme cyclooxygenase-2 (Cox-2); however, when THP-1 cells were stimulated by LPS in the presence of 2ccPA, the increase in the expression of pro-inflammatory cytokine and Cox-2 mRNA was attenuated. 2ccPA treatment also decreased the amount of prostaglandin E2 (PGE2) produced by LPS-stimulated THP-1 cells and decreased expression of the mRNA of prostaglandin E receptor 2 (EP2, PTGER2), a PGE2 receptor that mediates inflammation. These results indicate that 2ccPA has anti-inflammatory properties.     

Quantitative real-time imaging of molecular dynamics during cancer cell invasion and metastasis in vivo

Despite our advanced understanding of primary cancer development and progression, metastasis and the systemic spread of the disease to secondary sites remains the leading cause of cancer-associated death. The metastatic process is therefore a major potential therapeutic target area for cancer researchers and elucidating the key steps that are susceptible to therapeutic intervention will be critical to improve our treatment strategies. Recent advances in intravital imaging are rapidly improving our insight into this process and are helping in the design of stage-specific drug regimes for the treatment of metastatic cancer. Here we discuss current developments in intravital imaging and our recent use of photobleaching and photoactivation in the analysis of dynamic biomarkers in living animals to assess the efficacy of therapeutic intervention on early stages of tumor cell metastasis.Key words: in vivo imaging, photobleaching, photoactivation, biomarkersMetastasis is a complex process consisting of interactions between cancer cells and their surrounding extra-cellular matrix and stroma. To give rise to a secondary tumor, a primary tumor cell undergoes alterations to its cell-cell and cell-ECM contacts, allowing it to breach the basement membrane and intravasate into the vasculature or the lymphatic system. A tumor cell must survive in the circulation before extravasating at a secondary site and initiating new tumor growth and the development of its own blood supply. Imaging this process in live animals under native physiological conditions is inherently difficult due to poor sample stability, tissue penetration and autofluorescence of the tissue. However, new advances in fluorescent imaging, including the continued development of green fluorescent protein (GFP) and its variants, have facilitated the observation of this process and shed light on some key mechanisms that determine how and why cells metastasise. The use of fluorescent probes for in vivo imaging can be divided into two types (1) ‘passive’ markers or reporters used for direct visualization and tracking of cell movement in relation to extracellular structures and (2) more complex, ‘active’ reporters or biosensors for monitoring detailed processes such as biochemical activity or protein-protein interactions during metastasis.^1,2 In some cases there can be overlap between both types of imaging which will be addressed here.The majority of early intravital imaging studies focused on the stages of metastasis that occur after dissemination from the primary tumor and predominantly used a ‘passive’ reporter approach to assess tumor cell behavior. Models of circulating tumor cells have allowed for analysis at the single cell level of tumor cell velocity, persistence, shape change and interactions with the ECM and stroma in secondary tissue.^3–5 The use of fluorescently-labelled cells has also revealed some limiting factors that cause the arrest of cancer cells in target tissue such as trapping in small capillary networks due to tumor cell size or adhesion to surrounding vessel walls.^6,7 Furthermore, experimental models of metastasis such as intra-splenic, intra-cardial and tail vein injections in combination with fluorescently-tagged tumor cells has provided information on the colonisation, extravasation and dormancy of tumor cells in secondary sites (Fig. 1 and refs. ^{5, 8} and ⁹). Collectively, along with the rapid increase in tissue specific expression of GFP in mouse cancer models,¹⁰ a wealth of information on different steps of the metastatic process has begun to emerge.Open in a separate windowFigure 1(A) Whole body optical imaging of mCherry-expressing SW 620 colon cancer cell metastases after approximately six weeks post intra-splenic injection. Images were obtained using the Olympus OV100 whole body imaging system with an Olympus MT10, 150 w, Xenon light source, using a low magnification objective (macro lens) with a magnification of 0.14× and numerical aperture of 0.04. (B) mCherry expressing SW 620 colon cancer cells colonizing the liver 30 mins after intra-splenic injection. 1 × 10⁶ cells were injected into the spleen of an anesthetised CD-1 nude mouse and the incision sealed using ‘Clay Adams’ vetinary clips (VetTec). The mouse was placed on a heat pad for 30 mins then sacrificed. An incision was made in the abdomen to expose the liver and images of fluorescent cells within the liver were obtained using a 0.8× (0.22 NA) objective lens with variable zoom on the Olympus OV100.The departure of individual cells away from solid primary tumors into the blood stream has been a more difficult process to study using intravital imaging. It is a rare, sporadic event, requiring long acquisition and the inherent density and complex nature of the tumor tissue poses problems for imaging. Overcoming autofluorescence and light scattering has recently been improved due to advances in fluorophores^1,11 and the combined use of long-term multiphoton microscopy¹² has allowed greater resolution and tissue penetration than before. Multiphoton imaging can also provide additional detail regarding the interaction between cells and the surrounding extracellular environment using second harmonic signal generation (SHG) from collagen, elastin and other matrix proteins found in connective tissue.^13,14 In this regard, imaging the interaction of cancer cells with extracellular matrix has revealed distinct modes of cell locomotion adopted by cancer cells in vivo, such as ameboid or mesenchymal invasion, that depend upon the topography or density of the surrounding matrix.^3,13,15 A greater understanding of the initial cell movement and interaction with the extracellular environment will enhance our ability to pin-point cell-ECM targets that may be of clinical relevance in the future.Concurrent with the use of GFP as a ‘passive’ marker, a number of techniques have been developed that facilitate the visualization and localisation of GFP-tagged fusion proteins to quantify changes in protein expression, mobility and sub-cellular interactions during various processes in vitro. These include photobleaching (PB), photoactivation (PA), fluorescence resonance energy transfer (FRET) and fluorescent life time imaging microscopy (FLIM).^2,16,17 The adaptation of these techniques for in vivo imaging to examine the activity of key molecules will provide new ‘active’ markers or reporters that can be correlated with biological processes important in disease progression such as migration, proliferation and cell death. Other fluorescent probes such as MMPsense or Apotrace that measure ‘active’ processes such as metalloproteinase activity or apoptosis have also recently been used in animals.^18,19 In this way we can get closer to understanding how subcellular components or signal transduction pathways interact in real-time. The improved spatial and temporal detail will facilitate the ‘when and where’ we should target metastatic cancer cells for therapy.¹²In our recent paper we have adapted two techniques, photobleaching and photoactivation, for in vivo imaging and used them to assess the potential of E-cadherin as a molecular biosensor for cell migration in live tumors.²⁰ E-cadherin-based cell-cell contacts are prominent sites of remodelling during early stages of epithelial to mesenchymal transition (EMT). The disruption or deregulation of E-cadherin-based adhesions leads to the collapse of normal epithelial architecture that precedes the initial intravasation of cells from tumors.^21–23 In vitro photobleaching analysis of E-cadherin can be used as an ‘active’ molecular read-out of cell migration, as cells within a stationary colony show significantly reduced E-cadherin mobility compared to collectively migrating cells.²⁰ Moreover, as demonstrated in Figure 2 (reviewed in ref. ²⁰), E-cadherin mobility can also be spatially regulated within a population of tumor cells, as cells at the rear of a wound show impaired E-cadherin mobilisation compared to cells at the leading edge of the wound. This suggests a gradient of E-cadherin mobilisation within the local environment of a tumor may exist and could potentially be used in the future to map areas of weakened cell-cell adhesion from which cells are more likely to migrate. In vivo analysis of E-cadherin dynamics showed that changes in the mobility of E-cadherin can also be used as an ‘active’ marker of cell behavior in live animals, and may be useful in predicting cell mobilisation from primary tumors.²⁰Open in a separate windowFigure 2FRAP of GFP-E-cadherin at the rear or front of a wound heal assay. (A and B) Schematic and representative images of a wound heal assay depicting the area of cells selected for E-cadherin-based cell-cell junction FRAP analysis (red broken line). (C and D) Representative images of FRAP experiments performed at the rear or front of a wound heal assay respectively. White solid arrows represent area of photobleaching at the rear and white broken arrows represent area of photobleaching at the front of the wound. Red arrows indicate dynamics of cells at the front of the wound. Cells were classed to be at the front of the wound within the first three cells from the wound border (reviewed in ref. ²⁰).We also demonstrated the subcellular tracking of plasma membrane dynamics in vivo using the membrane-targeting sequence of H-Ras fused to photoactivatable-GFP.^24,25 Importantly, both the dynamics of cell-cell junctions, as visualised using E-cadherin:GFP, and the dynamics of the plasma membrane, which also plays a fundamental role in cell invasion and metastasis, are significantly different in vivo than in vitro.²⁰ Critically, this raises the possibility that many signalling axes and networks may function differently in vivo and therefore care must be taken when correlating in vitro information to the live setting. Lastly, we demonstrated the benefits of in vivo imaging in the assessment of molecular-based targeted therapeutics by using the Src inhibitor dasatinib, which impaired E-cadherin cell mobility in vivo but not in vitro.^20,26In the context of previous intravital imaging studies, our work suggests that we are at the beginning of a new stage of intravital imaging in which ‘active’ probes can help predict the efficacy of novel therapeutic treatments and also provide a context dependent read-out of oncogene-induced biological behavior in live animals. Importantly, not all molecules are adaptable for this type of in vivo imaging. Careful selection of candidate molecular markers that demonstrate clear changes attributable to a biological function, for example, subcellular relocalization or compartmentalisation, will be ideally suited for this type of intravital examination in the future.Here we have adopted two key fluorescent imaging techniques typically used in vitro and combined them with a fundamental biological question in vivo. The adaptation of other techniques for in vivo imaging such as FRET or FLIM-FRET probes will provide a detailed pixel by pixel map of the activity and behavior of key signalling proteins in live animals.^2,27 The use of these ‘active’ probes in vivo may hold further surprises concerning differences in molecular behavior in live animals compared to the traditional ‘snap-shot’ approach in vitro. Finally, one of the major challenges of in vivo imaging during drug discovery is the need for repeated imaging of the same animal in the presence or absence of drugs. The continued development of optical windows and observation chambers for non-invasive real-time imaging will facilitate this and allow for the assessment of drug response at the single cell level.²⁸ This, when combined with the subcellular optical techniques described here, will prove very useful in the future for in vivo imaging when evaluating the aetiology of the disease or during the drug discovery process.     

Cyclopentanoid analogs of dipalmitoyl phosphatidic acid: effect of backbone geometry on thermotropic properties

Seven geometrical or positional isomers of dipalmitoyl cyclopentanophosphoric acid (DPCPA) have been synthesized and studied: 1,3/2-1P (I); 1,2/3-1P (II); 1,2/3-3P (III), 1,2,3/0-1P (IV); 1,2,3/0-2P (V); 1,3/2-2P (VI); 1,2/3-2P (VII). When dispersed in 0.1 M Tris-HCl at pH 7.4, I-VII gave thermal transitions (Tc) of 60.0 degrees, 59.0 degrees, 56.8 degrees, 55.3 degrees, 38.3 degrees, 36.8 degrees and 34.0 degrees C, respectively, as measured by differential scanning calorimetry (DSC). When the lipids were dispersed at pH 9.5 in 0.1 M borate, Tc of I-IV decreased, whereas Tc of V-VII increased. In contrast, at pH 1.5 in 0.1 M HCl/KCl, Tc of I-IV decreased slightly, but Tc of V-VII rose markedly. To determine the effect of head group geometry and substitution pattern on acyl chain motion, EPR spectra of 1-palmitoyl, 2-[16-doxylstearoyl]-glycero-3-phosphoric acid in bilayers of DPCPA isomers were acquired. Abrupt spectral changes occurred at temperatures closely correlating with transition temperatures observed by DSC. These results have led to the conclusions that: (i) isomers I-IV containing vicinal acyl chains form bilayers that exhibit structural transitions at temperatures higher than those at which transitions are exhibited by isomers V-VII which have a polar phosphate group interposed between the two chains; (ii) the effects of differences in backbone structure are transmitted down the entire length of the acyl chains; (iii) the orientation of the cyclopentane ring in the isomers I-IV is significantly different from that in isomers V-VII at pH values where the phosphate group is doubly negatively charged.     

Stimulation of phosphatidic acid of calcium influx and cyclic GMP synthesis in neuroblastoma cells

Phosphatidic acid added to the medium markedly elevated intracellular cyclic GMP content in cultured neuroblastoma N1E 115 cells. There was a significant elevation of cyclic GMP with 1 micrograms/ml and a maximum (70-fold) elevation with 100 micrograms/ml of phosphatidic acid. Other natural phospholipids did not increase, or increased only slightly, the cyclic GMP content in the cells. The elevation of cyclic GMP content by phosphatidic acid was absolutely dependent on extracellular calcium. Phosphatidic acid stimulated the influx of calcium into neuroblastoma cells 2- to 5-fold. The pattern of the calcium influx induced by phosphatidic acid was comparable to that of cyclic GMP elevation. The stimulation of calcium influx by phosphatidic acid was also observed in cultured heart cells, indicating that phosphatidic acid acts as a calcium ionophore or opens a specific calcium-gate in a variety of cell membranes. Treatment of neuroblastoma cells with phospholipase C increased 32Pi labeling of phosphatidic acid, stimulated the influx of calcium, and elevated the cyclic GMP content in the cells. Thus exogenous as well as endogenous phosphatidic acid stimulates the translocation of calcium across cell membranes and, as a consequence, induces the synthesis of cyclic GMP in the neuroblastoma cells.     

Ionophorous functions of phosphatidic acid in the plant cell

Effects of phosphatidic acid (PA), a product of phospholipase D activity, on Ca²⁺ and H⁺ transport were investigated in membrane vesicles obtained from roots and coleoptiles of maize (Zea mays L.). Calcium flows were measured with fluorescent probes indo-1 and chlorotetracycline loaded into the vesicles and added to the incubation medium, respectively. Phosphatidic acid (50–500 μM) was found to induce downhill flow of Ca²⁺ along the concentration gradient into the plasma membrane vesicles and endomembrane vesicles (tonoplast and endoplasmic reticulum). Protonophorous functions of PA were probed with acridine orange. First, the ionic H⁺ gradient was created on the tonoplast vesicles by means of H⁺-ATPase activation with Mg-ATP addition. Then, the vesicles were treated with 25–100 μM PA, which induced the release of protons from tonoplast vesicles and dissipation of the proton gradient. Thus, PA could function as an ionophore and was able to transfer Ca²⁺ and H⁺ across plant cell membranes along concentration gradients of these ions. The role of PA in mechanisms of intracellular signaling in plants is discussed.     

Conformationally-restricted cyclic sulfones as potent and selective mTOR kinase inhibitors

Novel conformationally-restricted mTOR kinase inhibitors with cyclic sulfone scaffold were designed. Synthesis and structure-activity relationship (SAR) studies are described with emphasis on optimization of the mTOR potency and selectivity against class I PI3Kα kinase. PF-05139962 was identified with excellent mTOR biochemical inhibition, cellular potency, kinase selectivity and in vitro ADME properties.     

Design and synthesis of 3,3-piperidine hydroxamate analogs as selective TACE inhibitors

Structure-based methods were used to design beta-sulfone 3,3-piperidine hydroxamates as TACE inhibitors with the aim of improving selectivity for TACE versus MMP-13. Several compounds in this series were synthesized and evaluated in enzymatic and cell-based assays. These analogs exhibit excellent in vitro potency against isolated TACE enzyme and show good selectivity for TACE over the related metalloproteases MMP-2, -13, and -14.