Rebecca Salmonsen

PTEN in Neural Precursor Cells: Regulation of Migration, Apoptosis, and Proliferation

PTEN is a lipid phosphatase, and PTEN mutations are associated with gliomas, macrocephaly, and mental deficiencies. We have used PTEN +/- mice to assess PTENs role in subventricular zone (SVZ) precursor cells. For cultured SVZ neurosphere cells, haploinsufficiency for PTEN increases phosphorylation of Akt and forkhead transcription factor and slightly enhances proliferation. Based on a filter penetration assay, PTEN +/- cells are substantially more migratory and invasive than +/+ cells. The +/- cells also are more resistant to H(2)O(2)-induced apoptosis. Analysis of PTEN +/- and +/+ mice by BrdU labeling reveals no difference in the rate of cell proliferation in the SVZ. Exit of BrdU-labeled cells from the SVZ and radial migration to the outer layers of the olfactory bulb are more rapid for +/- cells. These observations indicate that PTEN regulates SVZ precursor cell function and is particularly important for migration and apoptosis in response to oxidative stress.

Antitumor effects of ajulemic acid (CT3), a synthetic non-psychoactive cannabinoid.

One of the endogenous transformation products of tetrahydrocannabinol (THC) is THC-11-oic acid, and ajulemic acid (AJA; dimethylheptyl-THC-11-oic acid) is a side-chain synthetic analog of THC-11-oic acid. In preclinical studies, AJA has been found to be a potent anti-inflammatory agent without psychoactive properties. Based on recent reports suggesting antitumor effects of cannabinoids (CBs), we assessed the potential of AJA as an antitumor agent. AJA proved to be approximately one-half as potent as THC in inhibiting tumor growth in vitro against a variety of neoplastic cell lines. However, its in vitro effects lasted longer. The antitumor effect was stereospecific, suggesting receptor mediation. Unlike THC, however, whose effect was blocked by both CB(1) and CB(2) receptor antagonists, the effect of AJA was inhibited by only the CB(2) antagonist. Additionally, incubation of C6 glioma cells with AJA resulted in the formation of lipid droplets, the number of which increased over time; this effect was noted to a much greater extent after AJA than after THC and was not seen in WI-38 cells, a human normal fibroblast cell line. Analysis of incorporation of radiolabeled fatty acids revealed a marked accumulation of triglycerides in AJA-treated cells at concentrations that produced tumor growth inhibition. Finally, AJA, administered p.o. to nude mice at a dosage several orders of magnitude below that which produces toxicity, inhibited the growth of subcutaneously implanted U87 human glioma cells modestly but significantly. We conclude that AJA acts to produce significant antitumor activity and effects its actions primarily via CB(2) receptors. Its very favorable toxicity profile, including lack of psychoactivity, makes it suitable for chronic usage. Further studies are warranted to determine its optimal role as an antitumor agent.

Resolution of inflammation by N-arachidonoylglycine

N‐arachidonoylglycine (NAgly) is an endogenous signaling lipid that is a member of the eicosanoid super family and is related to anandamide. It shows anti‐inflammatory activity in vivo in the mouse peritonitis model where it reduces migration of inflammatory leukocytes following injection of pro‐inflammatory agents into the peritoneal cavity. Using cell culture models, including GPR18 transfected HEK‐293 cells, evidence is presented that the orphan receptor GPR18 is involved in this action. Increases in free arachidonic acid, and robust stimulation of anti‐inflammatory eicosanoids were observed at low micromolar concentrations. These included 15‐deoxy‐delta‐13,14‐PGJ2 and lipoxin A4 both of which are believed to mediate the resolution stage of inflammation. It was further shown that NAgly might act via GPR18 activation in promoting the number of Trypan Blue stained cells, a possible indicator of programmed cell death. Thus, we hypothesize that NAgly induces the death of inflammatory cells, a process that is considered to be important for the resolution of inflammation. J. Cell. Biochem. 112: 3227–3233, 2011.

Neurotensin elevates hematocrit and plasma levels of the leukotrienes, LTB4, LTC4, LTD4 and LTE4, in anesthetized rats

The IV injection of neurotensin (NT) into anesthetized rats produced a marked increase in hematocrit, labored breathing and peripheral blood stasis with cyanosis. This effect could also be produced by the NT-related peptides, neuromedin-N and xenopsin; however, it was not observed when nine other biologically active peptides, including bradykinin and substance P, were tested. Associated with these responses were increases in the plasma levels of histamine (measured radioenzymatically) and the leukotrienes, LTB4, LTC4, LTD4, and LTE4 (measured by RIA and HPLC). The increment in hematocrit after varying doses of NT correlated to the increase in plasma levels of LTC4. Histamine and LTC4 were both capable of elevating hematocrit when given IV; however, LTC4 was approximately 1000 times more potent than histamine and active doses of histamine elevated LTC4 levels. Furthermore, the effects of NT on plasma LTC4 and hematocrit were reduced by pretreating animals with antagonists to histamine and serotonin. Pretreatment with the specific mast cell degranulating agent, compound 48/80, also blocked NTs ability to elevate plasma levels of histamine, LTB4 and LTC4 and prevented the increased hematocrit and cyanosis. These results indicate that NT-related peptides are very potent and specific stimulators of leukotriene release and that this action is mediated by mast cells and associated with loss of plasma volume and blood stasis. A working hypothesis is that histamine, released from mast cells in response to NT, stimulates LTC4 production by other cells.

Acylamido analogs of endocannabinoids selectively inhibit cancer cell proliferation

A series of amide derivatives of long-chain fatty acids has been studied for their effects on the proliferation of cancer cells in vitro. Fatty acids ranged from palmitic to higher polyunsaturated types containing 22 carbon atoms. The amino portions of the molecules included ammonia, ethanolamine, various amino acids and dopamine. Several cell lines were used as models and these included HTB-125 (normal human breast cells), HTB-126 (human breast cancer cells), HeLa (cervical cancer cells), WI-38 (human embryonic lung cells), RAW264.7 (mouse macrophage tumor cells) and RBL-2H3 (rat basophilic leukemia cells). The HTB lines were obtained from the same donor, so, could be considered a matched pair, that is, normal control versus cancer cells and thus, provide a model for testing specificity of action for the acylamido analogs. While many compounds were efficacious in inhibiting the proliferation of various cell lines, only two analogs showed a high degree of specificity in the matched HTB cell lines. N-palmitoyl dopamine and N-palmitoyl tyrosine each demonstrated complete specificity of action at a concentration of 10muM and were highly efficacious in both cases. No clear structure-activity pattern could be derived from these studies since the intensity of the inhibitory action seemed to depend on three factors, namely, the fatty acid, the amine group and the cell type.

N-Amino acid linoleoyl conjugates: anti-inflammatory activities.

Several N-linked amino acid-linoleic acid conjugates were studied for their potential as anti inflammatory agents. The parent molecule, N-linoleoylglycine was tested in an in vivo model, the mouse peritonitis assay where it showed activity in reducing leukocyte migration at doses as low as 0.3mg/kg when administered by mouth in safflower oil. Harvested peritoneal cells produced elevated levels of the inflammation-resolving eicosanoid 15-deoxy-Δ(13,14)-PGJ(2). These results are similar to those obtained in earlier studies with N-arachidonoylglycine. An in vitro model using mouse macrophage RAW cells was used to evaluate a small group of structural analogs for their ability to stimulate 15-deoxy-Δ(13,14)-PGJ(2) production. The d-alanine derivative was the most active while the d-phenylalanine showed almost no response. A high degree of stereo specificity was observed comparing the d and l alanine isomers; the latter being the less active. It was concluded that linoleic acid conjugates could provide suitable templates in a drug discovery program leading to novel agents for promoting the resolution of chronic inflammation.

Isolation and quantitation of several new peptides from the canine neurotensin/neuromedin N precursor

Using antisera towards the bioactive peptides, neurotensin and neuromedin N, as well as towards the N-terminal and C-terminal regions of their shared 170-residue precursor, peptides representing various portions of the precursor were isolated from extracts of canine ileum. In total, seven peptides were isolated, two of which had not been previously identified. One was the C-terminal tail of the precursor (Gly-Ser-Tyr-Tyr-Tyr) and the other was the tail peptide extended N-terminally to include neurotensin (Glp-Leu-Tyr-Glu-Asn-Lys-Pro-Arg-Arg-Pro-Tyr-Ile-Leu-Lys-Arg-Gly-Ser-Tyr -Tyr- Tyr). By comparing the measured concentrations for each of the identified peptides, it was established that processing at the three Lys-Arg cleavage sites within the precursor did not occur to the same extent. Cleavage at the N-terminus of neuromedin N was approximately 10% complete, that between neurotensin and the tail was approximately 90% complete, and that between neuromedin N and neurotensin was approximately 95% complete. Three immunoreactive proteins were also identified by immunochemical and chromatographic analyses: N-terminally extended neuromedin N (125 residues), N-terminally extended neurotensin (140 residues), and the entire 147-residue precursor. It was concluded that neurotensin, tail and large molecular neuromedin N were the primary products of processing for this precursor in canine ileum, while minor products included neuromedin N, neurotensin tail, and large molecular neurotensin.

Differentiation prevents assessment of neural stem cell pluripotency after blastocyst injection.

Earlier studies reported that neural stem (NS) cells injected into blastocysts appeared to be pluripotent, differentiating into cells of all three germ layers. In this study, we followed in vitro green fluorescent protein (GFP)–labeled NS and embryonic stem (ES) cells injected into blastocysts. Forty‐eight hours after injection, significantly fewer blastocysts contained GFP‐NS cells than GFP‐ES cells. By 96 hours, very few GFP‐NS cells remained in blastocysts compared with ES cells. Moreover, 48 hours after injection, GFP‐NS cells in blastocysts extended long cellular processes, ceased expressing the NS cell marker nestin, and instead expressed the astrocytic marker glial fibrillary acidic protein. GFP‐ES cells in blastocysts remained morphologically undifferentiated, continuing to express the pluripotent marker stage‐specific embryonic antigen‐1. Selecting cells from the NS cell population that preferentially formed neurospheres for injection into blastocysts resulted in identical results. Consistent with this in vitro behavior, none of almost 80 mice resulting from NS cell–injected blastocysts replaced into recipient mothers were chimeric. These results strongly support the idea that NS cells cannot participate in chimera formation because of their rapid differentiation into glia‐like cells. Thus, these results raise doubts concerning the pluripotency properties of NS cells.

Increased Plasma Levels of Leukotrienes in Response to Neurotensin

Neurotensin (NT) was discovered and first isolated using a bioassay based on NTs unique ability to induce hemoconcentration, blood stasis, and cyanosis in rats. NT can also elicit the release of histamine and serotonin from perfused rat hindquarter and can trigger their secretion from isolated mast cells. Given the ability of these autocoids to increase vascular permeability, we wondered whether they were responsible for the hypovolemia and stasis seen after NT. Here we report that NT also elevates plasma levels of the leukotrienes, LTB4/C4/D4/E4, and we provide evidence that leukotrienes, LTC4 in particular, mediate some of the vascular effects of NT. The i.v. injection of NT (6 nmol/kg) into anesthetized rats increased hematocrit from 37 f 2 to 62 f 3 (mean f SEM, n = 4) and produced cyanosis. Associated with these effects were marked increases in plasma histamine and leukotrienes (TABLE 1). Histamine levels were highest at the first time point, 5 min, while LTC4 plateaued at 20 to 30 min. By means of an assay that recognized LTC4 and its metabolites, 5to 10-fold higher levels of leukotriene were measured and HPLC separation indicated that LTC4/D4/E4 were all elevated. Total leukotrienes were elevated from -1 ng/ml to -70 n g / d at 5 rnin and to -140 ng/ml at 20 min. FIGURE 1A shows the dose-response relationship between the amount of NT injected and the plasma level of LTC4. Doses of NT as low as 0.8 nmol/kg led to a significant increase in plasma LTC4, and doses higher than 8 nmol/kg usually led to death. As shown in FIGURE lB, the increase in plasma LTC4 measured after varying doses of NT was correlated to the increase in hematocrit. The increases in plasma LTC4, histamine, and hematocrit appeared specific to NT and its relatives, neuromedinN and xenopsin (TABLE 1). Pretreatment with 48/80, which depletes mast cell mediators, or with cromoglycate, an inhibitor of degranulation, blocked these effects of NT, and pretreatment with antihistamines and antiserotonin greatly diminished them. Given intravenously (10-40 nmoledkg), LTC4 significantly increased hematocrit. In this regard, LTC4 was 15 to 20 times less potent than NT. Given intravenously (5-500 pmoles/kg), histamine elevated plasma levels of LTC4/D4/E4 and increased hematocrit in a timeand dose-dependent fashion.

Asymmetric synthesis of novel N-(1-phenyl-2,3-dihydroxypropyl)arachidonylamides and evaluation of their anti-inflammatory activity.

AIMS To design and synthesize novel N-(1-phenyl-2,3-dihydroxypropyl)arachidonylamides and evaluate their analgesic and anti-inflammatory potential. MAIN METHODS The murine macrophage cell line RAW 264.7 has been widely used as a model for inflammatory responses in vitro. Our model consists of cultured monolayers of RAW 264.7 cells in which media concentrations of 15-deoxy-Δ(13,14)-PGJ2 (PGJ) are measured by ELISA following LPS (10ng/ml) stimulation and treatment with 0.1, 0.3, 1.0, 3.0 and 10μM concentrations of the compounds. KEY FINDINGS Our data indicate that several of our compounds have the capacity to increase production of PGJ and may also increase the occurrence of programmed cell death (apoptosis). SIGNIFICANCE Thus these agents are potential candidates for the therapy of conditions characterized by ongoing (chronic) inflammation and its associated pain.