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PubMed1982

Tryptic peptide analysis of the human apolipoprotein E isomorphs.

The nature of the polypeptide backbone of human apolipoprotein (apo) E present in the very low density lipoproteins (VLDL) of normal and homozygous type III hyperlipoproteinemic patients was investigated by tryptic cleavage fingerprinting and specific chemical modification studies. Apo E from normal subjects was resolved on polyacrylamide isoelectric focussing gels into five bands (apo E-I', E-I, E-II, E-III, and E-IV), whereas apo E from type III patients was resolved into three bands (apo E-I3', E-I3, and E-II3). The apo E isoforms, contained within unstained polyacrylamide gel slices, were washed to remove ampholytes, desialylated, and digested with L-1-tosylamido-2-phenylethylchloromethyl ketone treated trypsin. Autoradiography of 125I-labelled tryptic apo E peptides showed complete identity between all isoforms from normal subjects. High performance liquid chromatographic (HPLC) analysis showed that complete peptide identity exists between apo E-I', E-I, and E-II and between apo E-I3', E-I3, and E-II3. Distinct HPLC peptide profiles were found for apo E-II, E-III, E-IV, and E-II3. These resolved peak differences were reproducible between runs, between digests, and between apo E isolations, suggesting that the distinct profiles were neither a result of artifacts nor of contamination. Specific chemical modification studies revealed that human apo E isomorphism is due, in part, to differences in arginine and cysteine residues but not to lysine residues. These findings indicate that human apo E isomorphism results from differences in the primary amino acid sequence of the individual isoforms in addition to charged carbohydrate heterogeneity. Furthermore, the apo E isomorphic profile observed in homozygous type III hyperlipoproteinemic patients reflects both a deficiency of apo E-III and E-IV and the presence of the altered apo E-II isoprotein (apo E-II3).

Acrylonitrile↗

PubMed1987

Angiotensin II stimulates phospholipases C and A2 in cultured rat mesangial cells.

Angiotensin II stimulates prostaglandin (PG) E2 formation in mesangial cells cultured from rat renal glomeruli. The interactions between angiotensin II and PGE2 are important in modulating glomerular function. We examined the mechanism for stimulation of PGE2 production in mesangial cells using the putative diacylglycerol-lipase inhibitor RHC 80267 and trifluoperazine (TFP), an agent interfering with Ca2+-CaM-mediated processes. Although RHC 80267 inhibited diacylglycerol-lipase activity in mesangial cells, it did not influence PGE2 production in response to either angiotensin II or A23187. In contrast, TFP (50 microM) inhibited basal PGE2 production and stimulation by angiotensin II and A23187. TFP also decreased 14C release in response to angiotensin from cells prelabeled with [14C]arachidonic acid, which was associated with inhibition of 14C loss from phosphatidylinositol. In cells prelabeled with 32P, orthophosphate angiotensin II caused a rapid hydrolysis of phosphatidylinositol 4,5-bisphospate. TFP enhanced 32P labeling of phosphatidylinositides, but did not prevent the loss of phosphatidylinositol 4,5-bisphosphate in response to angiotensin. This was verified in cells prelabeled with myo-[3H]inositol where angiotensin stimulated formation of [3H]inositol trisphosphate. TFP enhanced formation of [3H]inositol trisphosphate both under basal- and angiotensin II-stimulated conditions. Thus TFP did not inhibit phospholipase C activation by angiotensin. Angiotensin II caused marked increases in [32P]lysophospholipids, indicating activation of also phospholipase A2. This process was inhibited by TFP. Taken together, these results are consistent with stimulation of both phospholipase C and A2 by angiotensin, the latter step responsible for the release of arachidonic acid and PGE2 formation. The activation of phospholipase A2, but not that of phospholipase C, is inhibited by TFP, perhaps by interference with calmodulin-dependent steps.

Angiotensin II↗

PubMed1993

Metabolism of a long-chain diacylglycerol by permeabilized A10 smooth muscle cells.

The regulatory effects of diacylglycerol (DAG) second messengers will be terminated by metabolism. A long-chain DAG, 1-palmitoyl-2-[1-14C]oleoyl-sn-glycerol (2-[14C]POG), was metabolized by cultured A10 smooth muscle cells after permeabilization by preincubation with 340 U/ml alpha-toxin from Staphylococcus aureus. In contrast to results with the cell-permeable DAG analogue, dioctanoyl-glycerol ([3H]diC8), no appreciable 2-[14C]POG degradation could be detected in control A10 cells not treated with alpha-toxin. With permeabilized A10 cells, 2-[14C]POG was mainly converted into lipolytic products of a lipase pathway, monoacylglycerol (MG) and fatty acid (FA); very little radioactivity was incorporated into triacylglycerol (TG) or phospholipid (PL) via reactions catalyzed by either DAG acyltransferase, cholinephosphotransferase, or DAG kinase. Similar results were obtained in experiments with 1-stearoyl-2-[1-14C]arachidonoyl-sn-glycerol. The conversion of 2-[14C]POG into PL and TG was not enhanced by the addition of 1 mM ATP-MgCl2, 1 mM CDP-choline, or 1 mM oleoyl-CoA to the alpha-toxin-treated A10 cells. The formation of FA and MG by permeabilized A10 cells was inhibited by DAG lipase inhibitors, U-57,908 (50 microM) and tetrahydrolipstatin (1-25 nM). The predominant contribution of the lipase pathway to the metabolism of a long-chain DAG, 2-[14C]POG, by alpha-toxin-treated A10 cells is similar to results for the degradation of [3H]diC8 by intact A10 cells.

Animals↗

PubMed1996

Caerulein-stimulated arachidonic acid release in rat pancreatic acini: a diacylglycerol lipase affair.

This study was performed to evaluate the effect of caerulein, a cholecystokinin analogue, on arachidonic acid (AA) release in rat pancreatic acini and to determine the cellular mechanism involved. Caerulein did not stimulate phospholipase A2 (PLA2); however, diacylglycerol (DAG) lipase activity was increased. Validity of PLA2 or DAG lipase inhibitors was confirmed by their ability to selectively inhibit PLA2 or DAG lipase activities. Caerulein increased AA release from acini prelabeled with [3H]AA both dose and time dependently. Inhibitors were used to evaluate the involvement of different signaling pathways. Mepacrine and aristolochic acid, two PLA2 inhibitors, did not inhibit caerulein-induced AA release, whereas the DAG lipase inhibitor RHC-80267 did. The phospholipase C (PLC) inhibitor U-73122 totally inhibited caerulein-induced AA release, whereas the phospholipase D (PLD) inhibitor wortmannin had no effect. Our data indicate that caerulein-induced AA release results from the combined action of PLC and DAG lipase without PLA2 or PLD activation.

Androstadienes↗

PubMed1985

Involvement of arachidonate metabolism in neurotensin-induced prolactin release in vitro.

Neurotensin increased in a concentration-dependent manner the level of hypophyseal [3H]arachidonic acid in vitro as well as prolactin release from hemipituitary glands. The effect of 1 microM neurotensin on arachidonate release was already present at 2.5 min, maximal at 5, and disappeared after a 10-min incubation. Neurotensin analogues produced an enhancement of hypophyseal arachidonate similar to their relative potencies in other cellular systems, whereas other peptides (somatostatin and vasoactive intestinal peptide) were devoid of any effect on the concentration of the fatty acid in the pituitary. Seventy micromoles RHC 80267, a rather selective inhibitor of diacylglycerol lipase, completely prevented the neurotensin-stimulated prolactin release and decreased arachidonate release both in basal or in neurotensin-induced conditions. Similar results were obtained with 50 microM quinacrine, a phospholipase A2 inhibitor. To clarify whether arachidonate released by neurotensin requires a further metabolism through specific pathways to stimulate prolactin release, we used indomethacin and BW 755c, two blockers of cyclooxygenase and lipoxygenase pathways. Thirty micromoles indomethacin, a dose active to inhibit cyclooxygenase, did not affect unesterified arachidonate levels either in basal or in neurotensin-induced conditions; moreover, the drug did not modify basal prolactin release but slightly potentiated the stimulatory effect of neurotensin on the release of the hormone. On the other hand, 250 microM BW 755c, an inhibitor of both cyclooxygenase and lipoxygenase pathways, significantly inhibited both basal and neurotensin-stimulated prolactin release and further potentiated the increase of the fatty acid concentrations produced by 1 microM neurotensin.(ABSTRACT TRUNCATED AT 250 WORDS)

4,5-Dihydro-1-(3-(trifluoromethyl)phenyl)-1H-pyraz↗

PubMed1986

A possible role of arachidonate metabolism in the mechanism of prolactin release.

The cleavage of arachidonate from pituitary phospholipids may contribute to the process that regulates the release of prolactin. To test this hypothesis, primary cultures of anterior pituitary cells from female rats were preincubated with [3H]arachidonate to label their phospholipid-containing components. The cells were then washed and incubated with vehicle or test agents and the release into the medium of prolactin and [3H]arachidonate cleaved from the phospholipids was measured. Thyrotropin-releasing hormone (TRH) and neurotensin significantly increased the release of both [3H]arachidonate and prolactin. Although basal [3H]arachidonate release was not affected by dopamine or somatostatin, both of these agents reduced [3H]arachidonate release induced by TRH. The relationship between calcium mobilization and arachidonate release was investigated by exposing the cells to agents that modify calcium balance. Maitotoxin, a calcium channel activator, stimulated prolactin and arachidonate release. In contrast cobalt, a calcium channel blocker, penfluridol, a calcium-binding protein inhibitor, and low-calcium medium decreased basal and TRH-induced prolactin release and diminished the TRH-induced release of arachidonate. RHC 80267, an inhibitor of diacylglycerol lipase, decreased TRH-induced prolactin and arachidonate release. BW755c, an inhibitor of the conversion of arachidonate to its metabolites, decreased TRH-induced prolactin release but predictably increased arachidonate release. These findings support the hypothesis that arachidonate metabolites may be involved in the process regulating prolactin release.

4,5-Dihydro-1-(3-(trifluoromethyl)phenyl)-1H-pyraz↗

PubMed1986

Involvement of glutathione oxidation reduction in parathyroid hormone secretion.

This study determines whether calcium affects glutathione metabolism and whether glutathione metabolism may influence parathyroid (PTH) secretion in collagenase dispersed bovine parathyroid cells. Reduced glutathione (GSH) and glutathione disulfide (GSSG) were measured fluorometrically and enzymatically while PTH secretion was determined by radioimmunoassay. The total GSH and GSSG content of parathyroid cells was found to range from 1.59 to 1.71 micrograms/mg cell protein, and this did not vary significantly with changes in extracellular calcium. An increase in the medium calcium concentration from 0.5 to 2.0 mM did, however, cause an increase in GSSG from 0.43-0.54 to 1.19-1.20 micrograms/mg protein with a concomitant decrease in GSH. The compound 2-cyclohexen-1-one was used to deplete the cells of GSH at a low-calcium medium (0.5 mM) to levels seen in high-calcium medium (2.0 mM). This treatment was found to inhibit PTH secretion in the low-calcium medium, as if the cells were incubated in high medium calcium. Both 2-cyclohexen-1-one and calcium caused a rapid decrease in reduced GSH levels and in hormone secretion. The ketone was not found to affect cellular protein synthesis, indicating that there was no nonspecific toxic effect of this treatment on the cells. These results suggest that changes in the calcium concentration of the medium affect the GSH/GSSG ratio of dispersed parathyroid cells. Changes in the GSH/GSSG ratio induced by calcium may be related to changes in PTH secretion.

Animals↗

PubMed1983

Glutathione depletion inhibits amylase release in guinea pig pancreatic acini.

Isolated guinea pig pancreatic acini were specifically depleted of glutathione by treatment with 2-cyclohexene-1-one (2-CHX-1). Untreated acini contained 4.3 +/- 0.6 micrograms of glutathione per milligram protein. Incubation with 1 mM 2-CHX-1 for 5 min at 37 degrees C depleted glutathione to 17% of control values; 5 mM 2-CHX-1 depleted glutathione to less than 4% of control values. Incubation with 2-CHX-1 also impaired the ability of the isolated acini to secrete amylase in response to stimulation with carbachol and the ionophore A23187. The depletion of glutathione and the inhibition of amylase secretion by 2-CHX-1 were both dose dependent and time dependent. Incubation of acini with 2 mM 2-CHX-1 for 15 min at 37 degrees C reduced glutathione levels to 6.6% of control and reduced carbachol-stimulated amylase release to 63% of control. Higher doses of 2-CHX-1 or longer incubations resulted in greater depletion of glutathione and greater inhibition of carbachol-induced amylase release. These data indicate that specific depletion of glutathione impairs the ability of isolated acini to secrete amylase in response to physiological and pharmacologic stimuli and suggest that glutathione has a role in stimulus-secretion coupling in the exocrine pancreas.

Amylases↗

PubMed1984

Role of glutathione in gastric mucosal cytoprotection.

Exogenous thiol compounds have been reported to protect the stomach from ethanol-induced necrotic lesions. The gastric mucosa contains high levels of an endogenous thiol, glutathion (GSH). Because of the known role of glutathione in protecting against hepatic injury, its role in gastric mucosal cytoprotection was of interest. By use of an animal model for acute gastric injury from ethanol, a close parallel relation between depletion of endogenous mucosal GSH and induction of mucosal protection was demonstrated. Surprisingly, mucosal protection varied inversely with the level of mucosal GSH obtained after treatment with specific GSH-depleting agents (diethyl maleate and cyclohexene-1-one). Depletion of gastric mucosal GSH was associated with an increase in the mucosal content of prostaglandins 6-keto F1 alpha and F2 alpha but not E2. The protective effect induced by GSH-depleting agents was partially reversed by indomethacin in some but not all studies. Although GSH depletors increased gastric juice volume, protection with these agents persisted after the volume and mucosal GSH had returned to control levels and also was not reversed by increasing the dose of ethanol threefold to overcome a possible dilutional effect. We conclude that, contrary to apparent predictions, depletion of endogenous gastric GSH protects the stomach from acute ethanol-induced injury. Although the mechanism of this protection is unknown, a mediation by endogenous release of prostaglandins seems to play a minor role since diethyl maleate was protective even in indomethacin-treated animals.

6-Ketoprostaglandin F1 alpha↗

PubMed1991

Dual pathways for agonist-stimulated arachidonic acid release in pancreatic acini: roles in secretion.

The present experiments were performed to determine pathways responsible for arachidonic acid release stimulated by cholecystokinin (CCK) and phorbol ester, 4 beta-phorbol 12-myristate 13-acetate (PMA), and the roles of pathways in the secretory response in dispersed acini from guinea pig pancreas. Both CCK-octapeptide (CCK-OP) and PMA increased intracellular arachidonic acid. To determine the source of released arachidonic acid, we measured the effects of PMA and CCK-OP on cellular 1,2-diacylglycerol and lysophosphatidylcholine (LPC) and of diglyceride lipase inhibitor RHC 80267 on [3H]arachidonic acid release. Both PMA and CCK-OP increased 1,2-diacylglycerol and LPC. RHC 80267 had no effect on LPC but inhibited the increase in [3H]arachidonic acid release with a concentration of CCK-OP that was maximal for enzyme secretion. The increase in [3H]arachidonic acid release with PMA or a supramaximal concentration of CCK-OP was not inhibited by RHC 80267. In parallel fashion, RHC 80267 inhibited amylase release caused by maximally effective concentrations of CCK-OP but not that caused by PMA or by supramaximally effective concentrations of CCK-OP. Arachidonic acid stimulated amylase release. Exogenous addition of phospholipase A2 caused increases in [3H]arachidonic acid release, LPC formation, and amylase release. The results indicate that there are at least two pathways responsible for the increase in free cellular arachidonic acid stimulated by pancreatic agonists. One is sequential action of phospholipase C and diglyceride lipase on phosphatidylinositol. The other is a phospholipase A action on phosphatidylcholine. The results also suggest a stimulatory role for both pathways in the secretory response.

Amylases↗

PubMed2005

Role of phospholipase C and diacylglyceride lipase pathway in arachidonic acid release and acetylcholine-induced vascular relaxation in rabbit aorta.

ACh stimulates arachidonic acid (AA) release from membrane phospholipids of vascular endothelial cells (ECs). In rabbit aorta, AA is metabolized through the 15-lipoxygenase pathway to form vasodilatory eicosanoids 15-hydroxy-11,12-epoxyeicosatrienoic acid (HEETA) and 11,12,15-trihydroxyeicosatrienoic acid (THETA). AA is released from phosphatidylcholine (PC) and phosphatidylethanolamine (PE) by phospholipase A2 (PLA2), or from phosphatidylinositol (PI) by phospholipase C (PLC) pathway. The diacylglycerol (DAG) lipase can convert DAG into 2-arachidonoylglycerol from which free AA can be released by monoacylglycerol (MAG) lipase or fatty acid amidohydrolase (FAAH). We used specific inhibitors to determine the involvement of the PLC pathway in ACh-induced AA release. In rabbit aortic rings precontracted by phenylephrine, ACh induced relaxation in the presence of indomethacin and N(omega)-nitro-L-arginine (L-NNA). These relaxations were blocked by the PLC inhibitor U-73122, DAG lipase inhibitor RHC-80267, and MAG lipase/FAAH inhibitor URB-532. Cultured rabbit aortic ECs were labeled with [14C]AA and stimulated with methacholine (10(-5) M). Free [14C]AA was released by methacholine. Methacholine decreased the [14C]AA content of PI, DAG, and MAG fractions but not PC or PE fractions. Methacholine-induced release of [14C]AA was blocked by U-73122, RHC-80267, and URB-532 but not by U-73343, an inactive analog of U-73122. The data suggested that ACh activates PLC, DAG lipase, and MAG lipase pathway to release AA from membrane lipids. This pathway is important in regulating vasodilatory eicosanoid synthesis and vascular relaxation in rabbit aorta.

Acetylcholine↗

PubMed1995

Albumin reduces basement membrane hydraulic conductance in part due to arginyl side groups.

Albumin reduces capillary hydraulic conductance (Lp) even at low concentrations. To determine if part of this barrier protective effect might be extracellular, we studied the effects of bovine serum albumin (BSA) on Lp of self-assembled basement membrane (Matrigel). Lp with tris(hydroxymethyl)aminomethane (Tris) buffer superfusate was stable at 1.77 +/- 0.22 x 10(-5) (SE) cm.s-1.cmH2O-1 over several hours. At 0.1 g/dl BSA, experimental/control (Tris) Lp fell to 83.1 +/- 6.0% (2P < 0.025), with decreases to 72.4 +/- 3.7% at 1 g/dl (2P < 0.005), 45.3 +/- 5.1% at 2.5 g/dl (2P < 0.001), and 45.0 +/- 4.8% at 4.0 g/dl (2P < 0.001). In separate experiments, BSA arginine groups were neutralized by 1,2-cyclohexanedione (CHD), and experimental/control Lp values were measured. At 2.5 g/dl, CHD-BSA depressed Lp to 54.4 +/- 4.8%, while unmodified BSA reduced Lp to 40.8 +/- 3.5% of Tris control (2P = 0.05). Finally, soluble arginine at three- and sixfold the arginine in BSA was added to BSA superfusate. For threefold, Lp rose to 120 +/- 8% of BSA level and for sixfold to 129 +/- 9% (2P < 0.05). We conclude that some part of the albumin protective effect is very likely due to consequences on extracellular matrix and that at least 18-22% of this effect is related to arginine groups on albumin when computed from Lp, and up to 34% when viscosity is taken into account. Membrane-saturable arginine-binding sites can be unbound with arginine, thus nullifying part of the barrier protective effect of BSA.

Animals↗

PubMed2004

Capsazepine, a vanilloid antagonist, abolishes tonic responses induced by 20-HETE on guinea pig airway smooth muscle.

The aim of this study was to delineate the mode of action of 20-hydroxy-eicosatetraenoic acid (20-HETE) in airway smooth muscle (ASM) cells. ASM metabolizes arachidonic acid by various enzymatic pathways, including the cytochrome P-450 (CYP-450) omega-hydroxylase, which leads to the production of 20-HETE, a bronchoconstrictive eicosanoid. The present study demonstrated that 20-HETE induced concentration-dependent tonic responses in ASM, whereas transient responses were recorded in Ca2+-free solution, suggesting an intracellular Ca2+ release process. 20-HETE inotropic responses were abolished by 36 microM 2-aminoethoxydiphenyl borate or 1 microM thapsigargin but were insensitive to 10 microM ryanodine, indicating that inositol triphosphate receptors likely control the release of intracellular Ca2+. Sustained tension, which required Ca2+ entry, was partially blocked by 1 microM nifedipine (an L-type) and 100 microM Gd3+ (a nonselective cationic channel blocker). Moreover, in the absence of selective 20-HETE receptor antagonists, 20-HETE tonic responses were inhibited in a concentration-dependent manner (0.1-10 microM) by capsazepine, a well-characterized vanilloid receptor antagonist. Capsazepine was also observed to reverse cumulative responses to 20-HETE and capsaicin, a TRPV1 agonist. In addition, capsazepine pretreatment largely modified the sustained inotropic responses to 20-HETE, suggesting that 20-HETE cross-reacted with TRPV1 receptors with a low affinity (microM) or that its specific receptor was inhibited by the vanilloid antagonist. Data obtained using RHC-80267, ONO-RS-082, and eicosatetraynoic acid, respective inhibitors of diacylglycerol-lipase, phospholipase A2, and CYP-450 omega-hydroxylase, reveal that intracellular arachidonic acid production and its 20-HETE metabolite may be responsible for the activation of nonselective cationic channels and tonic responses.

5,8,11,14-Eicosatetraynoic Acid↗

PubMed1991

Release of arachidonic acid by complement C5b-9 complex in glomerular epithelial cells.

In experimental membranous nephropathy, C5b-9 induces noncytolytic glomerular epithelial cell (GEC) injury and proteinuria, which in some models is partially mediated by metabolites of arachidonic acid. In cultured GEC, sublytic C5b-9 increases cytosolic Ca2+ concentration ([Ca2+]i), activates phospholipase C (PLC), and releases arachidonic acid and eicosanoids. This study examined mechanisms of arachidonic acid production by C5b-9. In GEC labeled with [3H]arachidonate C5b-9 increased free [3H]arachidonic acid and 1,2-[3H]-arachidonoyl-diacylglycerol (DAG), an endogenous activator of protein kinase C (PKC). Elevated [Ca2+]i was not sufficient to account for increased free arachidonic acid. Moreover, in GEC that had been depleted of PKC by preincubation for 18 h with 2 microM phorbol myristate acetate, the C5b-9-induced arachidonate release was inhibited by greater than 75%. Reacylation of phospholipids was not decreased by C5b-9. Homogenates of GEC that had been stimulated with C5b-9 released more [14C]arachidonate from exogenously added 2-[14C]arachidonoyl-phosphatidyl-ethanolamine or 2-[14C]arachidonoyl-phosphatidylcholine than homogenates of unstimulated cells (assayed at a Ca2+ concentration of 2 mM). These experiments demonstrate directly that C5b-9 increased phospholipase A2 (PLA2) activity. PLA2 appeared to be stimulated as a result of PKC activation (probably secondary to increased DAG) in association with elevated [Ca2+]i. The C5b-9-induced activation of PLA2 may lead to release of eicosanoids, which may contribute toward impaired glomerular capillary wall permselectivity in experimental membranous nephropathy.

Animals↗

PubMed1997

Bradykinin-stimulated cPLA2 phosphorylation is protein kinase C dependent in rabbit CCD cells.

We have used an established cell line of rabbit cortical collecting duct (RCCD) epithelial cells representing a mixed population of principal and intercalated cell types to determine which phospholipase A2 (PLA2) enzyme therein is responsible for bradykinin (BK)-stimulated arachidonic acid (AA) release and how its activation is regulated. BK-stimulated AA release was reduced 92% by arachidonyl trifluoromethyl ketone, an inhibitor of cytosolic PLA2 (cPLA2). Examination of PLA2 activity in vitro demonstrated that BK stimulation resulted in a greater than twofold increase in PLA2 activity and that this activity was dithiothreitol insensitive and was inhibited by an antibody directed against cPLA2. To determine a possible role for protein kinase C (PKC) in the BK-mediated activation of cPLA2, we used the PKC-specific inhibitor Ro31-8220 and examined its effects on AA release, cPLA2 activity, and phosphorylation. Ro31-8220 reduced BK-stimulated AA release and cPLA2 activity by 51 and 58%, respectively. cPLA2 activity stimulated by phorbol ester [phorbol 12-myristate 13-acetate (PMA)] displayed a similar degree of activation and was associated with an increase in serine phosphorylation identical to that caused by BK. The phosphorylation-induced activation of this enzyme was confirmed by the phosphatase-mediated reversal of both BK- and PMA-stimulated cPLA2 activity. In addition, we have also found that PMA stimulation did not cause a synergistic potentiation of BK-stimulated AA release as did calcium ionophore. This occurred despite membrane PKC activity increasing 93% in response to PMA vs. 42% in response to BK. These data, taken together, indicate that cPLA2 is the enzyme responsible for BK-mediated AA release, and, moreover, they indicate that PKC is involved in the onset responses of cPLA2 to BK.

Animals↗

PubMed2004

2-arachidonoylglycerol: a novel inhibitor of androgen-independent prostate cancer cell invasion.

Endocannabinoids have been implicated in cancer. Increasing endogenous 2-arachidonoylglycerol (2-AG) by blocking its metabolism inhibits invasion of androgen-independent prostate cancer (PC-3 and DU-145) cells. Noladin ether (a stable 2-AG analog) and exogenous CB1 receptor agonists possess similar effects. Conversely, reducing endogenous 2-AG by inhibiting its synthesis or blocking its binding to CB1 receptors with antagonists increases the cell invasion. 2-AG and noladin ether decrease protein kinase A activity in these cells, indicating coupling of the CB1 receptor to downstream effectors. The results suggest that cellular 2-AG, acting through the CB1 receptor, is an endogenous inhibitor of invasive prostate cancer cells.

Androgens↗

PubMed2005

Elucidation of thioredoxin as a molecular target for antitumor quinols.

Heteroaromatic quinols 4-(benzothiazol-2-yl)-4-hydroxycyclohexa-2,5-dienone (1) and 4-(1-benzenesulfonyl-1H-indol-2-yl)-4-hydroxycyclohexa-2,5-dienone (2) exhibit potent and selective antitumor activity against colon, renal, and breast carcinoma cell lines in vitro (GI50 < 500 nmol/L). In vivo growth inhibition of renal, colon, and breast xenografts has been observed. Profound G2-M cell cycle block accompanied down-regulation of cdk1 gene transcription was corroborated by decreased CDK1 protein expression following treatment of HCT 116 cells with growth inhibitory concentrations of 1 or 2. The chemical structure of the quinol pharmacophore 4-(hydroxycyclohexa-2,5-dienone) suggested that these novel agents would readily react with nucleophiles in a double Michael (beta-carbon) addition. Indeed, COMPARE analysis within the National Cancer Institute database revealed a number of chemically related quinone derivatives that could potentially react with sulfur nucleophiles in a similar manner and suggested that thioredoxin/thioredoxin reductase signal transduction could be a putative target. Molecular modeling predicted covalent irreversible binding between quinol analogues and cysteine residues 32 and 35 of thioredoxin, thereby inhibiting enzyme activity. Binding has been confirmed, via mass spectrometry, between reduced human thioredoxin and 1. Microarray analyses of untreated HCT 116 cells and those exposed to either 1 (1 micromol/L) or 2 (500 nmol/L and 1 micromol/L) determined that of > or =10,000 cancer-related genes, expression of thioredoxin reductase was up-regulated >3-fold. Furthermore, quinols 1 and 2 inhibited insulin reduction, catalyzed by thioredoxin/thioredoxin reductase signaling in a dose-dependent manner (IC50 < 6 micromol/L). Results are consistent with a mechanism of action of novel antitumor quinols involving inhibition of the small redox protein thioredoxin.

Amino Acid Sequence↗

PubMed2004

Induction of thyroid cancer cell apoptosis by a novel nuclear factor kappaB inhibitor, dehydroxymethylepoxyquinomicin.

PURPOSE: The objective of the study was to determine the effects of a novel selective nuclear factor kappaB (NF-kappaB) inhibitor, dehydroxymethylepoxyquinomicin (DHMEQ), in thyroid carcinoma cells in vitro and in vivo and to additionally elucidate the molecular mechanisms underlying the action of this chemotherapeutic agent. EXPERIMENTAL DESIGN: In the in vitro experiments, the induction of apoptosis by DHMEQ in various human thyroid carcinoma cell types was determined by flow cytometry analysis of annexin-V binding and the caspase activation by Western blotting. For the in vivo study, female nu/nu mice were xenografted with s.c. FRO thyroid tumors. DHMEQ solution was injected i.p. at a dose of 8 mg/kg/day for two weeks. Tumor dimensions were monitored twice weekly, and apoptosis in tumor specimens was determined by terminal deoxynucleotidyl transferase-mediated nick end labeling staining. RESULTS: Treatment with DHMEQ substantially inhibited the translocation of p65 and p50 NF-kappaB subunits to the nucleus, the DNA-binding activity of the RelA/p65, NF-kappaB-dependent expression of the inhibitor of apoptosis (IAP)-family proteins, cIAP-1, cIAP-2, and XIAP, and the de novo synthesis of inhibitor of nuclear factor kappaB alpha. At concentration levels ranging from 0.1 to 5 microg/ml, DHMEQ induced a caspase-mediated apoptotic response that could be abrogated by the c-Jun NH(2)-terminal kinase inhibitor SP600125 but not by either mitogen-activated protein/extracellular signal-regulated kinase kinase or p38 inhibitors. In contrast, normal human thyrocytes were resistant to DHMEQ-induced apoptosis. At higher doses of DHMEQ we observed the necrotic-like killing of both normal and malignant thyrocytes, which was resistant to mitogen-activated protein kinase inhibitors. In nude mice DHMEQ substantially inhibited tumor growth without observable side effects, and increased numbers of apoptotic cells were observed in the histologic sections of tumors treated with DHMEQ. CONCLUSIONS: Our results show the potential usefulness of the novel NF-kappaB inhibitor, DHMEQ, in future therapeutic strategies for the treatment of thyroid cancers that do not respond to conventional approaches.

Apoptosis↗