Cycloheximide reduces PGD2 or Δ12-PGJ2 cytotoxicity on NCG cells

To study the precise mechanism of cytotoxic activity of PGD₂ or Δ¹²-PGJ₂ (a biological active metabolite of PGD₂), we examined the effect of various compounds on PGD₂ or Δ¹²-PGJ₂ cytottoxic, using a human neuroblastoma cell line (NCG). Cycloheximide (CHM) specifically protected PGD₂ cytotoxicity on NCG cells. When Δ¹²-PGJ₂ was tested, CHM exhibited a similar rescue effect. Puromycin, mitomycin C, and α-amanitin did not affect PGD₂ or Δ¹²-PGJ₂ cytotoxicity. Emetine showed a variable and no consistent rescue effect CHM may have been active at the primary site where PGD₂ or Δ¹²-PGJ₂ exerts its cytotoxicity. This is the first report indicating that CHM reduces the cytotoxicity induced by PGD₂ or Δ¹²-PGJ₂.     

Intracellular glutathione level modulates the induction of apoptosis by Δ-prostaglandin J2

We studied the effect of intracellular glutathione (GSH), which was known to conjugate readily with an α, β-unsaturated carbonyl of 9-deoxy-Δ^9,12-13,14-dihydro PGD₂ (Δ¹²-PGJ₂), on the cytotoxicity of Δ¹²-PGJ₂. Δ¹²-PGJ₂ caused DNA fragmentation in human hepatocellular carcinoma Hep 3B cells, which was blocked by cycloheximide (CHX). The Δ¹²-PGJ₂-induced apoptosis was augmented by GSH depletion resulted from pretreatment with buthioninine sulfoximine (BSO), an inhibitor of γ-glutamylcysteine synthetase. On the contrary, N-acetyl-cysteine (NAC), a precursor of cysteine, elevated the GSH level and protected cells from initiating apoptosis by Δ¹²-PGJ₂. Sodium arsenite, a thiol-reactive agent, also induced apoptosis, which was potentiated or attenuated by BSO or NAC treatment respectively. These results suggest that the apoptosis-inducing activity of Δ¹²-PGJ₂ is due to thiol-reactivity and intracellular GSH modulates the Δ¹²-PGJ₂-induced apoptosis by regulating the accessibility of Δ¹²-PGJ₂ to target proteins containing thiol groups.     

Prostaglandin D2 metabolite stimulates collagen synthesis by human osteoblasts during calcification

We investigate the effect of the prostaglandin D₂ metabolite Δ¹²−PGD₂ (9−Deoxy−Δ⁹, Δ¹²−13,14-dihydroprostaglandin D₂) on collagen synthesis in human osteoblast. Δ¹²-PGJ₂ at 10⁻⁵M enhanced collagen synthesis in the presence of 2 mM α-glycerophosphate-2Na. The stimulative effect appeared as early as 3 days after addition and continued until 22 days. The enhancement of type I collagen synthesis was confirmed by polyacrylamide gel electrophoresis. The potency was the same as 10^1t-8M 1 α, 25 dihydroxy vitamine D₃ (1,25(OH)₂D₃). Northern blot analysis showed that 10⁻⁵M Δ ¹²-PGD₂ and 10⁻⁸M 1,25(OH)₂D₃ enhanced the transcribtion of type 1 procollagen (α₁) mRNA levels in osteoblasts.     

Selective synthesis and retention of 66k protein in a human neuroblastoma cell line (NCG) treated with a cytotoxic dosage of δ12-prostaglandin J2

δ¹²-prostaglandin(PG)J₂ (7.5μg/ml) significantly inhibited protein synthesis and cell growth in a human neuroblastoma cell line (NCG), decreasing these factors by 31.5% and 78.2% of the control values, respectively. Two protein synthesis inhibitors, cycloheximide (CHM)_and emetine, exhibited a dose-dependent protective effect for neuroblastoma cells against δ¹²-PCJ₂ cytotoxicity. At a concentration of 15μ/ml CHM, the number of viable cells increased from 21.8% to 36.7% of the control value (p<0.01). The sodium dodecyl sulfate-polyacrylamide gel analysis of [³⁵S]methionine-incorporated proteins revealed an increased synthesis of 86k, 70k and 66k proteins in the δ¹²-PGJ₂-treated NCG cells under the condition that δ¹²-PGJ₂ exerts cytotoxicity. Of these proteins, the amount of 66k protein was particularly increased in cell cytosol; however, its synthesis did not occur when CHM prohibited the δ¹²-PGJ₂ cytotoxic effect. When emetine was used instead of CHM, similar results were obtained.These results strongly suggest that the 66k protein plays a critical role in the °¹²-PGJ₂ cytotoxicity.     

Cycloheximide reduces PGD2 or delta 12-PGJ2 cytotoxicity on NCG cells

To study the precise mechanism of cytotoxic activity of PGD2 or delta 12-PGJ2 (a biologically active metabolite of PGD2), we examined the effect of various compounds on PGD2 or delta 12-PGJ2 cytotoxicity, using a human neuroblastoma cell line (NCG). Cycloheximide (CHM) specifically protected PGD2 cytotoxicity on NCG cells. When delta 12-PGJ2 was tested, CHM exhibited a similar rescue effect. Puromycin, mitomycin C, and alpha-amanitin did not affect PGD2 or delta 12-PGJ2 cytotoxicity. Emetine showed a variable and no consistent rescue effect CHM may have been active at the primary site where PGD2 or delta 12-PGJ2 exerts its cytotoxicity. This is the first report indicating that CHM reduces the cytotoxicity induced by PGD2 or delta 12-PGJ2.     

Prostaglandins and renin release: III. Effects of PGE1, E2 F2α and D2 on renin release from rabbit renal cortical slices

We have investigated the direct effects of prostaglandins E₁, E₂, F_2α and D₂ on renin release from rabbit renal cortical slices. Prostaglandin E₁ (PGE₁) was the most potent stimulant of renin release, while PGE₂ was 20–30 fold less active. PGF_2α was found not to be an inhibitor of renin release as reported by others, but rather a weak agonist. PGD₂ up to a concentration of 10 μg/ml had no activity in this system. That the stimulation of renin release by PGE₁ is a direct effect is supported by the finding that PGE₁-induced release is not blocked by L-propranolol or by Δ^5,8,11,14-eicosatetraynoic acid (ETYA), a prostaglandin synthesis is inhibitor. The fatty acid precursor of PGE₁, Δ^8,11,14-eicosatrienoic acid, also stimulated renin release, an effect which was blocked by ETYA. In addition to the above findings, ethanol, a compound frequently used to dissolve prostaglandins, was shown to inhibit renin release.     

PGJ2 and ΔPGJ2 inhibit cell growth accompanied with inhibition of phosphoinositide turnover in human astrocytoma cells

PGJ₂ and Δ¹²PGJ₂ (1 μM to 30 μm) inhibited the growth of human astrocytoma cells (1321N1) in a time-dependent manner within 48 hrs, determined by [³H]thymidine incorporation into acid-insoluble fraction or amounts of protein. The EC₅₀ values for PGJ₂ and Δ¹²PGJ₂ were approximately 8 μM and 6 μM, respectively. [³H]Thymidine incorporation to acid insoluble fraction was inhibited by these PGs within 1 hr, indicating that these PGs rapidly affect cell functions. Although it has been reported that an increase in cyclic AMP inhibits cell growth, PGJ₂ and Δ¹²PGJ₂, but not PGE₁, reduced isoproterenol (10 μM)-induced accumulation of cyclic AMP, suggesting that PGJ₂ and Δ¹²PGJ₂ may disturb adenylate cyclase system, which might be independent on cell growth. On the other hand, these PGs inhibited the incorporation of [³H]inositol into phospholipid fraction within 6 hrs. Furthermore, PGJ₂ and Δ¹²PGJ₂ inhibited carbachol- and/or histamine-induced accumulation of inositol phosphates with a similar dose-dependency to their inhibitions of cell growth. In membrane preparations, however, PGJ₂ and Δ¹²PGJ₂ failed to inhibit GTPγS (10 μM)- nor Ca²⁺ (1mM)-induced accumulation of inositol phosphate. The site of PGJ₂ or Δ¹²PGJ₂ in inhibition of inositol phosphate accumulation would not be phospholipase C nor a putative GTP binding protein involved in activation of phospholipase C. The present results indicate that PGJ₂ and Δ¹²PGJ₂ inhibit cell growth in human astrocytoma cells and the inhibition of phosphoinositide turnover by these PGs might be involved in the inhibition of cell growth.     

The effects of PGD2 and 9-deoxy-Δ-PGD2 on colony formation of murine osteosarcoma cells

Effects of antineoplastic prostaglandins (PG), PGD₂ and 9-deoxy-Δ⁹-PGD₂, on colony formation of cloned Dunn osteosarcoma (TA 102), normal Swiss 3T3 and V-79 cell lines were evaluated. PGD₂ significantly inhibited the colony formation of TA 102 cells in a dose-dependent manner at concentrations between 0.5 and 5 ug/ml. The IC₅₀ value was calculated to be 0.72 ug/ml. A dose-dependent inhibition of TA 102 colony formation was also observed with 9-deoxy-Δ⁹-PGD₂ between 0.01 to 1 ug/ml, the IC₅₀ value being 0.22 ug/ml. These prostaglandins did not exert cytocidal effects in vitro on Swiss 3T3 cells at concentrations between 0.01 to 1 ug/ml. The two agents had no significant cytocidal effects on V-79 cells except for 9-deoxy-Δ⁹-PGD₂ at a concentration of 5 ug/ml. These results suggest that PGD₂ and 9-deoxy-Δ⁹-PGD₂ are considered to have cytocidal activity on Dunn osteosarcoma cells in dosages which do not affect non-malignant cells.     

Effects of E2 levuglandins on the contractile activity of the rat uterus

The effects of E₂ levuglandins on the contractile activity of rat uterine horns were studied. LGE₂, AnLGE₂, Δ⁹-LGE₂ and the synthetic epimer, 8-epi-Δ⁹-LGE₂ all induced contractions in a dose-response fashion. AnLGE₂ gave decreased response with increased bath concentrations. Paired comparision showed potent and selective inhibitory effects of AnLGE₂ on the uterotonic activity of prostaglandins. An LGE₂ inhibited the uterotonic activity of PGe₂ at a 0.1:1 ratio, of PGD₂ at 1:1 ratio, but did not inhibit the activity of PGF_2α. Exposure of sponteneously contracting uteri to high concentrations of AnLGE₂, or prolonged exposure to lower concentrations, suppressed contractions.     

Intracellular glutathione level modulates the induction of apoptosis by Δ-prostaglandin J2

We studied the effect of intracellular glutathione (GSH), which was known to conjugate readily with an α, β-unsaturated carbonyl of 9-deoxy-Δ^9,12-13,14-dihydro PGD₂ (Δ¹²-PGJ₂), on the cytotoxicity of Δ¹²-PGJ₂. Δ¹²-PGJ₂ caused DNA fragmentation in human hepatocellular carcinoma Hep 3B cells, which was blocked by cycloheximide (CHX). The Δ¹²-PGJ₂-induced apoptosis was augmented by GSH depletion resulted from pretreatment with buthioninine sulfoximine (BSO), an inhibitor of γ-glutamylcysteine synthetase. On the contrary, N-acetyl-cysteine (NAC), a precursor of cysteine, elevated the GSH level and protected cells from initiating apoptosis by Δ¹²-PGJ₂. Sodium arsenite, a thiol-reactive agent, also induced apoptosis, which was potentiated or attenuated by BSO or NAC treatment respectively. These results suggest that the apoptosis-inducing activity of Δ¹²-PGJ₂ is due to thiol-reactivity and intracellular GSH modulates the Δ¹²-PGJ₂-induced apoptosis by regulating the accessibility of Δ¹²-PGJ₂ to target proteins containing thiol groups.     

Evaluation of the Stability,Bioavailability, and Hypersensitivity of the Omega-3 Derived Anti-Leukemic Prostaglandin: Δ12-Prostaglandin J3

Previous studies have demonstrated the ability of an eicosapentaenoic acid (EPA)-derived endogenous cyclopentenone prostaglandin (CyPG) metabolite, Δ¹²-PGJ₃, to selectively target leukemic stem cells, but not the normal hematopoietic stems cells, in in vitro and in vivo models of chronic myelogenous leukemia (CML). Here we evaluated the stability, bioavailability, and hypersensitivity of Δ¹²-PGJ₃. The stability of Δ¹²-PGJ₃ was evaluated under simulated conditions using artificial gastric and intestinal juice. The bioavailability of Δ¹²-PGJ₃ in systemic circulation was demonstrated upon intraperitoneal injection into mice by LC-MS/MS. Δ¹²-PGJ₃ being a downstream metabolite of PGD₃ was tested in vitro using primary mouse bone marrow-derived mast cells (BMMCs) and in vivo mouse models for airway hypersensitivity. ZK118182, a synthetic PG analog with potent PGD₂ receptor (DP)-agonist activity and a drug candidate in current clinical trials, was used for toxicological comparison. Δ¹²-PGJ₃ was relatively more stable in simulated gastric juice than in simulated intestinal juice that followed first-order kinetics of degradation. Intraperitoneal injection into mice revealed that Δ¹²-PGJ₃ was bioavailable and well absorbed into systemic circulation with a C_max of 263 µg/L at 12 h. Treatment of BMMCs with ZK118182 for 12 h resulted in increased production of histamine, while Δ¹²-PGJ₃ did not induce degranulation in BMMCs nor increase histamine. In addition, in vivo testing for hypersensitivity in mice showed that ZK118182 induces higher airways hyperresponsiveness when compared Δ¹²-PGJ₃ and/or PBS control. Based on the stability studies, our data indicates that intraperitoneal route of administration of Δ¹²-PGJ₃ was favorable than oral administration to achieve effective pharmacological levels in the plasma against leukemia. Δ¹²-PGJ₃ failed to increase histamine and IL-4 in BMMCs, which is in agreement with reduced airway hyperresponsiveness in mice. In summary, our studies suggest Δ¹²-PGJ₃ to be a promising bioactive metabolite for further evaluation as a potential drug candidate for treating CML.     

Neuroeffector actions of prostaglandin D2 on isolated dog mesenteric arteries

Treatment with prostaglandin (PG) D₂ in concentrations (10⁻⁸ to 10⁻⁷ M) insufficient to alter the basal tone potentiated the contractile response of helical strips of dog mesenteric arteries to transmural electrical stimulation but did not influence the response to norepinephrine. The potentiating effect of PGD₂ was not prevented by treatment with diphloretin phosphate, a PG antagonist, whereas contractions of dog cerebral arteries induced by PGD₂ were suppressed. The ³H-overflow evoked by transmural stimulation in superfused mesenteric arterial strips previously soaked in ³H-norepinephrine containing media was significantly increased by PGD₂. It is concluded that PGD₂ increases the stimulation-evoked release of norepinephrine from adrenergic nerves innervating the arterial wall. PGD₂ appears to act differently on receptive sites responsible for increasing the release of norepinephrine and for producing arterial contraction.     

Bio-synthesis of PGD2 and TXB2 by rabbit blood monocytes

A method for the preparation of a highly purified sample of rabbit blood monocytes is described. The metabolism of arachidonic acid (AA) in these cells was studied. Mononuclear cells were prepared by centrifugation on Ficoll-Paque gradients and the monocytes were obtained by further centrifugation and adherence onto plastic culture dishes. These procedures provided a preparation which contained 95% monocytes (non-specific esterase positive). Incubation of [1-¹⁴C]-AA with these cells produced four major metabolites which were separated by TLC; these corresponded to prostaglandin (PG) D₂, thromboxane (TX) B₂, 12-hydroxyheptadecatrienoic acid (HHT) and 12-/15- hydroxyeicosatetraenoic acid (HETE). A minor product which co-migrated with PGE₂ was also detected but neither 6-keto-PGF_1α nor PGF_2α were detected. Also, there was no evidence of the formation of 5-lipoxygenase products (5-HETE and LTB₄) by rabbit monocytes with or without calcium-ionophore A23187-stimulation. The production of PGD₂, TXB₂ and PGE₂ was further confirmed by analyzing [³H]-AA metabolites using high-performance liquid chromatography (HPLC) with tritiated standards as references. The biosynthesis of these compounds from endogenous substrate in A23187-stimulated monocytes was confirmed by specific radioimmunoassays with or without prior HPLC separation. The synthesis of immunoreactive LTB₄ and LTC₄ by A23187-stimulated cells was also monitored and found to be relatively low. The synthesis of PGD₂, TXB₂ and PGE₂ from both exogenous and endogenous substrate was suppressed by treatment of the monocytes with indomethacin (10⁻⁶ M).     

Evidence for separate PGD2 and PGF2α receptors in the canine mesenteric vascular bed

Potential interactions between PGD₂ and PGF_2α in the mesenteric and renal vascular beds were investigated in the anesthetized dog. Regional blood flows were measured with electromagnetic flow probes. PGD₂, PGF_2α and Norepinephrine (NE) were injected as a bolus directly into the appropriate artery, and responses to these agents were obtained before, during and after infusion of either PGD₂ or PGF_2α into the left ventricle. In each case, the infused prostaglandin caused vascular effects of its own. Left ventricular infusion of PGD₂ reduced responses to local injections of PGD₂ in the intestine, and a similar effect was observed for PGF_2α, suggesting significant receptor or receptor-like interactions for each of the prostanoids. However, systemic infusion of prostaglandin F_2α (20–100 ng/kg/min) had no effect on renal or mesenteric vascular responses to local injection of prostaglandin D₂. Similarly, PGD₂ administration (100 ng/kg/min) did not affect responses to PGF_2α in the intestine. The present results therefore suggest that these prostaglandins, i.e., D₂ and F_2α, act through separate receptors in the mesenteric and renal vascular beds. In addition, increased prostaglandin F_2α levels produced by infusion of F_2α reduced mesenteric but not renal blood flow, suggesting that redistribution of cardiac output might participate in side effects often observed with clinical use of this prostaglandin, such as nausea and abdominal pain.     

Cardiovascular effects of prostaglandin D3 and D2 in anesthetized dogs

Prostaglandin (PG) D₃ has been identified as an inhibitor of human platelet aggregation, but little is known of the hemodynamic activity of this material. In morphine pretreated, chloralose-urethan anesthetized dogs, bolus intravenous injections (1, 3.2 and 10 μg/kg) of PGD₃ and also PGD₂ were associated with marked, dose-related increases in pulmonary arterial pressure. Cardiac index and rate increased, while peripheral vascular resistance decreased in response to injections of PGD₃. A biphasic (depressor followed by a pressor phase) effect on systemic arterial pressure was observed after PGD₂, while PGD₃ was associated with dose-related depressor responses. Graded intravenous infusions (0.25, 0.50 and 1.0 μg/kg/min) of PGD₃ and PGD₂ were associated with qualitatively similar cardiovascular responses. Quantitatively, PGD₃ infusions were associated with greater decreases in peripheral vascular resistance and greater increases in cardiac output, heart rate, and peak left ventricular dp/dt than were infusions of PGD₂. In contrast, PGD₃ was less potent than PGD₂ as a pulmonary pressor material. Systemic arterial pressure responses to infusions of the prostaglandins were variable. In these experiments, PGD₃ and PGD₂ were associated with qualitatively similar cardiovascular responses characterized by peripheral vasodilatation.     

Bronchopulmonary and cardiovascular effects of prostaglandin D2 in the dog

The effects of intravenously administered prostaglandin D₂ (PGD₂) on bronchopulmonary and cardiovascular functions were examined in the dog. PGD₂ (0.03–1.0 μg/kg) was shown to be more active than PGF_2α, a known bronchoconstrictor, in decreasing dynamic lung compliance, tidal volume, and expiratory airflow rate, as well as in elevating lung resistance. PGD₂ demonstrated a potency approximately 4–6 times that of PGF_2α on pulmonary mechanics. Atropine sulfate infusions reduced significantly the resistance and compliance responses to PGF_2α, but only the resistance responses to PGD₂, thereby suggesting that part of the bronchoconstrictor activities of these agents involved a cholinergic component.In another series of anesthetized dogs, PGD₂ (0.1–10.0 μg/kg) increased pulmonary arterial pressure (comparable to PGF_2α) and heart rate (greater than PGF_2α, but less than PGE₂), while concomitantly decreasing systemic arterial pressure in a dose-related manner ( that of PGE₂). Qualitatively similar alterations in cardiovascular parameters were obtained for PGD₂ in conscious dogs.Therefore, potent biologica activity of PGD₂ has been shown in the dog. No physiologic or pathologic role for PGD₂ has yet been demonstrated, but nonetheless, since it is a naturally occurring PG derived from arachidonic acid, further studies are warranted.     

Effects of PGD2 and PGF2α on longitudinal and circular muscles of guinea-pig isolated proximal colon

The mechanical effects of PGD₂ and PGF_2α on longitudinal and circular muscles of the guinea-pig isolated proximal colon were investigated. PGD₂ and PGF_2α (1 nM – 10 μM) produced a dose-dependent contraction in longitudinal and circular muscles. The contractile action of PGD₂ was more potent than that of PGF_2α in circular muscle and was less potent in longitudinal muscle.Contractions induced by PGD₂ or PGF_2α(1 μM) were unaffected by atropine (1 μM) in both muscles, but tetrodotoxin (1 μM) slightly inhibited these contractions in longitudinal muscle.The results suggest that in longitudinal muscle PGD₂ and PGF_2α have largely a direct action on the muscle cells and a partial neuronal action on the non-cholinergic intrinsic nerves, whereas in circular muscle these PGs have only a direct action on the muscle cells.     

Prostaglandins and renin release: II. Assessment of renin secretion following infusion of PGI2, E2 and D2 into the renal artery of anesthetized dogs

The influence of intra-renal infusions of prostaglandin (PG) I₂, PGE₂ and PGD₂ on renin secretion and renal blood flow was investigated in renally denervated, beta-adrenergic blocked, indomethacin treated dogs with unilateral nephrectomy. All three prostaglandins when infused at doses of 10⁻⁸ g/kg/min and 10⁻⁷ g/kg/min resulted in marked renal vasodilation. Renin secretory rates increased significantly with both PGI₂ and PGE₂ at the 10⁻⁸ g/kg/min and 10⁻⁷ g/kg/min infusion rates in a dose dependent manner. However, PGD₂ was inactive. At 10⁻⁷ g/kg/min, PGI₂ infusions resulted in systemic hypotension indicating recirculation of this prostaglandin. These findings suggest that PGI₂ should be included among the cyclooxygenase derived metabolites of arachidonic acid to be considered as possible mediators of renin release.     

A comparison of the contractile activity fo PGD2 and PGF2α on human isolated bronchus

Prostaglandins may be implicated in the bronchoconstriction which occurs in asthma. Prostaglandins F_2α (PGF_2α and D₂ (PGD₂) have been reported to produce bronchoconstriction in asthmatic subjects in vivo and PGF_2α cotnracts human isolated airway smooth muscle. We examined the relative efficacy and potency of PGF_2α and PGD₂ on human bronchial spiral strips taken from 6 patients at thoracotomy. PGF_2α had greater efficacy than PGD₂. The mean % T_max (percentage of maximal contractile response) ± s.e. mean were 84 ± 7 and 54 ±7 respectively (P < 0.05). PGF_2α (mean pD₂ ± s.e. mean = 6.39 ± 0.6) tended to be more potent than PGD₂ (5.68 ± 0.2). Since, in vivo, PGD₂ has greater efficacy and potency than PGF_2α, our results suggest that the in vivo effect of these prostaglandins does not result solely from an action on airway muscle.