The effects of PGD2 and 9-deoxy-Δ9-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.     

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

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

Cycloheximide reduces PGD2 or Δ-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.     

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.     

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.     

Prostaglandin D2 inhibits the proliferation of human malignant tumor cells

The cytotoxic effect of prostaglandin (PG) D₂, PGE₁ and PGF_2α was examined on human osteosarcoma cells (KSu cell line) , and PGD₂ was most effective. DNA, RNA and protein syntheses of KSu cells were also found to be inhibited by PGD₂ at a concentration of 5μg/ml. Furthermore, the proliferation of various human malignant tumor cells was inhibited by PGD₂ without exception so far. These results suggest that PGD₂ shows an antineoplastic effect on a variety of human malignant tumor 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.     

Thromboxane A2 and prostaglandin H2: Potent stimulators of the swine coronary artery

Thromboxane A₂ was generated by incubation of arachidonic acid with a suspension of human platelets. The filtrate contained 266 ± 46 ng/ml (n=10) of thromboxane A₂ and 25 ng/ml or less of prostaglandin endoperoxides (prostaglandins G₂+H₂). Thromboxane A₂ was 2–10 times more potent than prostaglandin H₂ and 9–102 times and 26–308 times more potent than prostaglandins E₂ and F₂α, respectively, in causing contractions of the superfused swine coronary artery.     

7beta-hydroxy-epiandrosterone modulation of 15-deoxy-delta12,14-prostaglandin J2, prostaglandin D2 and prostaglandin E2 production from human mononuclear cells

7β-hydroxy-epiandrosterone (7β-OH-EPIA) has been shown to be cytoprotective in various organs including the brain. It has also been shown that prostaglandin D₂ (PGD₂) and its spontaneous metabolite 15-deoxy-Δ^12,14-prostaglandin J₂ (15d-PGJ₂) are also cytoprotective. It is possible that these prostaglandins derived from circulating mononuclear cells may mediate the actions of 7β-OH-EPIA. The aim of this study, therefore, was to ascertain the effect of 7β-OH-EPIA (in the absence or presence of tumour necrosis factor-α (TNF-α)), a pro-inflammatory stimulus, on the biosynthesis of PGD₂, PGE₂ and 15d-PGJ₂ from human mononuclear cells. Prostaglandins were measured by enzyme immunoassay (EIA). 7β-OH-EPIA alone induced a concentration-dependant increase in the production of PGD₂. TNF-α increased PGD₂ levels which were enhanced by 7β-OH-EPIA. 7β-OH-EPIA increased 15d-PGJ₂ levels both in the absence and presence of TNF-α. 7β-OH-EPIA alone had no effect on PGE₂ biosynthesis but suppressed TNF-α-induced PGE₂ circa 50%. 7β-OH-EPIA also increased the level of free arachidonic acid and radiolabelled prostaglandins in cells pre-incubated with radiolabelled arachidonic acid, indicating that the increase may occur via the enhanced release of substrate arachidonic acid. 7β-OH-EPIA did not affect levels of the anti-inflammatory cytokine IL-10 indicating that this is an unlikely mechanism by which 7β-OH-EPIA induces its actions but more likely exerts its effects via the production of cytoprotective prostaglandins.     

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.     

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.     

3-(1′,1′-Dimethylbutyl)-1-deoxy-Δ-THC and related compounds: synthesis of selective ligands for the CB2 receptor

The synthesis and pharmacology of 15 1-deoxy-Δ⁸-THC analogues, several of which have high affinity for the CB₂ receptor, are described. The deoxy cannabinoids include 1-deoxy-11-hydroxy-Δ⁸-THC (5), 1-deoxy-Δ⁸-THC (6), 1-deoxy-3-butyl-Δ⁸-THC (7), 1-deoxy-3-hexyl-Δ⁸-THC (8) and a series of 3-(1′,1′-dimethylalkyl)-1-deoxy-Δ⁸-THC analogues (2, n=0–4, 6, 7, where n=the number of carbon atoms in the side chain−2). Three derivatives (17–19) of deoxynabilone (16) were also prepared. The affinities of each compound for the CB₁ and CB₂ receptors were determined employing previously described procedures. Five of the 3-(1′,1′-dimethylalkyl)-1-deoxy-Δ⁸-THC analogues (2, n=1–5) have high affinity (K_i=<20 nM) for the CB₂ receptor. Four of them (2, n=1–4) also have little affinity for the CB₁ receptor (K_i=>295 nM). 3-(1′,1′-Dimethylbutyl)-1-deoxy-Δ⁸-THC (2, n=2) has very high affinity for the CB₂ receptor (K_i=3.4±1.0 nM) and little affinity for the CB₁ receptor (K_i=677±132 nM).

Scheme 3. (a) (C₆H₅)₃PCH₃⁺ Br⁻, n-BuLi/THF, 65°C; (b) LiAlH₄/THF, 25°C; (c) KBH(sec-Bu)₃/THF, −78 to 25°C then H₂O₂/NaOH.     

Antihypertensive activity of 16,16-dimethyl-oxa-alkyl-prostaglandins of the PGA2, PGE2 and trans-Δ 2-11-deoxy-PGE1 series: Structure-activity relationships

Utilizing Corey's synthesis, a variety of prostaglandins (PGs) with a modified ω-side chain were prepared. The 16,16-dimethyl-oxa-alkyl analogues of PGA₂ had potent antihypertensive activity. HR 466 (16,16-dimethyl-18-oxa-PGA₂), the best compound out of this series was active for 5–6 hours after oral administration of 0,1 mg/kg to conscious renal hypertensive dogs. The corresponding analogues of PGE₂ were also potent antihypertensive compounds, but were much more spasmogenic. Structural variations within the trans-Δ2-11-deoxy-PGE₁-series, in both side chains, gave HR 601 (trans-Δ2-15α-acetoxy-16,16-dimethyl-18-oxa-11-deoxy-PGE₁-methylester) which was orally active in the hypertensive dog with similar activity to HR 466.     

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.     

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.     

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).     

Synthesis of [17,18-3H]trans-13-azaprostanoic acid. A labeled probe for the PGH2/TXA2 receptor

Because of its highly unstable nature, TXA₂, produced by platelet metabolism of arachidonic acid, does not lend itself to use as a receptor probe for its own receptor. As such, the stable TXA₂/PGH₂ antagonist, trans-13-azaprostanoic acid (trans-13-APA, 12b), was prepared as the [17,18 ³H] derivative ([³H] trans-13-APA, 12c) to study this receptor and to better evaluate the mechanism of action of these azaprostanoids. Tritiated trans-13-APA, 12c, was prepared in nearly theoretical specific activity (57 Ci/mmole) from (17z)-trans-13-azaprost-17-enoic acid (11b) by catalytic tritiation. The unsaturated 11b was prepared by condensation of cis-7-amino-3-heptene (8) with 2-(6-carboxyhexyl) cyclopentanone (9), NaBH₄ reduction, chromatography, and hydrolysis of the trans isomer so isolated. The olefins 11a and b were also of biochemical interest because of the unsaturation in the lower side chain. The presence of similar unsaturation in PGH₃ (4) and TXA₃ (3) renders these prostaglandins inactive as proaggregatory agents. Evaluation of the antiaggregatory activity of 11a and b indicated it to be about the same potency in inhibiting human platelet aggregation as the parent cis and trans-13-APAs, suggesting that introduction of a double bond at the 17 position in platelet prostaglandin antagonists is unlikely to result in enhanced antiplatelet activity.     

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.     

Prostaglandin E1 and F2α specific binding in bovine corpora lutea: Comparison with luteolytic effects

Preliminary characterization indicated the presence of separate prostaglandin (PG)E₁ and (PG)F_2α binding sites in membrane fractions prepared from bovine corpora lutea. These differ in the rate and temperature dependence of the specific binding. Equilibrium binding data indicate the apparent dissociation constants as 1.32 × 10⁻⁹M and 2.1 × 10⁻⁸M for PGE₁ and PGF_2α, respectively. Competition of several natural prostaglandins for the PGE₁ and PGF_2α bovine luteal specific binding sites indicates specificity for the 9-keto or 9α-hydroxyl moiety, respectively. Differences in relative ability to inhibit ³H-PG binding were found due to sensitivity to the absence or presence of the 5,6-cis-double bond as well.Bovine luteal function was affected following treatment of heifers with 25 mg PGF_2α as measured by reduced estrous cycle length, decreased corpus luteum size and significantly decreased plasma progesterone levels. In contrast, treatment with 25 mg PGE₁ resulted in cycle lengths comparable to those of non-treated herdmates with no apparent modification in corpus luteum size. However, plasma progesterone levels were increased significantly following PGE₁ treatment compared to pretreatment values. In so far as data obtained in vitro on PGF_2α relative binding affinity to the bovine CL can be compared to data obtained independently in vitro on PGF_2α induced luteolysis in the bovine, PGF_2α relative binding to the CL and luteolysis appeared to be associated. By similar reasoning, there was no apparent relationship between PGE₁ relative binding affinity in the luteal fractions and luteolysis in estrous cyclic cattle.