Inhibitory Properties of Antitumor Prostaglandins against Topoisomerases

Prostaglandins (PGs) having antitumor activity such as Δ^12,14-PGJ₂, Δ¹²-PGJ₂, PGA₂ and PGA₁ strongly inhibited topoisomerase II (topo II) from human placenta, the potential order of inhibitory activity of the PGs resembling that of the antitumor activity. PGs having no antitumor activity did not inhibit topo II. Δ^12,14-PGJ₂ to be a potent inhibitor showed inhibitions to some extent against topo I from wheat germ, NIH3T3 and calf thymus gland, and showed no inhibition against the enzymes from Vero, A549, HeLa and COLO 201 cells. Δ^12,14-PGJ₂ differentially inhibited topo I from different sources. Δ^12,14-PGJ₂ was a topo inhibitor of the cleavable complex-nonforming type without DNA intercalation.     

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.     

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 and J2 induce apoptosis in human leukemia cells via activation of the caspase 3 cascade and production of reactive oxygen species

The presence of prostaglandins (PGs) has been demonstrated in the processes of carcinogenesis and inflammation. In the present study, we found that 12-o-tetradecanoylphorbol 13-acetate (TPA) induced cyclooxygenase 2 (COX-2), but not COX-1, protein expression in HL-60 cells, and the addition of arachidonic acid (AA) in the presence or absence of TPA significantly reduced the viability of HL-60 cells, an effect that was blocked by adding the COX inhibitors, NS398 and aspirin. The AA metabolites, PGD₂ and PGJ₂, but not PGE₂ or PGF_2α, reduced the viability of the human HL60 and Jurkat leukemia cells according to the MTT assay and LDH release assay. Apoptotic characteristics including DNA fragmentation, apoptotic bodies, and hypodiploid cells were observed in PGD₂- and PGJ₂-treated leukemia cells. A dose- and time-dependent induction of caspase 3 protein procession, and PARP and D4-GDI protein cleavage with activation of caspase 3, but not caspase 1, enzyme activity was detected in HL-60 cells treated with PGD₂ or PGJ₂. Additionally, DNA ladders induced by PGD₂ and PGJ₂ were significantly inhibited by the caspase 3 peptidyl inhibitor, Ac-DEVD-FMK, but not by the caspase 1 peptidyl inhibitor, Ac-YVAD-FMK, in accordance with the blocking of caspase 3, PARP, and D4-GDI protein procession. An increase in intracellular peroxide levels by PGD₂ and PGJ₂ was identified by the DCHF-DA assay, and anti-oxidant N-acetyl cysteine (NAC), mannitol (MAN), and tiron significantly inhibited cell death induced by PGD₂ and PGJ₂ by reducing reactive oxygen species (ROS) production. The PGJ₂ metabolites, 15-deoxy-Δ^12,14-PGJ₂ and Δ¹²-PGJ₂, exhibited effective apoptosis-inducing activity in HL-60 cells through ROS production via activation of the caspase 3 cascade. The proliferator-activated receptor-γ (PPAR-γ) agonists, rosiglitazone (RO), troglitazone (TR), and ciglitazone (CI), induced apoptosis in cells which was blocked by the addition of the PPAR-γ antagonists, GW9662 and BADGE, via blocking of caspase 3 and PARP cleavage. However, neither GW9662 nor BADGE showed any protective effect on PGD₂- and PGJ₂-induced apoptosis. A differential apoptotic effect of PGs through ROS production, followed by activation of the caspase 3 cascade, was demonstrated.     

Effects of estradiol and progesterone on uterine prostaglandin levels in the pregnant rat

Prostaglandin D₂ was found to be a potent inhibitor of B-16 melanoma cell replication $\text{in vitro}$. The inhibition was dose-dependent between 3×10^?9M and 3×10^?6M (IC_50～ 0.3 μM after 6 days). On a molar basis, PGD₂ was a better inhibitor than PGA₂ or 16,16-dimethyl-PGE₂-methyl ester (di-M-PGE₂) and in higher concentrations (10^?6?10^?7M), comparable to retinoic acid. In higher concentrations, PGD₂ inhibited DNA, RNA and protein synthesis. The B-16 melanoma cell line which we used synthesized arachidonic acid metabolites which comigrated with PGA₂, PGD₂, PGE₂ and PGF_2α on a thin layer chromatography system.     

The effect of PGE2 on the activation of quiescent lung fibroblasts

The effect of prostaglandin E₂ (PGE₂) on fibroblast proliferation was examined. The presence of PGE₂ for 24 h inhibited the growth of quiescent cells stimulated with serum, platelet-derived growth factor and macrophage-derived factors. Maximal inhibition of nuclear labeling with [³H]thymidine occurred at concentrations greater than 10⁻⁷ M. The inhibitory effect of PGE₂ was less potent in exponentially growing cells and was not the result of conversion of PGE₂ to PGA₂ during incubation in growth medium. The G₁ phase was determined to be 12–14 h in untreated cultures. The extent of growth inhibition by PGE₂ was similar with addition of PGE₂ at 0, 3, 6, or 9 h following restimulation of quiescent cell cultures. Approximately 25% of the cells that enter S phase are refractory to PGE₂-induced growth inhibition. Short-term exposure to PGE₂ (5 min and 30 min) caused substantial growth inhibition. The serum-induced proliferation was also inhibited by the cAMP analogue, dibutyrl cAMP. Our results suggest that PGE₂ affects a distinct subpopulation of cells. Restimulation of quiescent cells treated with PGE₂ for 24 h, indicated that release from PGE₂ exposure is associated with prolongation of the G₁ phase of the cell cycle.     

The effect of prostaglandins on cultured lapine articular chondrocytes

The effect of 8 prostaglandins (PG) on growth and sulfate incorporation by monolayer and spinner-cultured rabbit articular chondrocytes has been measured. PGA₁, PGB₁, PGE₁ and PGE₂ reduced synthesis of sulfated glycosaminoglycans (GAG) but the PGF series did not. PGA₁ was the most potent, being effective at a concentration of 2.5 μg/ml [6.8 μM] while the others required 25 μg/ml. These compounds had no effect on degradation of GAG. All 8 PGs augmented growth slightly but significantly at 2.5 μg/ml. At the higher concentration, PGA₁ was highly cytotoxic, and PGB₁ as well as PGE₂ reduced cell growth. The cytotoxicity of PGA₁ was also observed in two additional types of cultured connective tissue cells, but the inhibition of sulfated-GAG synthesis by PGA₁ and PGB₁ was confined to the chondrocytes. The response of cultured chondrocytes to exogenous PGs, albeit at apparently unphysiologically high concentrations, together with other evidence, suggests that these compounds may conceivably play a direct role in cartilage metabolism in vivo.     

Prostaglandin E1 specific binding in rhesus myometrium

Myometrial low speed supernatant prepared from non-pregnant rhesus uteri was incubated with ³H-Prostaglandin (PG)E₁ with or without addition of unlabelled prostaglandins. The uptake of ³H-PGE₁ was inhibited in a dose dependent fashion by PGE₂>PGE₁>PGA₁>PGF_2α=PGA₁>PGB₁=PGB₂≥PGD₂. PGE₁ metabolites inhibited ³H-PGE₁ binding in the following order: 13,14-dihydro-PGE₁>13,14-dihydro-15-keto-PGE₁=15-keto-PGE₁. The specific binding of ³H-PGE₁ and ³H-PGF_2α was similarly affected by the temperature and time of incubation. Equilibrium binding constants determined using rhesus uteri obtained during the luteal phase of the menstrual cycle indicate the presence of high affinity PGE₁ binding sites with an average (n=3) apparent dissociation constant of 2.2 × 10⁻⁹M and a lower affinity PGE₁ binding site with a K_d ≅ 1 × 10⁻⁸M. No high affinity — low capacity ³H-PGF_2α sites could be demonstrated.Relative uterine stimulating potencies of some natural prostaglandins and prostaglandin analogs tested after acute intravenous administration in mid-pregnant rhesus monkeys corresponded with the PGE₁ binding inhibition of the respective compound. The uterine stimulating potencies of the prostaglandin analogs tested were: (15S)-15-methyl-PGE₂=16,16-dimethyl-PGE₂>17-phenyl-18,19,20-trinor-PGE₂>16 phenoxy-17,18,19,20-tetranor-PGF_2α=PGE₂=PGE₁=(15S)-15-methyl-PGF_2α>PGF_2α.     

The synthesis and biological activities of some 12-Aza-prostaglandin analogues

12-Aza-prostaglandin (PG) analogues containing the pyrrolidine-2,4-dione ring system have been synthesized from ,2-disubstituted glycine esters via cyclisation of their -ethoxycarbonylacetyl derivatives. 5-(6-Carboxyhexyl)-1-octylpyrrolidine-2,4-dione (5) had little or no PG-like activity on superfused intestinal or vascular smooth muscle preparations but it selectively antagonised smooth muscle responses to PGE₂, PGE₁, PGF_2α and PGA₂in vitro. At a concentration of 10⁻⁵ g/ml it reduced responses of the rat stomach strip to PGE₂ by over 80% but did not affect responses of this tissue to acetylcholine, 5-hydroxytryptamine (5-HT) or bradykinin. Polyphloretin phosphate (PPP), the known PG antagonist, had a similar effect at the same concentration (10⁻⁵ g/ml).5-(6-Carboxyhexyl)-1-(3-hydroxyoctyl)pyrrolidine-2,4-dione (12) had the same profile of activity on superfused smooth muscle preparations as PGE₂ or PGA₂. On intravenous injection into anaesthetised rats it caused dose-dependent falls in arterial blood pressure with associated tachycardias, which is typical of the response to PGE₂. The smooth muscle activity of (12) was not reduced by passage through isolated perfused guinea-pig lungs nor was its potency as a vasodepressor increased when given intra-arterially to rats. These results suggest that, unlike PGE₂, this analogue is not removed by the pulmonary circulation.     

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.     

THE DEPRESSION OF PHAGOCYTOSIS BY EXOGENOUS CYCLIC NUCLEOTIDES, PROSTAGLANDINS, AND THEOPHYLLINE

The effects of agents that elevate intracellular cyclic adenosine 3',5'-monophosphate (cAMP) have been studied with respect to phagocytosis by guinea pig polymorphonuclear leukocytes. The investigation depends upon the use of a precise method for following ingestion. Theophylline, dibutyryl cAMP, and prostaglandins inhibited the phagocytosis of starch particles. The inhibitions caused by prostaglandins E₁, E₂, and F_2α (PGE₁, PGE₂, and PGF_2α) were synergistic with that due to theophylline. Inhibition by PGA₁ and PGA₂ was not. At equal concentrations the order of increasing inhibition of phagocytosis (assayed at 10 min) by the prostaglandins was PGE₁ < PGF_2α < PGE₂ < PGA₁ = PGA₂. Our results are consistent with the hypothesis that increased intracellular levels of cAMP impair the phagocyte's ability to ingest particles. The mechanism of the inhibition has not been defined. The increment in oxidation of [1-¹⁴C]glucose to ¹⁴CO₂ that normally accompanies phagocytosis was found to be depressed in the presence of PGE₁ or theophylline, together or individually as expected from the inhibition of phagocytosis. Paradoxically, oxygen consumption although depressed by theophylline or PGE₁ plus theophylline, was stimulated by PGE₁ alone.     

Enzymatic formation of prostaglandin F2α in human brain

Prostaglandin (PG)E₂ 9-ketoreductase, which catalyzes the conversion of PGE₂ to PGF₂, was purified from human brain to apparent homogeneity. The molecular weight, isoelectric point, optimum pH, Km value for PGE₂, and turnover number were 34,000, 8.2, 6.5–7.5, 1.0 mM, and 7.6 min^–1, respectively. Among PGs tested, the enzyme also catalyzed the reduction of other PGs such as PGA₂, PGE₁, and 13,14-dihydro-15-keto PGF₂, but not that of PGD₂, 11-PGE₂, PGH₂, PGJ₂, or ¹²-PGJ₂. The reaction product formed from PGE₂ was identified as PGF₂, by TLC combined with HPLC. This enzyme, as is the case for carbonyl reductase, was NADPH-dependent, preferred carbonyl compounds such as 9,10-phenanthrenequinone and menadione as substrates, and was sensitive to indomethacin, ethacrynic acid, and Cibacron blue 3G-A. The reduction of PGE₂ was competitively inhibited by 9,10-phenanthrenequinone, which is a good substrate of this enzyme, indicating that the enzyme catalyzed the reduction of both substrates at the same active site. These results suggest that PGE₂ 9-ketoreductase, which belongs to the family of carbonyl reductases, contributes to the enzymatic formation of PGF₂ in human brain.Special issue dedicated to Dr. Sidney Udenfriend.     

Determination of Branched β-Cyclodextrin–Prostaglandin Complexes Using Electrospray Ionization Mass Spectrometry

Mass spectral measurements by electrospray ionization mass spectrometry (ESI-MS) detected the ions of β-cyclodextrin (βCD) or branched βCDs (glucosyl-, galactosyl-, mannosyl- and maltosyl-βCD)–prostaglandins (PGs: PGA₂, PGD₂, PGE₁, PGE₂, PGF_2α and PGJ₂) complexes, i.e., βCD–PG complexes, with a host:guest ratio of 1:1 in the negative ion mode. This is the first study to report the ions of branched βCD–PG complexes using ESI-MS. The inclusion complexes were determined by a flow injection analysis using acetonitrile/water. We could confirm by this method the presence of a βCD–PGE₂ complex with a host:guest ratio of 1:1 in a solution-dissolved pharmaceutical formulation consisting of βCD–PGE₂ (Prostarmon^TM E tablet).     

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.     

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.     

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.     

Effects of various prostanoids on th in vitro metabolism of bovine articular chondrocytes

The dose-dependent effects of 9 prostanoids (PGA₁, PGA₂, PGE₁, PGE₂, PGF_1α, PGF_2α, PGD₂, PGI₂, 6 keto- PGF_1α) on metabolism of cultured bovine articular chrondrocytes were investigated. Most prostanoids dose-dependently inhibited ³⁵SO₄⁼ and ³H-glycine incorporation. At 25 μg/ml, the inhibitory sequence was A₂D₂>E₂ = E₁ = A₁>6 keto-F_1α>F₁>F₂, but sensivity (lowest dose eliciting inhibition) followed the sequence E₂ > 6 keto-F_1α = F₁ > A₂ = D₂>E₁>A₁. At 25 μg/ml PGA₂ also inhibited incorporation of ³H-cytidine and ^#H-thymidine, but had no significant effect on ³H-glucose or ¹⁴C-xylose incorporation. The inhibitory effect of PGA₂ was apparent after 30 minutes exposure for ³⁵SO₄= and after 60 minutesd for ³H-cytidine, and was still present up to 72 hours following incubation in fresh non-PG-containing medium. PGI₂ had no significant effect of ³⁵SO₄⁼ incorporation but at concentrations below 10 μg/ml enhanced uptake of ³H-glycine.The PG-induced inhibitory effect was apparently not due to cell damage as indicated by measurement of ³H-glucose metabolism and lactate production.     

Prostaglandin binding in human serum follows the polarity rule

Human serum binds PGA₁ > PGE₁ > PGF_2α. This is in inverse order of their polarity. Approximately 90% of PGA₁ is bound. By analogy with steroid and iodothyronine metabolism, it is likely that the tighter binding of PGA₁ accounts for its relatively slow clearance.     

Prostaglandin inhibition of cartilage chondromucoprotein synthesis: Concept of “intrinsic activity”

We have recently reported that cartilage has two sites for prostaglandin (PG) action. One site (S₁) is stimulated by PGA₁, PGE₁ and PGF_1α and elevates tissue cyclic 3′5′adenosine monophosphate (cAMP). A second site (S₂) is activated by PGA₁ (but not PGE₁ or PGF_1α) and inhibits the synthesis of cartilage macromolecules. The present study is an investigation of the effects of PGB₁ on embryonic chicken cartilage chondromucoprotein synthesis in vitro. PGB₁ was found to inhibit chondromucoprotein synthesis with an apparent affinity for S₂ which was similar to that of PGA₁. The maximal inhibition produced by PGB₁ was, however, approximately one-half the maximal inhibition caused by PGA₁. Studies of the combined effects of PGB₁ and PGA₁ were consistent with the hypothesis that both classes of prostaglandins act at a common site (S₂) with about equal affinity but that PGB₁ has a lower intrinsic activity than PGA₁. Similar studies of the combined effects of PGE₁ or PGF_1α with PGA₁ indicate that neither PGE₁ nor PGF_1α binds significantly to S₂. An independent effect of PGB₁ to activate S₁ and elevate tissue cAMP was also found.