Inhibitory properties of antitumor prostaglandins against topoisomerases

Prostaglandins (PGs) having antitumor activity such as delta12,14-PGJ2, delta12-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. Delta12,14-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. Delta12,14-PGJ2 differentially inhibited topo I from different sources. Delta12,14-PGJ2 was a topo inhibitor of the cleavable complex-nonforming type without DNA intercalation.     

Interactions of prostaglandins with hepatic microsomal cytochrome P-450

The spectral changes associated with the addition of prostaglandins (PGs) to hepatic microsomes from guinea pigs and rats were examined. PGA₁, PGA₂, PGE₁, PGE₂, PGF_1α and PGF_2α when added to guinea pig liver microsomes exhibited type I spectra. The binding affinities as determined from spectral dissociation constants (K_s) were highest with PGA₁ and PGA₂. With liver microsomes from control or 3-methyl-cholanthrene (MC)-treated rats, PGs did not yield type I spectra; however, in this case a $\text{weak}$ spectrum, designated here as type “II” was at times observed, With microsomes from phenobarbital (Pb)-treated rats only PGA₁ and PGA₂ yielded type I spectra; again in absence of type I spectrum, a weak type “II” was occasionally observed. The addition of PGA₁ and PGA₂ to liver microsomes from Pb-treated rats inhibited the microcomal mediated hydroxylation of hexobarbital. The inhibition by PGA₁ was competitive; the K_i = 8.2 × 10^?4 M was found to be similar in magnitude to the K_s = 7.3 × 10^?4 M of PGA₁ observed with rat liver microsomes. These observations suggested that PGs particularly of the A series interact with the hepatic microsomal cytochrome P-450 monooxygenase system.     

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.     

Inhibition of growth in oral squamous carcinoma cells by cyclopentenone prostaglandins: comparison with chemotherapeutic agents

Four cyclopentenone prostaglandins (CPPGs) and PGE₂ caused significant dose-dependent inhibition in growth of human oral squamous carcinoma cells (SCC-15). The rank order of their potency was PGJ₂>PGA₁>16, 16-dimethyl PGA₁>PGA₂>PGE₂. In a follow-up experiment it was found that the mean per cent inhibition in cell growth by PGJ₂ and Δ¹²-PGJ₂ at 10⁻⁵ M was 61.22 and 63.81, while that of 5-fluorouracil and methotrexate was 36.67 and 38.86, respectively. Δ¹²-PGJ₂ and PGJ₂ induced significant dose-dependent inhibition in nuclear DNA synthesis (i.e. cell proliferation). Combining vitamin E succinate with lower concentrations of CPPGs enhanced significantly their inhibitory effect on nuclear DNA synthesis of cancer cells.     

Alteration in morphology and induction of glutamine synthetase in rat glioma C6BU-1 cells cultured with prostaglandin D2-supplemented media

We evaluated the effects of prostaglandins (PGs) on rat glioma C6BU-1 cells by supplementing the culture media with PGs. In the medium containing PGD₂ (15 or 20 μM), the glial cells showed altered morphology from an elongated fibroblastic form to a spreading multipolar one within 24 h, and their growth rate was suppressed to half of that of control cultures. In these cultures, the specific activity of glutamine synthetase (GS) increased approximately twofold within 48 h in comparison to the value for vehicle-treated controls. Simultaneous treatment with actinomycin D or cycloheximide completely blocked the PGD₂-elicited increase in GS specific activity, suggesting that the increase was due to de novo synthesis of the enzyme. PGD₂-like prostanoids such as PGD₁ and 9-deoxy-Δ⁹, Δ¹²-13,14-dihydro-PGD₂ (Δ¹²-PGJ₂), when added to the culture medium, mimicked the actions of PGD₂ on the C6BU-I cells, though their effective concentrations were not necessarily identical. PGs of the E- and F-series had almost no discernible effect on the glioma. These results might imply a possibility that PGD₂ plays a regulatory effect in growth and/or differentiation of rat glioma C6BU-1 cells.     

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.     

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.     

Novel prostaglandin D2-derived activators of peroxisome proliferator-activated receptor-γ are formed in macrophage cell cultures

Incubation of RAW 264.7 murine macrophages with 9,15-dihydroxy-11-oxo-, (5Z,9α,13E,15(S))-Prosta-5,13-dien-1-oic acid [prostaglandin D₂ (PGD₂)] induced formation of considerable peroxisome proliferator-activated receptor-γ (PPARγ) activity [Nature 391 (1998) 79]. Because PGD₂ itself is a poor PPARγ ligand, we incubated RAW 264.7 macrophage cultures with prostaglandin D₂ for 24 h and studied the ability of the metabolites formed to activate PPARγ. PGD₂ products were extracted and fractionated by reverse phase high-performance liquid chromatography. Chemical identification was achieved by UV spectroscopy, gas–liquid chromatography/mass spectrometry and chemical syntheses of reference compounds. PGD₂ was converted to eight products, six of which were identified. Ligand-induced interaction of PPARγ with steroid receptor coactivator-1 was determined by glutathione-S-transferase pull-down assays and PPARγ activation was investigated by transient transfection of RAW 264.7 macrophages. In addition to the previously known ligand 11-oxo-(5Z,9,12E,14Z)-Prosta-5,9,12,14-tetraen-1-oic acid (15-deoxy-Δ^12,14-PGJ₂), a novel PPARγ ligand and activator viz. 9-hydroxy-11-oxo-, (5Z,9α,12E,14Z)-Prosta-5,12,14-trien-1-oic acid (15-deoxy-Δ^12,14-PGD₂) was identified. The biological significance of these results is currently under investigation.     

Selective,potent PPARγ agonists with cyclopentenone core structure

A series of analogues of the PPARγ ligand 15-deoxy-Δ^12,14-PGJ₂ have been synthesized by functionalization of a 5-alkyl-4-hydroxycyclopentenone core structure obtained by Piancatelli rearrangement of precursor furylcarbinol. Transient transactivation assays indicate that analogues 18 and 20 are selective nanomolar agonists of PPARγ. This subtype selectivity is lost in derivatives (23, 24) with an alkynyl (oct-1-yn) chain at the C3 position, although the cyclopentenone derivative with cis relative configuration (23) showed greater affinity for PPARα.     

Presence of prostaglandins E2 and A2 in canine gastric secretions

The presence of prostaglandins (PGs) was determined in gastric juice obtained from 3 conscious dogs, provided with a chronic gastric fistula. Outputs of acid (mequiv min^?1) and PGs (pg min^?1) were measured in gastric secretions stimulated by pentagastrin (100 or 200 ng kg^?1min^?1). Prostaglandin activity was estimated, after extraction and thin layer chromatography, by radioimmuno-assay of the PGB formed by treatment with alkali. Tritiated PGs were added to gastric juice for the purpose of correcting for PGs recovery. Using this method, the minimum mass of PGB which could be satisfactorily distinguished from zero was 25 pg. Prostaglandins A₂ and E₂ were present in pentagastrin-activated gastric secretions and averaged (mean ± SE, n = 8) 200.7 ± 18.1 and 260.1 ± 18.0 pg min^?1 respectively. The identity of PGA₂ and PGE₂ was confirmed by gas liquid chromatography combined with mass spectrometry. The amount of PGE₂ converted to PGA₂ during extraction, separation and conversion procedures was estimated from the amount of [³H] PGA₂ found when only [³H] PGE₂ had been added to a sample of gastric juice and averaged 14.5% ± 2.0. Our preliminary results support the possibility that PGE₂ and PGA₂ may be of physiological importance in the regulation of canine gastric secretions.     

Prostaglandin-mediated inhibition of noradrenaline release: III. Separation of prostaglandins released from stimulated hearts and analysis of their neurosecretion inhibitory capacity

Isolated rabbit hearts were infused with ¹⁴C-arachidonic acid and subjected to sympathetic nerve stimulation. Prostaglandins in the cardiac effluent were extracted and separated using thin layer chromatography. Other hearts were infused with un-labelled arachidonic acid and the effluent was assayed for neurosecretion inhibitory capacity on the field-stimulated guinea pig vas deferens, and for anti-aggregatory activity on ADP-induced platelet aggregation. PGs in the effluent from hearts infused with un-labelled arachidonic acid were extracted and separated on TLC, and the different fractions were assayed for neurosecretion inhibitory activity.Sympathetic nerve stimulation after preincubation with ¹⁴C-AA elicited outflow of four different peaks of ¹⁴C-labelled PGs: one chromatographing close to PGF_2α (probably mainly 6-keto-PGF_1α), and three peaks corresponding to PGA₂/PGB₂, PGD₂, and PGE₂ respectively. The cardiac interstitial effluent contained anti-aggregatory material which was inactivated by heat treatment, and thus probably identical to PGI₂. The cardiac effluent also contained material with neuro-secretion inhibitory activity, which was resistant to heat treatment. Fractional assay of the TLC separated cardiac effluent demonstrated that the neurosecretion inhibitory activity chromatographed with PGE₂ only.It has earlier been observed that endogenous PGs inhibit trans-mitter release in sympathetically stimulated organs. On the basis of the current data we suggest that PGE₂ is the only physiological inhibitor of sympathetic transmitter release.     

Influences of prostaglandins on electrophoretic mobility and aggregation of rabbit platelets

Influences of prostaglandin(PG)s on electrophoretic mobilities and aggregation of rabbit platelets were studied. The PGs studied (PGI₂, PGE₁, PGD₂, PGE₂, PGF_2α, PGA₂ and PGA₁) had no effect on platelet electrophoretic mobility. However, both PGE₁ and PGI₂ in 0.3 and 3.0 μM inhibited ADP-induced aggregation and ADP-induced decrease in the mobility. PGD₂ in 0.3 and 3.0, and PGE₂ in 30 μM inhibited the aggregation but did not depress the ADP-induced decrease in the mobility. PGF_2α, PGA₂ and PGA₁ had no effect on the decrease in electrophoretic mobility and on the aggregation caused by ADP.     

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 inhibit testosterone secretion by mouse testes in vitro

Production of testosterone (T) by decapsulated mouse testes in vitro was significantly inhibited by adding prostaglanain (PG) A₁, PGA₂ or PGE₁ to the incubation medium. Prostaglandin A₁ at a concentration of 10^?6M inhibited T production in this system both in the presence of moderate amounts of hCG (12.5 or 25.0 mIU/ml), and in the absence of gonadotropins. However, in the presence of very high levels of hCG (125.0 mIU/ml), all PGs tested appeared to have had a slight potentiating effect on T production when added in concentrations ranging from 10^?7 to 10^?5M, and the inhibition of T accumulation in the medium was consistently observed only when the concentration of PGs was increased to 10^?3M. These results suggest that a direct effect of PGs on testicular steroidogenesis may account for, or contributes to, the decrease in peripheral T levels observed after administration of PGs in vivo.     

Comparison of the pulmonary vascular response to prostaglandin A1, prostaglandin F2α and angiotensin II; Metabolism of PGA1 in lung

A rabbit lung preparation, perfused $\text{in vitro}$, was used to examine pulmonary metabolism of prostaglandin A₁ (PGA₁) and to compare the vasoconstrictor actions of PGA₁, prostaglandin F_2α (PGF_2α) and angiotensin II. PGF_2α caused significantly more, and angiotensin II significantly less, vasoconstriction than did an equimolar concentration of PGA₁. Of three likely PGA₁ metabolites only 15-keto-PGA₁ had any significant vasoconstrictor action. Furosemide and aminophylline (10^?3 M) reduced PGA₁, PGF_2α or angiotensin II-induced vasoconstruction. Diphloretin phosphate potentiated the vascular effect of angiotensin II. Furosemide (10^?3 M) and DPP (9.5 × 10^?6 M) significantly reduced pulmonary metabolism of PGA₁ while aminophylline (10^?3 M) had no effect on this process. Perfusion of the lungs with a hypoxic medium had no effect on PGA₁ metabolism.     

15-deoxy-Δ12, 14-prostaglandin J2 enhances anticancer activities independently of VHL status in renal cell carcinomas

Renal cell carcinoma (RCC) is relatively resistant to chemotherapy and radiotherapy. Clear cell RCC (ccRCC) accounts for the majority of RCC, which have mutations or epigenetic silencing of the von Hippel–Lindau (VHL) gene. VHL-positive Caki-2 cells are killed by an endogenous anticancer substance, 15-deoxy-Δ^{12, 14}-prostaglandin J₂ (15d-PGJ₂). The MTT reduction assay reflecting mitochondrial succinate dehydrogenase activity was employed for assessment of cell viability. We confirmed anticancer activities of camptothecin (topoisomerase I inhibitor), etoposide (topoisomerase II inhibitor), doxorubicin (topoisomerase II inhibitor) in VHL-positive Caki-2 cells. Combination of topoisomerase inhibitors with 15d-PGJ₂ exhibited the synergistic effect in VHL-positive Caki-2 cells. However, 15d-PGJ₂ did not increase cytotoxicities of topoisomerase inhibitors on VHL-negative 786-O cells. In addition, the 15d-PGJ₂-enhanced antitumor activity of topoisomerase inhibitors was detected in neither VHL-positive nor VHL-negative RCC4 cells. Our finding indicated that 15d-PGJ₂ enhanced the antitumor activity of topoisomerase inhibitors independently of VHL.     

Suppression of adipogenesis program in cultured preadipocytes transfected stably with cyclooxygenase isoforms

Prostaglandins (PGs) are known to play a variety of roles in adipocytes and precursor cells, which have the arachidonate cyclooxygenase (COX) pathway to generate several series of PGs at different stages of life cycle of adipocytes. To gain a unique insight into the specific roles of the COX isoforms during the life cycle of adipocytes, 3T3-L1 preadipocytes were stably transfected with a mammalian expression vector harboring either cDNA coding for murine COX-1 or COX-2. The cloned stable transfectants with COX-1 or COX-2 exhibited higher expression levels of their corresponding mRNA and proteins, and greater production of PGE₂ upon stimulation with free arachidonic acid or A23187 than the parent cells and the transfectants with vector only. However, either type of transfectants brought about the marked reduction in the accumulation of triacylglycerols after the standard adipogenesis program. Unexpectedly, aspirin or other COX inhibitors at different phases of life cycle of adipocytes failed to reverse the reduced storage of fats. The transfectants with COX-2 were sensitive to exogenous 15-deoxy-Δ^12,14-PGJ₂ (15d-PGJ₂) and troglitazone as peroxisome proliferator-activated receptor γ (PPARγ) agonists during the maturation phase for restoring the adipogenesis. By contrast, the transfectants with COX-1 were much less sensitive, which was reflected by much lower gene expression levels of PPARγ and the related adipocyte-specific markers. Taken together, the results suggest that the sustained overexpression of either COX-1 or COX-2 resulted in the interference of adipogenesis program through a PG-independent mechanism with a different mode of action of COX isoforms.     

A PPAR-γ ligand, 15-deoxy-Δ12,14-prostaglandin J2, inhibited gastric mucosal injury induced by ischemia–reperfusion in rats

Abstract

Introduction: Recent studies have demonstrated the anti-inflammatory action of 15-deoxy-Δ12,14-prostaglandin J₂ (15d-PGJ₂), a derivative of the PGD₂ metabolic pathway. Acute inflammation, including neutrophil activation, plays a critical role in the pathogenesis of ischemia–reperfusion (I/R). The aim of the present study was to determine the effect of 15d-PGJ₂ on I/R-induced gastric mucosal injury in rats.

Methods: Gastric mucosal damage was induced in male Wistar rats by clamping the celiac artery for 30 min followed by reperfusion. 15d-PGJ₂ (0.01–1.0 mg/kg) was given to the rats intraperitoneally 1 h before the vascular clamping. The area of gastric mucosal erosions (erosion index) was measured. Thiobarbituric acid reactive substances (TBARS) and tissue-associated myeloperoxidase (MPO) activity were measured in the gastric mucosa as indices of lipid peroxidation and neutrophil infiltration. The expression of tumor necrosis factor-α (TNF-α) in gastric mucosa was measured by ELISA. In addition, to elucidate whether the protective effects of 15d-PGJ₂ are related to the activation of the PPAR-γ receptor, we also investigated the effects of a PPAR-γ antagonist, GW9662.

Results: After 60 min of reperfusion, the area of gastric erosion index had significantly increased from the mean basal levels. The increase in the erosion index was significantly inhibited by pretreatment with 15d-PGJ₂ in a dose-dependent manner. On the other hand, GW9662 reversed the protective effect of 15d-PGJ₂. The concentration of TBARS and MPO activity in the gastric mucosa were both significantly increased after I/R, and pretreatment with 15d-PGJ₂ significantly reduced these increases. The TNF-α content was significantly higher in the I/R group than in the sham-operated group. However, the increase in TNF-α was significantly inhibited by pretreatment with 15d-PGJ₂.

Conclusions: 15d-PGJ₂ significantly inhibited the severity of acute gastric mucosal injury induced by I/R in rats through PPAR-γ-dependent mechanisms. This effect may be due, in part, to a reduction in the infiltration of neutrophils into the gastric mucosa, possibly via the inhibition of inflammatory cytokine.