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.     

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.     

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.     

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.     

Proteomic identification of the candidate target proteins of 15‐deoxy‐delta12,14‐prostaglandin J2

15‐Deoxy‐delta12, 14‐prostaglandin J₂ (15d‐PGJ₂) is an endogenous anti‐inflammatory lipid derived from PGD₂. One potential mechanism for its activity is the covalent modification of cellular proteins, via a reactive α,β‐unsaturated carbonyl group in its cyclopentenone ring, which in turn alters protein function. In order to identify the candidate target proteins covalently modified by 15d‐PGJ₂ in human aortic endothelial cell (EC), EC was treated with biotinylated‐15d‐PGJ₂, the modified proteins extracted by Neutravidin affinity‐purification and the proteins identified by LTQ Orbitrap mass spectrometer. Classification of the 358 identified proteins was performed using PANTHER classification system ( www.pantherdb.org ), showing that the proteins mapped to metabolic process, cellular process, and transport activity. This protein data set highlights the potential for 15d‐PGJ₂ to covalently modify cellular proteins and provides a source of data that will aid further studies on the mechanism of action of this endogenous regulator of inflammation.     

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.     

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.     

Aldo-keto reductase 1C3 expression in MCF-7 cells reveals roles in steroid hormone and prostaglandin metabolism that may explain its over-expression in breast cancer

Aldo-keto reductase (AKR) 1C3 (type 5 17β-hydroxysteroid dehydrogenase and prostaglandin F synthase), may stimulate proliferation via steroid hormone and prostaglandin (PG) metabolism in the breast. Purified recombinant AKR1C3 reduces PGD₂ to 9α,11β-PGF₂, Δ⁴-androstenedione to testosterone, progesterone to 20α-hydroxyprogesterone, and to a lesser extent, estrone to 17β-estradiol. We established MCF-7 cells that stably express AKR1C3 (MCF-7-AKR1C3 cells) to model its over-expression in breast cancer. AKR1C3 expression increased steroid conversion by MCF-7 cells, leading to a pro-estrogenic state. Unexpectedly, estrone was reduced fastest by MCF-7-AKR1C3 cells when compared to other substrates at 0.1 μM. MCF-7-AKR1C3 cells proliferated three times faster than parental cells in response to estrone and 17β-estradiol. AKR1C3 therefore represents a potential target for attenuating estrogen receptor α induced proliferation. MCF-7-AKR1C3 cells also reduced PGD₂, limiting its dehydration to form PGJ₂ products. The AKR1C3 product was confirmed as 9α,11β-PGF₂ and quantified with a stereospecific stable isotope dilution liquid chromatography–mass spectrometry method. This method will allow the examination of the role of AKR1C3 in endogenous prostaglandin formation in response to inflammatory stimuli. Expression of AKR1C3 reduced the anti-proliferative effects of PGD₂ on MCF-7 cells, suggesting that AKR1C3 limits peroxisome proliferator activated receptor γ (PPARγ) signaling by reducing formation of 15-deoxy-Δ^12,14-PGJ₂ (15dPGJ₂).     

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.     

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.     

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.     

Select cyclopentenone prostaglandins trigger glutathione efflux and the role of ABCG2 transport

Electrophilic cyclopentenone prostaglandins (cyPGs), such as 15-deoxy-Δ^12,14-prostaglandin J₂ (15dPGJ₂), initiate redox-based cell signaling responses including increased intracellular glutathione (GSH) synthesis. We investigated whether cyPGs facilitated GSH efflux and if members of the ATP-binding cassette (ABC) protein family mediated the efflux. Four human cell lines were treated with 1–6 μM cyPGs for 48 h. Media and cells were harvested for GSH measurements using HPLC-EC. CyPG treatment increased extracellular GSH levels two- to threefold over controls in HN4 and C38 cells and five- to sixfold in SAEC and MDA 1586 cells and was dependent on increased GSH synthesis. Our studies show that prostaglandin D₂ and its metabolites, prostaglandin J₂ and 15dPGJ₂, specifically induce GSH efflux compared to other eicosanoids. These higher extracellular GSH levels were associated with protection from tert-butylhydroperoxide. Superarray analysis of ABC transporters suggested only ABCG2 expression had a positive relationship in the four cell types compared with extracellular GSH increases after cyPG treatment. The ABCG2 substrate Hoechst 33342 inhibited extracellular GSH increase after 15dPGJ₂ treatment. We report for the first time that ABCG2 may play a role in GSH efflux in response to cyPG treatment and may link inflammatory signaling with antioxidant adaptive responses.     

Effect of prolonged prostaglandin exposure on prostaglandin synthesis by human lung fibroblasts

Pretreatment of human lung fibroblasts with PGE₂ but not PGF_2α enhanced synthesis of prostaglandins (PGs). The effect of the pretreatment on PG synthesis was related to the concentration of PGE₂ that was added to the culture medium. Pretreatment with PGE₂ at 5 × 10⁻¹²M did not enhance PG synthesis whereas pretreatment with PGE₂ at 5 × 10⁻⁶M induced a maximal effect. Production of PGs was increased following 1 day of pretreatment with PGE₂ and was increased further following 3 days of pretreatment. The PGE₂ treated cells showed only a slight increase in the bradykinin-induced release of radioactivity from cells prelabeled with [³H]arachidonic acid but showed a dramatic increase in the bradykinin-induced synthesis of radio-labeled PGs. The conversion of free arachidonate to PGs in both intact cells and in a cell-free preparation was increased by PGE₂ pretreatment. The presence of cyclohexamide during the pretreatment did not inhibit the PGE₂-induced activation of PG synthesis. Taken together, the results indicate that pretreatment of cells with PGE₂ increased PG synthesis by augmenting the conversion of arachidonate to PGs.     

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 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.