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 A1 induces differentiation in friend erythroleukemia cells

The effect of different prostaglandins and prostaglandin-metabolites on the growth and differentiation of Friend erythroleukemia cells (FLC) was evaluated. The prostaglandin-metabolites, thromboxane B₂ and 6-keto PGF₁α, were completely inactive, while PGE₁ inhibited slightly and PGF₂α stimulated the replication of FLC. PGA₁ was found to be the most active compound. It profoundly inhibited the replication of both DMSO-treated and undifferentiated FLC. Most importantly, PGA₁ alone induced differentiation in FLC, stimulating hemoglobin production over a five-day period. PGA₁-stimulated differentiation was completely suppressed by the addition of 10⁻⁶M hydrocortisone. Finally, treatment of DMSO-differentiated cells with PGA₁ (but no DMSO) prevented the return to the undifferentiated state.     

Effect of prostaglandins on H-thymidine uptake into human epidermal cells

Prostaglandins A₂, B₁, E₁, E₂, F_1α and F_2α were added to cultures of human epidermal cells (keratinocytes) for 24 hours at 37°C, and the effects on ³H-thymidine uptake into DNA was measured. At 70 μg/ml all prostaglandins tested except PGF_2α inhibited the uptake of ³H-thymidine greater than 50%. However, at 35 μg/ml, PGA₂ and PGB₁ were the only two prostaglandins to show significant inhibition, 96% and 51% respectively. At 17.5 μg/ml only PGA₂ caused substantial inhibition, 68%. In order to determine if the PGA₂ action was mediated by membrane receptors propranolol, phentolamine, metiamide and prostynoic acid were added in conjunction with PGA₂. None of the above receptor antagonists were able to reduce the PGA₂-induced inhibition of ³H-thymidine uptake. These results indicate that the pre-incubation of human keratinocytes with prostaglandins for 24 hours results in a decrease of ³H-thymidine incorporation in DNA. The precise mechanism of action is unknown at this time.     

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 effect of PGA1on the immune response in B-16 melanoma-bearing mice

This study evaluated the effects of PGA₁ on B-16 melanoma-bearing mice. Intraperitoneal injection of PGA₁ (10 μg/day) significantly inhibited the rate of melanoma growth measured both as delay in the rate of appearance and decrease in the tumor volume. In contrast to the diluent control-treated mice, by 17 days, less than half of the PGA₁ — treated animals developed measurable (>2mm) subcutaneous tumors. In addition to its effect on tumor size, PGA₁ was also effective in stimulating both the humoral and cellular components of the immune response. B-16 tumor-bearing mice were shown to be immunosuppressed, in that they had decreased anti-sRBC hemagglutinin titers, decreased splenic plaque-forming cells, suppressed delayed hypersensitivity responses, and delayed rejection of skin allografts from BALB/c mice. Although, PGA₁ had relatively little effect on normal mice, this prostaglandin substantially improved all these immunologic parameters in tumor-bearing animals.     

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.     

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.     

Metabolism of prostaglndin A. II. Isolation, characterization, and synthesis of PGA1 renal.

Since the renal cortex has recently been shown to be a major site of prostaglandin A₁ (PGA₁) metabolism, studies were undertaken to isolate and characterize the major metabolites. Homogenates of rabbit cortex (500g) were incubated with ³H-PGA₁ (50mg) in the presence of NAD⁺ (50mg). Acidic lipid extracts were subjected to linear gradient silicic acid chromatography. Six radioactive peaks were recovered, of which peak 4 was unconverted PGA₁. The major metabolites (1,3) were further subjected to reversed phase partition chromatography and TLC with and without silver nitrate. Three PGA₁ analogs were then synthesized via oxidation of the secondary alcohol group at C-15 by manganese dioxide (15-keto-PGA₁). The second compound was synthesized by hydrogenation of 15-keto-PGA₁ (15-keto 13, 14-dihydro PGA₁). The third compound (13, 14-dihydro PGA₁) was obtained by direct catalytic hydrogenation of PGA₁. Purification of these substances were achieved by a combination of silicic acid and thin layer chromatography. It was found that metabolite 1 cochromatographed on TLC (AgNO3) with synthesized 15-keto 13, 14-dihydro PGA₁. Both compounds were 100 times less potent than PGA₁ in lowering rat blood pressure. Metabolite 3 cochromatographed on TLC (AgNO3) with synthesized 13, 14-dihydro PGA₁. Both were as potent as PGA₁ in lowering rat blood pressure. Metabolites 1 and 3 absorbed UV at 221 nm but not at 280 nm following alkali treatment. These studies suggest that rabbit renal cortex metabolizes PGA₁ to what appears to be biologically active 13, 14-dihydro PGA₁ and biologically inactive 15-keto 13, 14-dihydro PGA₁. It remains possible that the hypotensive effect of PGA₁ is the result of its conversion to its biologically active 13, 14-dihydro derivative.     

A biotinylated analog of the anti-proliferative prostaglandin A1 allows assessment of PPAR-independent effects and identification of novel cellular targets for covalent modification

The cyclopentenone prostaglandin (cyPG) PGA₁ displays potent anti-proliferative and anti-inflammatory effects. Therefore, PGA₁ derivatives are being studied as therapeutic agents. One major mechanism for cyPG action is the modification of protein cysteine residues, the nature of the modified proteins being highly dependent on the structure of the cyPG. Biotinylated cyPGs may aid in the proteomic identification of cyPG targets of therapeutic interest. However, for the identified targets to be relevant it is critical to assess whether biotinylated cyPGs retain the desired biological activity. Here we have explored the anti-inflammatory, anti-proliferative and cell stress-inducing effects of a biotinylated analog of PGA₁ (PGA₁-biotinamide, PGA₁-B), to establish its validity to identify cyPG–protein interactions of potential therapeutic interest. PGA₁ and PGA₁-B displayed similar effects on cell viability, Hsp70 and heme oxygenase-1 induction and pro-inflammatory gene inhibition. Remarkably, PGA₁-B did not activate PPAR. Therefore, this biotinylated analog can be useful to identify PPAR-independent effects of cyPGs. Protein modification and subcellular distribution of PGA₁-B targets were cell-type-dependent. Through proteomic and biochemical approaches we have identified a novel set of PGA₁-B targets including proteins involved in stress response, protein synthesis, cytoskeletal regulation and carbohydrate metabolism. Moreover, the modification of several of the targets identified could be reproduced in vitro. These results unveil novel interactions of PGA₁ that will contribute to delineate the mechanisms for the anti-proliferative and metabolic actions of this cyPG.     

Uptake and metabolism of prostaglandins by isolated perfused lung: Species comparison and the role of plasma protein binding

We have investigated the uptake and subsequent metabolism of the prostaglandins (PGs) PGE₁, PGA₁, and PGB₁ by rat, guinea pig and rabbit isolated perfused lungs (IPL). Significant species differences were not observed in the uptake or metabolism of any PG on passage through the IPL. However, differences in the uptake of PGA₁ and PGB₁ and in the metabolism of PGA₁ were observed with a given species when the composition of the perfusion medium was varied. The IPL removed minimal amounts (<20% of the supply rate) of PGA₁ and PGB₁ from the circulation when the perfusate contained 4.5% bovine serum albumin (BSA). In the absence of BSA, however, both PGA₁ and PGB₁ were substantially removed from circulation (～53% of the supply rate) and PGA₁ was also metabolized. The composition of the perfusate had no effect on the uptake and metabolism of PGE₁ which was always taken up and metabolized to a greater extent than was PGA₁ and PGB₁. Thus, the apparent species differences previously reported for the pulmonary biotransformation of PGA can result from differences in the perfusion medium used. Our data suggest that both plasma protein binding and a transport system play important roles in determining the selectivity of the uptake of PGs by the lung.     

Inhibition of DNA synthesis in Harding-Passey melanoma cells by prostaglandins A1 and A2: comparison with chemotherapeutic agents.

PGA₁ and PGA₂ significantly depressed melanoma cell DNA synthesis and cell proliferation in a dose related fashion. Inhibition of DNA synthesis was rapid in onset (0.5–1 hr) and sustained (12 hr). This was not due to general cytotoxicity or depression of substrate uptake. Comparison with known cancer chemotherapeutic agent revealed PGA₁ and PGA₂ effectiveness on a molar basis exceeded that of Adriamycin, cyclophosphamide and hydroxyurea. Actinomycin D, Mutamycin and 5-fluorouracil were more potent than PGA₁ and PGA₂ but consideration of their toxicities may outweigh this point. The findings suggest that the A series prostaglandins or their analogs may be efficacious in cancer chemotherapy.     

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.     

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.     

Induction of renin release by exogenous prostaglandins in hyporeninemic hypoaldosteronism

A deficiency in renal prostaglandin synthesis has been proposed as the cause of the syndrome of hyporeninemic hypoaldosteronism. To determine if renin release could be stimulated by pharmacologic infusions of PGA₁, we infused PGA₁ 0.075 to 0.60 μg/kg/min to nine patients with the syndrome. Total renal PGE production as measured by urinary PGE excretion was normal (650 ± 169 vs 400 ± 55 ng/24hr in normal subjects). Renin (PRA) was markedly depressed in all patients despite stimulation with upright posture and furosemide (1.0 ± 0.4 vs 9.3 ± 0.7 ng/ml/hr, p<0.001). But in two patients PGA₁ induced an increase in renin similar to that of normal subjects. PRA increased to a lesser degree in two other patients and plasma aldosterone slightly increased. Five showed no response. Infusions of nitroprusside in doses and duration that mimicked the hypotensive effects of PGA₁ failed to increase PRA or aldosterone. The data suggest that total renal PGE production is normal in patients with the syndrome of hyporeninemic hypoaldosteronism. Although orthostasis, furosemide and nitroprusside do not increase renin, prostaglandin A₁ infusion appears to be a potent stimulus to renin release in some of the patients.     

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.     

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.     

Influence of prostaglandins and thyrotropin releasing hormone (TRH) on hormone secretion and growth in wether lambs

A series of experiments were conducted in ewes and wether (castrate male) lambs to evaluate the influence of prostaglandins on secretion of anabolic hormones and to determine if repeated injections of prostaglandin (PG) F₂α would chronically influence the secretion of these hormones and perhaps growth rate as well.A single intravenous injection of PGA₁ and PGB₁ (100 μg/kg) exerted no significant (P > .10) influence on plasma concentrations of prolactin (PRL), growth hormone (GH) or thyrotropin (TSH) in ewes. PGA₁, but not PGB₁, stimulated an increase in the plasma concentration of insulin. Infusion of PGF₂α for 5.5 hr into ewes resulted in increased (P < .05) plasma concentrations of both GH and PRL while TSH and insulin were not significantly influenced. Prostaglandin F₂α, when injected subcutaneously into wether lambs (10 mg twice weekly) stimulated (P < .05) plasma GH concentrations after the first injection, but not after 3 weeks of treatment. Changes in plasma PRL or TSH were not observed consistently in the lambs treated chronically with PGF₂α or TRH.Prostaglandin F₂α, in the present studies, and PGE₁ in previously reported studies (1–3), has been demonstrated to be stimulatory to the secretion of PRL and GH. In contrast, PGA₁ and PGB₁, which lack an 11-hydroxyl group, failed to influence the secretion of either PRL or GH. It would, therefore, appear that the 11-hydroxyl group is a structural requirement for prostaglandins to influence the secretion of these two hormones in sheep.Treatment with thyrotropin releasing hormone (TRH), alone or in combination with PGF₂α, significantly (P < .05) increased growth rate (average daily gains) while PGF₂α did not, despite the fact that both compounds exerted similar effects on plasma GH.     

Comparison of the effects of prostaglandins E1 and A1 on coronary occlusion induced arrhythmia

The present study compares the effects of PGE₁ and PGA₁ on ventricular arrhythmias following coronary artery occlusion. The left anterior descending coronary artery (LAD) was occluded abruptly in 55 cats anesthetized with α-chloralose. Lead II of the ECG along with arterial blood pressure were monitored for one hour after occlusion. Either vehicle or prostaglandin was infused into the left atrium (LA) or femoral vein (IV) 15 min prior to and for 1 hour after LAD occlusion at a rate of 0.15 ml/min. Prostaglandin was infused at either a high dose (1.0 μg/kg/min) or a low dose (0.1 μg/kg/min). Infusion of either PGE₁ or PGA₁ produced a marked fall in blood pressure and heart rate which returned toward control before occlusion. Abrupt occlusion of the LAD produced ventricular arrhythmia in all cats ranging from ventricular premature beats to ventricular fibrillation (VF). The control animals had a 38% incidence of VF. VF occurred in 75% of the animals in which PGE₁ was administered into the LA at either the high or low dose while the occurrence in those administered PGA₁ was 67% and 50%, respectively. Intravenous administration of the high dose of PGE₁ or PGA₁ resulted in VF in 13% and 67% of the animals after LAD occlusion, respectively. These data indicate that the IV administration of PGE₁ may protect the heart from VF while the infusion of PGE₁ or PGA₁ into the LA may enhance VF after LAD occlusion.     

Effects of A and B prostaglandins on cAMP, cortisol, and aldosterone production by the human adrenal.

PGA₁ and PGA₂ (10, 100 μg/ml) significantly increased human adrenal cAMP levels and cortisol output but low doses (1 μg/ml) depressed both parameters. Only 1 μg/ml PGA₁ significantly increased aldosterone output while higher doses depressed same. The low PGA₂ dose (1 μg/ml) depressed aldosterone output. The glucocorticoid and mineralocorticoid outputs appear to be inversely modulated by prostaglandins. PGB₁ and PGB₂ behaved similarly to E type prostaglandins. However, like PGA₁, 1 μg/ml of PGB₁ or PGB₂ significantly increased aldosterone output. Higher doses were ineffective. The present findings reveal an increased complexity of prostaglandin modulation of cyclic nucleotides and steroid output.     

A comparison of the diuretic effects of prostaglandin A1, sodium ethacrynate,and placebo

The comparative diuretic activity of prostaglandin A₁ (PGA₁), sodium ethacrynate, and placebo was assessed in healthy volunteers. Known active dosages of the various agents were administered as intravenous infusions. In the first four hours after the start of the infusion, the ratio of urine output to liquid intake was higher for sodium ethacrynate than either PGA₁ or placebo. Sodium ethacrynate was the only treatment which significantly changed urinary electrolyte excretion. At the doses administered in this study, sodium ethacrynate was more effective as a diuretic than either PGA₁ or placebo. PGA₁ and placebo exerted nearly identical effects upon the variables studied. This study suggests that PGA₁ may be effective only in the presence of altered distribution of blood flow in renal disturbances where tubular functions are involved in maintaining electrolyte excretion.