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Prostaglandin and Thromboxane Synthesis by Human Intracranial Tumors1

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Prostaglandin and Thromboxane Synthesis by Human Intracranial Tumors1 (CANCER RESEARCH 49, 1505-1508, March 15. 1989] Prostaglandin and Thromboxane Synthesis by Human Intracranial Tumors1 Maria Grazia Castelli, Chiara Chiabrando,2 Roberto Fanelli, Luciana Martelli, Giorgio Butti, Paolo Gaetani, and Pietro Paoletti ¡MinilodiRicerche Farmacologiche "Mario Negri", Via Eritrea 62, 20157, Milan, Italy [M. G. C„C.C., R. F., L. M.J; and thèDipartimento di Chinirgia, Sezione di ClínicaNeurochirurgica, Centro "Enrico Grossi-Paoletti" per lo Studio e il Trattamento delle Neoplasie del Sistema Nervoso, Università ' di Pavía,27100 Paria, Italy {G. B.. P. G., P. P.] ABSTRACT daily TXÃœ2(the hydrolysis product of TXA2) and PGE2, are produced by short-term cell cultures of human meningiomas Prostaglandin (PC) and thromboxane (TX) production by homogenates and gliomas (23). Using a highly selective method such as high of human intracranial tumors (33 gliomas, 32 meningiomas, six brain métastases)and "normal" brain (n = 26) from tumor-bearing patients resolution gas chromatography-mass spectrometry to measure was studied. l'<;(•',..,PGE2,PGD2, 6-keto-PGFi„(the hydrolysis product the five stable cyclooxygenase metabolites of AA, we have of PGIj) and TXB2 (the hydrolysis product of TXA2) were determined by preliminarily reported on prostanoid production by homoge high-resolution gas chromatography-mass spectrometry after ex vivo nates of human meningiomas and gliomas (24). In that study metabolism of endogenous arachidonic acid. Prostanoid profiles (relative we found high synthesis capacity and characteristic metabolic abundance of each metabolite) were different for gliomas and meningio profiles for each tumor, as we had already shown for murine mas, but similar for gliomas and their nontumoral counterpart, i.e., tumors such as Lewis lung carcinoma and M5076 ovarian "normal11 brain. Mean overall prostanoid production was significantly reticulosarcoma (6, 7). higher in gliomas (539 ±95) and meningiomas (523 ±69) than in We have now extended our observation to a larger number "normal11brain (198 ±23). Prostanoid synthesis significantly increased of patients with gliomas, meningiomas, and brain métastases. with anaplastic grade (glioblastomas > anaplastic astrocytomas > slow- growing astrocytomas > "normal11brain), while profiles did not substan We also present a comparative view of AA metabolic profiles in apparently nonpathological brain tissue, considered as the tially change (TXB2 was the most and 6-keto-PGFia the least abundant "normal" counterpart for gliomas. Correlations between pros product). Meningioma profiles showed no marked prevalence of any particular metabolite and no major differences between histológica! tanoid production and other biochemical and proliferative char subgroups. All brain métastasesfromdifferent carcinomas (n = 5) showed acteristics of each tumor are currently under investigation. a prevalence of TXB2 and PGE2 and very low PGD2 synthesis. MATERIALS AND METHODS INTRODUCTION Standards. PGF2„,PGF2„-D4,PGE2, PGE2-D4, PGD2, 6-keto- PGF,a, 6-keto-PGF1(I-D4, and TXB2 were kindly supplied by Dr. J. Several experimental and human tumors synthesize prosta- Pike of The Upjohn Co. (Kalamazoo, MI). TXB2-D8 was a kind gift of noids (1-5), which can be increasingly produced during tumor development (6-9). These cyclooxygenase metabolites of AA3 Dr. G. Galli (Università degli Studi di Milano, Milan, Italy). (unirai Samples. Specimens of intracranial tumors (33 gliomas, 32 (PGF2o, PGE2, PGD2, PGI2, and TXA2) may influence physio- meningiomas, and six métastases)werecollected during surgery. Spec pathological processes related to tumor development and dis imens of brain tissue (n = 26) were collected from some of these semination (e.g., cell proliferation, migration and adherence, patients who underwent lobectomy as part of a correct surgical treat host immunity, angiogenesis, hemostatic mechanisms) (1-5, ment. We will hereafter use the term "normal brain*1 to indicate a 10, 11). Cyclooxygenase or thromboxane synthetase inhibitors specimen which: (a) has been collected from tumor-bearing patients in may reduce tumor growth and metastasis, although there are an area apart from the tumor; (¿>)hasbeen proven free of tumoral cells controversial reports on this issue (1, 2, 7, 12-16). by histológica!examination of a section immediately adjacent to that used for AA metabolism. The capacity of tumors to grow, disseminate, and influence Ex Vivo Metabolism of Endogenous AA. The ex vivo evaluation of host homeostasis has in some cases been related to the produc endogenous AA cyclooxygenase metabolism has been described in tion of elevated amounts of some prostanoids. In human cancer, previous papers (24, 25). Briefly, samples (0.1-2 g) were rapidly frozen high content or synthesis capacity of selected prostanoids have in liquid nitrogen at the time of surgery and stored (-80°C) until been by some authors related to high met astatic potential (17), analysis. The frozen samples were directly homogenized in 10 volumes tumor size and met astatic spread (18), and irresponsiveness to of 50 mM phosphate buffer (pH 7.4). The homogenates were incubated for 15 min at 37°C,then spun at 15,000 x g for 45 min. The samples chemotherapy (19). Others found that although prostanoid were maintained at 4°Cduring homogenization and centrifugation. The synthesis of selected products was higher in malignant than in supernatants were kept at —¿20"Cuntilanalyzed. This procedure allows benign lesions, this did not correlate either with metastatic spread (20), or with clinical staging and prognosis (21), and the ex vivo metabolism of endogenous AA to give reproducible meta bolic profiles as reported previously (24, 25). was not associated with favorable prognosis (22). Quantitäten of AA Metabolites by HRGC-MS. The five stable me Data is scant regarding prostanoid production by human tabolites of AA (PGF2a, PGE2, PGD2, 6-keto-PGF,0, and TXB2) were intracranial tumors. Elevated amounts of prostanoids, espe- assayed by HRGC-MS and quantified by stable isotope dilution assay. Supernatants were spiked with deuterium-labeled internal standards Received 7/28/88; revised 11/23/88; accepted 11/30/88. The costs of publication of this article were defrayed in part by the payment (PGF2a-D4, PGE2-D4, 6-keto-PGF,a-D4, and TXB2-D8, 30 ng each; of page charges. This article must therefore be hereby marked advertisement in PGE2-D4 was used as internal standard for PGD2). Samples were accordance with 18 U.S.C. Section 1734 solely to indicate this fact. acidified (pH 3.5) with l N HC1 and applied to a Baker ( ',„SPEcolumn 'This work was supported in part by a grant from the Italian Ministry of (J. T. Baker Chemicals, Phillipsburg, NJ) prewashed with methanol Education (Rome, Italy, 1986) and from the National Research Council (87.01204.44). and 1 mN HC1. After washing with water and petroleum ether (10 ml 2To whom requests for reprints should be addressed. each), the column was eluted with methyl formate (1.5 ml). The eluate 3The abbreviations used are: AA, arachidonic acid; PG, prostaglandin; TX, was dried under an air flow and then derivatized to give the pentafluo- thromboxane; PGF!o-D4, 3,3'A,4'-*HrPGFia; PGEj-D4, 3,3',4,4'-2H,-PGE2; 6-keto-PGF,„-D4, 3,3',4,4'-2H4-6-keto-PGF,„; TXB¡-D8,5, 6, 8, 9, 11, 12, 14, robenzyl ester derivatives of AA metabolites and their deuterated ana 15-2H8-TXBi; HRGC-MS, high resolution gas chromatography-mass spectrom logues. The reaction was carried out by adding 30 n\ of a mixture of etry; El, electron impact; MCI, negative ion chemical ionization. pentafluorobenzyl bromide (Fluka, Buchs, Switzerland) and acetonitrile 1505 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1989 American Association for Cancer Research. PROSTANOID SYNTHESIS BY HUMAN BRAIN TUMORS (1:2, v/v) and 10 p.\diisopropylethylamine (Fluka, Buchs, Switzerland) as preliminarily reported with fewer specimens from glioma- to the residue and heating at 40°Cfor5 min. The sample was thoroughly bearing patients (24). In the present set of data we obtained dried and the procedure repeated. The dried residue was then resus- similar profiles with "normal" brain from patients bearing pended in chloroform and applied to a Silica Bond-Elut column (An- gliomas, meningiomas, and métastases. alytichem International, Harbor City, CA), prewashed with chloroform. Glioma profiles were strikingly similar to those of their After washing with chloroform (10 ml), the column was eluted with 1.5 nonpathological counterpart, i.e., "normal" brain. This might ml chloroform:methanol (9:1, v/v). The eluate was taken to dryness under an air stream. indicate that the pattern of AA metabolism in tumoral cells is AA metabolites and their deuterated analogues were converted to strongly influenced by the enzymatic endowment of the tissue the methyloxime, trimethylsilyl ether derivatives with 50 ¿tlmethylox- from which they originate, rather than being specific to neo- ime hydrochloride (Pierce Chemicals, Rockford, IL) (30 min at 60°C) plastic cells in general. Mean synthesis capacity varied widely followed by 50 n\ bis(trimethylsilyl)trifluoroacetamide (Fluka, Buchs, in different specimens cf gliomas, possibly being influenced by Switzerland) (15 min at 60°C).AA metabolites were analyzed by factors such as tumor size, kariotypic heterogeneity, presence HRGC-MS in the selected ion-monitoring mode. All samples were of necrosis, edema, or hemorrhage. Nevertheless, average pros- analyzed in the El mode, and some selected samples were also analyzed tanoid production (539 ±95ng/g) was significantly higher than in the NICI mode to further confirm the identity of the compounds. in "normal" brain tissue (198 ±23ng/g). The increased capac A fully computerized VG TS-250 mass spectrometer equipped with a Hewlett-Packard 5890 gas Chromatograph was used. EI-MS operating ity of neoplastic cells to produce prostanoids has already been conditions were: electron energy, 22.5 eV; ionizer temperature, 200°C. seen in a number of human tumors (22, 26-29).
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