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Prostaglandin E2 levels in human brain tumor tissues and arachidonic acid levels in the plasma membrane of human brain tumors
Cancer Letters 132 (1998) 17–21
Prostaglandin E2 levels in human brain tumor tissues and arachidonic acid levels in the plasma membrane of human brain tumors ¨ zyurt c E. Ko¨kog˘lu a,*, Y. Tu¨ter a, K.S. Sandıkc¸ı a, Z. Yazıcı b, E.Z. Ulakog˘lu a, H. So¨nmez a, E. O a
Department of Biochemistry, Cerrahpas¸a Medical Faculty, University of Istanbul, Istanbul, Turkey Department of Pharmacology, Cerrahpas¸a Medical Faculty, University of Istanbul, Istanbul, Turkey c Department of Neurosurgery, Cerrahpas¸a Medical Faculty, University of Istanbul, Istanbul, Turkey
b
Received 2 September 1997; accepted 27 April 1998
Abstract Arachidonic acid is stored in the cell membrane and released when the cell is activated by appropriate stimuli. It is the substrate for prostaglandins. Both experimental and human tumors often synthesize high levels of prostaglandins, most notably prostaglandin E2 (PGE2). Some experiments suggest that these compounds increase tumor growth through their actions on host immunocytes. In this study, 22 patients with various brain tumors and 12 control brain tissues were studied. PGE2 levels in tissue samples were measured by ELISA. Arachidonic acid levels in the plasma membrane of tissue samples were analyzed by capillary gas chromatography. The levels of PGE2 were significantly higher in gliomas (n = 10) and meningiomas (n = 7) compared with control tissues (P = 0.000 and P = 0.000, respectively). Also, PGE2 levels in meningiomas were significantly higher than in gliomas (P = 0.000). Arachidonic acid levels in the plasma membrane of gliomas (n = 9) and meningiomas (n = 6) were significantly higher than in the control tissues (P = 0.000 and P = 0.000, respectively). These results suggest that the increased production of PGE2 may suppress the immune system and play an important role in tumor growth. 1998 Elsevier Science Ireland Ltd. All rights reserved Keywords: Arachidonic acid; Prostaglandin E2; Human brain tumors
1. Introduction Polyunsaturated fatty acids, especially docosahexaenoic acid and arachidonic acid, are enriched in brain lipids [22] where they are major components of excitable membranes [2]. Arachidonic acid is stored intracellularly in membrane phospholipids and released when the cell is activated by appropriate stimuli [27]. Arachidonic acid is further metabolized in a cascade through the cyclooxygenase and lipoxygenase * Corresponding author.
0304-3835/98/$ - see front matter PII S0304-3835 (98 )0 0127-X
pathways, eventually producing such physiologically active substances as prostaglandins and leukotrienes [13]. Changes in the membrane phospholipid fatty acid composition can alter the amount of arachidonic acid contained in the cell membrane. This can affect the amount of prostaglandins [27]. Increased biosynthesis of prostaglandins, particularly of the E series, has been demonstrated in a large number of human and experimental cancers, including breast cancer [8], colon cancer [25,31], head and neck cancer [16] and lung cancer [21]. These compounds may play an important role in
1998 Elsevier Science Ireland Ltd. All rights reserved
18
E. Ko¨kog˘lu et al. / Cancer Letters 132 (1998) 17–21
tumor growth and spread [7]. The growth of many experimental tumors is suppressed by the administration of low non-toxic doses of cyclooxygenase inhibitors such as indomethacin, aspirin and piroxicam [12]. This indicates that, in many cases, tumor prostaglandin synthesis contributes positively to tumor growth. The mechanisms involved are unclear, but considerable evidence implicates prostaglandin E2 (PGE2) as an immunosuppressive molecule that acts to downregulate host antitumor reactions [9]. Little is known of the production of prostaglandins by intracranial tumors. Certain authors reported that PGE2 levels increase in brain tumors and that PGE2 is produced in the greatest amount in meningiomas [7]. By contrast, other authors reported no increased production of PGE2 in brain tumors [11]. The present study was designed to investigate the levels of arachidonic acid in the cell membrane of human brain tumors and the levels of PGE2 in brain tumor tissues.
2. Materials and methods Tumor or brain tissue was obtained from patients undergoing craniotomy at the Department of Neurosurgery, Cerrahpas¸a Medical Faculty. Twenty-two tumor samples were studied, including 10 gliomas, seven meningiomas, two metastatic tumors, two schwannomas and one medulloblastoma. Histologically normal adjacent brain tissues to tumor tissues were studied as a control group. After removal, surgical specimens were immediately frozen in liquid nitrogen and stored at −70°C until they were assayed. A part of the tissue samples (100 mg) was trimmed on ice, homogenized in 1 ml buffer (0.05 M Tris and 0.1 M NaCl/0.02 M ethylendiamine tetraasetic acid at a 9:1 ratio (pH 7.0)) and centrifuged for 15 min at 4°C [6,26]. ELISA was performed an aliquots of the supernatants for PGE2 determination using a Neogen Corporation Prostaglandin E2 ELISA kit. Aliquots of the homogenate were taken to determine the protein content by the Lowry method [20]. PGE2 levels in the tumor or brain tissues are expressed per milligram of protein. Another part of the tissue samples was weighed and 1 g tissue was homogenized in 9 volumes of 0.5 mM CaCl2/1 mM NaHCO3 at pH 7.5 using a tissue grinder fitted with a teflon pestil. Plasma membranes were isolated according to the procedure of
Koizumi et al. [18]. Each plasma membrane of tumor or brain tissues was dissolved in 1 ml NaCl (154 mM) and mixed with 0.1 ml heptadecanoic acid (25 mg) dissolved in CHCl3 and used as an internal standard and 0.1 ml diluted butylated hydroxytoluene (20 g/l). Methanol (2 ml) and chloroform (3.9 ml) were then added to each sample. After vortex mixing for 2 min, the tubes were centrifuged (2000 × g for 10 min at 4°C). The chloroform phase was removed and evaporated to dryness at 37°C under a stream of N2. The lipid extract was dissolved in 0.8 ml methanol/KOH (98:2, v/w) and saponified by heating for 5 min in a boiling water bath. After cooling, the fatty acids were methylated with 14% BF3 in methanol. The fatty acid methyl esters were extracted with hexane and analyzed by capillary gas chromatography (column 50 × 0.25 mm WCOT fused Silica, CP-Sil 88; flame ionization detector temperature 300°C; carrier gas N2; splitter injector temperature 290°C, oven temperature program from 150 to 240°C at 2°C/min; Perkin Elmer 84240 Capillary Gas Chromatograph, Gouda, The Netherlands). Arachidonic acid levels of tumor or brain tissues are expressed as a percentage of total fatty acids. Data are presented as the mean ± SD. Analysis of variance followed by the Tukey-HSD test was performed and a P-value of ,0.05 was considered statistically significant.
3. Results and discussion Arachidonic acid levels of tumor and control brain tissues are shown in Table 1. The levels of arachidonic acid in gliomas (n = 9) and meningiomas (n = 6) Table 1 The levels of arachidonic acid in the plasma membrane of human brain tumors and control brain tissues Type of tissue
n
Arachidonic acid (% of total fatty acids)
Control Glioma Meningioma Schwannoma Metastatic tumor Medulloblastoma
12 9 6 2 1 1
7.45 10.50 10.96 13.15 10.40 10.30
a
In comparison with controls (P = 0.000).
± ± ± ±
1.00 1.31a 1.52a 2.89
E. Ko¨kog˘lu et al. / Cancer Letters 132 (1998) 17–21 Table 2 PGE2 levels of human brain tumors and control brain tissues Type of tissue
n
PGE2 (pg/mg protein)
Control Glioma Meningioma Metastatic tumor Schwannoma Medulloblastoma
12 10 7 2 2 1
118.27 ± 156.89 ± 233.12 ± 356.70 ± 242.59 ± 293.851
a
15.42 29.4a 49.67a,b 4.44 61.80
In comparison with controls (P = 0.000). In comparison with gliomas (P = 0.000).
b
were significantly higher than those of control tissues (n = 12) (P = 0.000 and P = 0.000, respectively). The levels of arachidonic acid in two schwannomas, one metastatic tumor and one medulloblastoma were found to be higher than those of control brain tissue, although the statistical significance could not be analyzed. PGE2 levels of tumor and control brain tissues are shown in Table 2. The levels of PGE2 in gliomas (n = 10) and meningiomas (n = 7) were significantly higher than those of control tissues (P = 0.000 and P = 0.000, respectively) and PGE2 levels of meningiomas were significantly higher than those of gliomas (P = 0.000). The levels of PGE2 in two schwannomas, two metastatic tumors and one medulloblastoma were found to be higher than those of control brain tissues. Brain lipids are rich in arachidonic acid which is esterified to the second position of phospholipids in the cell membranes [13]. Certain authors have reported that arachidonic acid promotes tumor proliferation [5]. It is released readily from cell membrane phospholipids in response to even mild damage and prostanoid production increases as a result [7]. It has been shown that prostaglandins can influence the carcinogenic process in many different ways. They can stimulate tumor growth [32], act as tumor promoters [19], or influence tumor migration and the metastatic potential [10]. We found that arachidonic acid levels were significantly higher in the plasma membrane of various brain tumors. The tumor-promoting action of arachidonic acid is due primarily to its effects on the capacity of the tissues to produce prostaglandins. There is very little published work on prostaglandin production by human brain tumors. In the study of Cooper et al. [7], PGE2 was produced in the greatest
19
amount in meningiomas and the prostaglandin profiles for gliomas did not have a recognizable pattern. According to Anagnostopoulos-Schleep et al. [1], PGE2 and PGF2a are synthesized and released by various brain tumors, including gliomas and meningiomas. Gaetani et al. [11] reported that the ex vivo capacity to synthesize PGE2 and PGD2 was not significantly different in each subgroup of brain tumors. In our study, PGE2 levels were significantly higher in gliomas and meningiomas than in control tissues and PGE2 levels of meningiomas were also significantly higher than those of gliomas. PGE2 has broad kinds of biological activities. Above all, the immunosuppressive action would be the most important for tumor growth and metastatic potential [23]. The immunosuppressive state frequently observed in tumor bearers was reported to be due to PGE2 secreted by tumor cells and the host macrophage acting to suppress Tlymphocytes and natural killer activities [15,33]. In addition, natural killer cells are important for limiting cancer metastatic formation [10,29]. In our study, although the statistical significance could not be analyzed, the metastatic tumors had the highest PGE2 level at 356.7 ± 4.44 pg/mg protein. Increased PGE2 production in brain tumors may play a role in tumor growth and metastatic formation as an immunosuppressive molecule. Cerebral edema, a common secondary form of brain damage in patients with intracranial neoplasms, is a potential cause of morbidity and mortality [1]. Excessive production and release of prostaglandins by neoplastic cells alter the peritumoral environment, inducing microcirculatory disturbances [24,30], decreasing cerebral blood flow [24], disrupting the blood–brain barrier and revealing perifocal ischemia and edema [4,14,17]. In addition, local hypoxia may enhance the prostaglandin synthesis rate of the surrounding cells [14] and maintain edema expansion [1]. In this study, PGE2 concentrations in brain tumors were higher than in controls, in agreement with the findings of Anagnostopoulos-Schleep et al. [1], which showed that expansion of peritumoral edema was associated with increased PGE2 levels in neoplastic and edematous tissue. Although the edema parameter was not investigated in this study, increased PGE2 levels can lead to the formation of brain edema surrounding brain tumors. Furthermore, prostaglandins are locally acting
20
E. Ko¨kog˘lu et al. / Cancer Letters 132 (1998) 17–21
agents. Approximately 20% of meningiomas invade bone with bone resorption and new bone formation occurring simultaneously [7,28]. Since PGE2 is a potent bone resorbing agent [3], increased PGE2 levels may play a role in invading bone. Further studies are needed to clarify the importance of prostaglandins in human brain tumors. Acknowledgements This work was supported by the Research Fund of The University of Istanbul. Project Number: T-15/ 170395.
[12]
[13]
[14]
[15]
[16]
References [17] [1] J. Anagnostopoulos-Schleep, W. Schlegel, K.H. Kra¨hling, H.J. Ko¨nig, Prostaglandins F2a and E2 as possible mediators of peritumoral brain edema, Adv. Neurosurg. 16 (1988) 243– 249. [2] K.R. Applegate, J.A. Glomset, Computer based modeling of the conformation and packing properties of docosahexaenoic acid, J. Lipid Res. 27 (1986) 658–680. [3] D. Atkins, M. Greaves, K.J. Ibbotson, T.J. Martin, Role of prostaglandins in bone metabolism: a review, J. R. Soc. Med. 72 (1979) 27–34. [4] K.K. Bhakoo, H.A. Crockard, P.C. Lascelles, S.F. Avery, Prostaglandin synthesis and oedema formation during reperfusion following experimental brain ischemia in the gerbil, Stroke 15 (1984) 891–895. [5] S.A. Broitman, J.J. Vitale, E. Vavrousek-Jakuba, L.S. Gottlieb, Polyunsaturated fat, cholesterol and large bowel tumorigenesis, Cancer 40 (1977) 2455–2460. [6] S. Constantini, J. Tamir, M.J. Gomori, E. Shohami, Tumor prostaglandin levels correlate with edema around supratentorial meningiomas, Neurosurgery 33 (2) (1993) 204– 211. [7] C. Cooper, H.G. Jones, R.O. Weller, V. Walker, Production of prostaglandins and thromboxane by isolated cells from intracranial tumours, J. Neurol. Neurosurg. Psychiatry 47 (1984) 579–584. [8] A.M. Fulton, S. Zhang, Y.C. Chong, Role of prostaglandin E2 receptor in mammary tumor metastasis, Cancer Res. 51 (1991) 2047–2050. [9] A.M. Fulton, The role of eicosanoids in tumor metastasis, Prostaglandins Leukotrienes Essential Fatty Acids 34 (1988) 229–237. [10] A.M. Fulton, G.H. Heppner, Relationships of prostaglandin E and natural killer sensitivity to metastatic potential in murine mammary adenocarcinomas, Cancer Res. 45 (1985) 4779– 4784. [11] Gaetani, ‘Ex vivo’ release of eicosanoid from human brain
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tissue: its relevance in the development of brain edema. Neurosurgery, 28 (6) (1991) 853–858. J.S. Goodwin, Prostaglandin synthetase inhibitors as immunoadjuvants in the treatment of cancer, J. Immunopharmacol. 2 (1980) 397–424. T. Hanai, T. Hashimoto, K. Nishiwaki, M. Ono, Y. Akomo, M. Tanaka, I. Mizuno, J. Yura, Comparison of prostanoids and their precursor fatty acids in human hepatocellular carcinoma and carcinoma and noncancerous reference tissues, J. Surg. Res. 54 (1993) 57–60. F. Ianotti, A. Crockard, G. Ladds, L. Symon, Are prostaglandins involved in experimental ischemic edema in gerbils? Stroke 12 (1981) 301–306. J.M. Jessup, S.J. Legrue, B.D. Kahan, N.R. Pellis, Induction of suppressor cells by a tumor-derived suppressor factor, Cell Immunol. 93 (1985) 9–25. I. Klapan, V. Katic, F. Culo, V. Cuk, Prognostic significance of plasma prostaglandin E concentration in patients with head and neck cancer, J. Cancer Res. Clin. Oncol. 118 (1992) 308– 313. T. Koide, O. Gotoh, T. Asano, K. Takakura, Alterations of the eicosanoid synthetic capacity of rat brain microvessels following ischemia: relevance to ischemic brain edema, J. Neurochem. 44 (1985) 85–93. K. Koizumi, Y. Ito, K. Kozima, T. Fuji, Isolation and characterization of the plasma membranes from rat ascites hepatomas and from normal rat livers, including newborn, regenerating and adult livers, J. Biochem. 79 (1976) 739– 748. L. Levine, Arachidonic acid transformation and tumor production, Adv. Cancer Res. 35 (1981) 49–79. O.H. Lowry, N.J. Rosebrough, A.L. Farr, R.J. Randall, Protein measurement with the Folin phenol reagent, J. Biol. Chem. 193 (1951) 265. T.L. Mc lemore, W.C. Hubbard, C.L. Litterst, M.C. Liu, S. Miller, N.A. Mc Mahon, J.C. Eggleston, M.R. Boyd, Profiles of prostaglandin biosynthesis in normal lung and tumor tissue from lung cancer patients, Cancer Res. 48 (1988) 3140–3147. S.A. Moore, E. Yoder, S. Murphy, G.R. Dutton, A.A. Spector, Astrocytes, not neurons, produce docosahexaenoic acid and arachidonic acid, J. Neurochem. 56 (1991) 518–524. I. Nakazawa, M. Iwaizumi, K. Ohuchi, Some features in prostaglandin synthesis of the cancer cells which metastasized into liver from intestinal cancer lesions, Tohoku J. Exp. Med. 170 (1993) 131–133. J.D. Pickard, Role of prostaglandin and arachidonic acid derivatives in the coupling of cerebral blood flow to cerebral metabolism, J. Cereb. Blood Flow Metabol. 1 (1981) 361– 384. B. Rigas, I.S. Goldman, L. Levine, Altered eicosanoid levels in human colon cancer, J. Lab. Clin. Med. 122 (5 )(1993) 518–523. E. Shohami, Y. Shapira, G. Yadid, N. Reisfeld, S. Yedgar, Brain phospholipase A2 is activated after experimental closed head injury in the rat, J. Neurochem. 53 (5) (1989) 1541– 1546. A.A. Spector, C.P. Burns, Biological and therapeutic poten-
E. Ko¨kog˘lu et al. / Cancer Letters 132 (1998) 17–21 tial of membrane lipid modification in tumors, Cancer Res. 47 (1987) 4529–4537. [28] J.N. Walton, Rev. Brain’s Diseases of the Nervous System, eighth ed., Oxford University Press, Oxford, 1977, pp. 239– 240. [29] R.H. Wiltrout, R.B. Herberman, S.R. Zhang, M.A. Chirigos, J.R. Ortaldo, K.M. Grenn Jr., J.E. Talmadge, Role of organassociated NK cell in decreased formation of experimental metastases in lung and liver, J. Immunol. 134 (1985) 4267– 4275. [30] L. Wolfe, Eicosanoids: prostaglandins, thromboxanes, leukotrienes, and other derivates of carbon-20 unsaturated fatty acids, J. Neurochem. 38 (1987) 1–14.
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[31] A. Yamaguchi, T. Ishida, G. Nishimura, M. Katoh, I. Miyazaki, Investigation of colonic prostaglandins in carcinogenesis in the rat colon, Dis. Colon Rectum 34 (7) (1991) 572– 576. [32] T. Yoneda, M. Kitamura, T. Ogawa, S. Aya, M. Sakuda, Control of VX2 carcinoma cell growth in culture by calcium, calmodulin and prostaglandins, Cancer Res. 45 (1985) 398– 405. [33] M.R. Young, E. Wheeler, M. Newby, Macrophage-mediated suppression of natural killer cell activity in mice bearing Lewis carcinoma, J. Natl. Cancer Inst. 76 (1986) 745–750.
Prostaglandin E2 levels in human brain tumor tissues and arachidonic acid levels in the plasma membrane of human brain tumors ¨ zyurt c E. Ko¨kog˘lu a,*, Y. Tu¨ter a, K.S. Sandıkc¸ı a, Z. Yazıcı b, E.Z. Ulakog˘lu a, H. So¨nmez a, E. O a
Department of Biochemistry, Cerrahpas¸a Medical Faculty, University of Istanbul, Istanbul, Turkey Department of Pharmacology, Cerrahpas¸a Medical Faculty, University of Istanbul, Istanbul, Turkey c Department of Neurosurgery, Cerrahpas¸a Medical Faculty, University of Istanbul, Istanbul, Turkey
b
Received 2 September 1997; accepted 27 April 1998
Abstract Arachidonic acid is stored in the cell membrane and released when the cell is activated by appropriate stimuli. It is the substrate for prostaglandins. Both experimental and human tumors often synthesize high levels of prostaglandins, most notably prostaglandin E2 (PGE2). Some experiments suggest that these compounds increase tumor growth through their actions on host immunocytes. In this study, 22 patients with various brain tumors and 12 control brain tissues were studied. PGE2 levels in tissue samples were measured by ELISA. Arachidonic acid levels in the plasma membrane of tissue samples were analyzed by capillary gas chromatography. The levels of PGE2 were significantly higher in gliomas (n = 10) and meningiomas (n = 7) compared with control tissues (P = 0.000 and P = 0.000, respectively). Also, PGE2 levels in meningiomas were significantly higher than in gliomas (P = 0.000). Arachidonic acid levels in the plasma membrane of gliomas (n = 9) and meningiomas (n = 6) were significantly higher than in the control tissues (P = 0.000 and P = 0.000, respectively). These results suggest that the increased production of PGE2 may suppress the immune system and play an important role in tumor growth. 1998 Elsevier Science Ireland Ltd. All rights reserved Keywords: Arachidonic acid; Prostaglandin E2; Human brain tumors
1. Introduction Polyunsaturated fatty acids, especially docosahexaenoic acid and arachidonic acid, are enriched in brain lipids [22] where they are major components of excitable membranes [2]. Arachidonic acid is stored intracellularly in membrane phospholipids and released when the cell is activated by appropriate stimuli [27]. Arachidonic acid is further metabolized in a cascade through the cyclooxygenase and lipoxygenase * Corresponding author.
0304-3835/98/$ - see front matter PII S0304-3835 (98 )0 0127-X
pathways, eventually producing such physiologically active substances as prostaglandins and leukotrienes [13]. Changes in the membrane phospholipid fatty acid composition can alter the amount of arachidonic acid contained in the cell membrane. This can affect the amount of prostaglandins [27]. Increased biosynthesis of prostaglandins, particularly of the E series, has been demonstrated in a large number of human and experimental cancers, including breast cancer [8], colon cancer [25,31], head and neck cancer [16] and lung cancer [21]. These compounds may play an important role in
1998 Elsevier Science Ireland Ltd. All rights reserved
18
E. Ko¨kog˘lu et al. / Cancer Letters 132 (1998) 17–21
tumor growth and spread [7]. The growth of many experimental tumors is suppressed by the administration of low non-toxic doses of cyclooxygenase inhibitors such as indomethacin, aspirin and piroxicam [12]. This indicates that, in many cases, tumor prostaglandin synthesis contributes positively to tumor growth. The mechanisms involved are unclear, but considerable evidence implicates prostaglandin E2 (PGE2) as an immunosuppressive molecule that acts to downregulate host antitumor reactions [9]. Little is known of the production of prostaglandins by intracranial tumors. Certain authors reported that PGE2 levels increase in brain tumors and that PGE2 is produced in the greatest amount in meningiomas [7]. By contrast, other authors reported no increased production of PGE2 in brain tumors [11]. The present study was designed to investigate the levels of arachidonic acid in the cell membrane of human brain tumors and the levels of PGE2 in brain tumor tissues.
2. Materials and methods Tumor or brain tissue was obtained from patients undergoing craniotomy at the Department of Neurosurgery, Cerrahpas¸a Medical Faculty. Twenty-two tumor samples were studied, including 10 gliomas, seven meningiomas, two metastatic tumors, two schwannomas and one medulloblastoma. Histologically normal adjacent brain tissues to tumor tissues were studied as a control group. After removal, surgical specimens were immediately frozen in liquid nitrogen and stored at −70°C until they were assayed. A part of the tissue samples (100 mg) was trimmed on ice, homogenized in 1 ml buffer (0.05 M Tris and 0.1 M NaCl/0.02 M ethylendiamine tetraasetic acid at a 9:1 ratio (pH 7.0)) and centrifuged for 15 min at 4°C [6,26]. ELISA was performed an aliquots of the supernatants for PGE2 determination using a Neogen Corporation Prostaglandin E2 ELISA kit. Aliquots of the homogenate were taken to determine the protein content by the Lowry method [20]. PGE2 levels in the tumor or brain tissues are expressed per milligram of protein. Another part of the tissue samples was weighed and 1 g tissue was homogenized in 9 volumes of 0.5 mM CaCl2/1 mM NaHCO3 at pH 7.5 using a tissue grinder fitted with a teflon pestil. Plasma membranes were isolated according to the procedure of
Koizumi et al. [18]. Each plasma membrane of tumor or brain tissues was dissolved in 1 ml NaCl (154 mM) and mixed with 0.1 ml heptadecanoic acid (25 mg) dissolved in CHCl3 and used as an internal standard and 0.1 ml diluted butylated hydroxytoluene (20 g/l). Methanol (2 ml) and chloroform (3.9 ml) were then added to each sample. After vortex mixing for 2 min, the tubes were centrifuged (2000 × g for 10 min at 4°C). The chloroform phase was removed and evaporated to dryness at 37°C under a stream of N2. The lipid extract was dissolved in 0.8 ml methanol/KOH (98:2, v/w) and saponified by heating for 5 min in a boiling water bath. After cooling, the fatty acids were methylated with 14% BF3 in methanol. The fatty acid methyl esters were extracted with hexane and analyzed by capillary gas chromatography (column 50 × 0.25 mm WCOT fused Silica, CP-Sil 88; flame ionization detector temperature 300°C; carrier gas N2; splitter injector temperature 290°C, oven temperature program from 150 to 240°C at 2°C/min; Perkin Elmer 84240 Capillary Gas Chromatograph, Gouda, The Netherlands). Arachidonic acid levels of tumor or brain tissues are expressed as a percentage of total fatty acids. Data are presented as the mean ± SD. Analysis of variance followed by the Tukey-HSD test was performed and a P-value of ,0.05 was considered statistically significant.
3. Results and discussion Arachidonic acid levels of tumor and control brain tissues are shown in Table 1. The levels of arachidonic acid in gliomas (n = 9) and meningiomas (n = 6) Table 1 The levels of arachidonic acid in the plasma membrane of human brain tumors and control brain tissues Type of tissue
n
Arachidonic acid (% of total fatty acids)
Control Glioma Meningioma Schwannoma Metastatic tumor Medulloblastoma
12 9 6 2 1 1
7.45 10.50 10.96 13.15 10.40 10.30
a
In comparison with controls (P = 0.000).
± ± ± ±
1.00 1.31a 1.52a 2.89
E. Ko¨kog˘lu et al. / Cancer Letters 132 (1998) 17–21 Table 2 PGE2 levels of human brain tumors and control brain tissues Type of tissue
n
PGE2 (pg/mg protein)
Control Glioma Meningioma Metastatic tumor Schwannoma Medulloblastoma
12 10 7 2 2 1
118.27 ± 156.89 ± 233.12 ± 356.70 ± 242.59 ± 293.851
a
15.42 29.4a 49.67a,b 4.44 61.80
In comparison with controls (P = 0.000). In comparison with gliomas (P = 0.000).
b
were significantly higher than those of control tissues (n = 12) (P = 0.000 and P = 0.000, respectively). The levels of arachidonic acid in two schwannomas, one metastatic tumor and one medulloblastoma were found to be higher than those of control brain tissue, although the statistical significance could not be analyzed. PGE2 levels of tumor and control brain tissues are shown in Table 2. The levels of PGE2 in gliomas (n = 10) and meningiomas (n = 7) were significantly higher than those of control tissues (P = 0.000 and P = 0.000, respectively) and PGE2 levels of meningiomas were significantly higher than those of gliomas (P = 0.000). The levels of PGE2 in two schwannomas, two metastatic tumors and one medulloblastoma were found to be higher than those of control brain tissues. Brain lipids are rich in arachidonic acid which is esterified to the second position of phospholipids in the cell membranes [13]. Certain authors have reported that arachidonic acid promotes tumor proliferation [5]. It is released readily from cell membrane phospholipids in response to even mild damage and prostanoid production increases as a result [7]. It has been shown that prostaglandins can influence the carcinogenic process in many different ways. They can stimulate tumor growth [32], act as tumor promoters [19], or influence tumor migration and the metastatic potential [10]. We found that arachidonic acid levels were significantly higher in the plasma membrane of various brain tumors. The tumor-promoting action of arachidonic acid is due primarily to its effects on the capacity of the tissues to produce prostaglandins. There is very little published work on prostaglandin production by human brain tumors. In the study of Cooper et al. [7], PGE2 was produced in the greatest
19
amount in meningiomas and the prostaglandin profiles for gliomas did not have a recognizable pattern. According to Anagnostopoulos-Schleep et al. [1], PGE2 and PGF2a are synthesized and released by various brain tumors, including gliomas and meningiomas. Gaetani et al. [11] reported that the ex vivo capacity to synthesize PGE2 and PGD2 was not significantly different in each subgroup of brain tumors. In our study, PGE2 levels were significantly higher in gliomas and meningiomas than in control tissues and PGE2 levels of meningiomas were also significantly higher than those of gliomas. PGE2 has broad kinds of biological activities. Above all, the immunosuppressive action would be the most important for tumor growth and metastatic potential [23]. The immunosuppressive state frequently observed in tumor bearers was reported to be due to PGE2 secreted by tumor cells and the host macrophage acting to suppress Tlymphocytes and natural killer activities [15,33]. In addition, natural killer cells are important for limiting cancer metastatic formation [10,29]. In our study, although the statistical significance could not be analyzed, the metastatic tumors had the highest PGE2 level at 356.7 ± 4.44 pg/mg protein. Increased PGE2 production in brain tumors may play a role in tumor growth and metastatic formation as an immunosuppressive molecule. Cerebral edema, a common secondary form of brain damage in patients with intracranial neoplasms, is a potential cause of morbidity and mortality [1]. Excessive production and release of prostaglandins by neoplastic cells alter the peritumoral environment, inducing microcirculatory disturbances [24,30], decreasing cerebral blood flow [24], disrupting the blood–brain barrier and revealing perifocal ischemia and edema [4,14,17]. In addition, local hypoxia may enhance the prostaglandin synthesis rate of the surrounding cells [14] and maintain edema expansion [1]. In this study, PGE2 concentrations in brain tumors were higher than in controls, in agreement with the findings of Anagnostopoulos-Schleep et al. [1], which showed that expansion of peritumoral edema was associated with increased PGE2 levels in neoplastic and edematous tissue. Although the edema parameter was not investigated in this study, increased PGE2 levels can lead to the formation of brain edema surrounding brain tumors. Furthermore, prostaglandins are locally acting
20
E. Ko¨kog˘lu et al. / Cancer Letters 132 (1998) 17–21
agents. Approximately 20% of meningiomas invade bone with bone resorption and new bone formation occurring simultaneously [7,28]. Since PGE2 is a potent bone resorbing agent [3], increased PGE2 levels may play a role in invading bone. Further studies are needed to clarify the importance of prostaglandins in human brain tumors. Acknowledgements This work was supported by the Research Fund of The University of Istanbul. Project Number: T-15/ 170395.
[12]
[13]
[14]
[15]
[16]
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