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Prostaglandin E2concentrations in naturally occurring canine cancer
Prostaglandins, Leukotrienes and Essential FattyAcids (2001) 64(1),1^ 4 & 2001 Harcourt Publishers Ltd doi:10.1054/plef.2000.0231, available online at http://www.idealibrary.com on
Prostaglandin E2 concentrations in naturally occurring canine cancer S. I. Mohammed,1 K. Coffman,1 N.W. Glickman,2 M. G. Hayek,3 D. J.Waters,1 D. Schlittler,1 D. B. DeNicola,4 D.W. Knapp1 1
Department of Veterinary Clinical Sciences, Purdue University,West Lafayette, IN, USA Research Programs, Purdue University,West Lafayette, IN, USA 3 The Iams Company, Dayton, OH, USA 4 Department of Veterinary Pathobiology, Purdue University,West Lafayette, IN, USA 2
Summary The purpose of this study was to determine the PGE2 concentration in naturally-occurring cancer in pet dogs and in canine cancer cell lines in order to identify specific types of canine cancer with high PGE2 production which could serve as preclinical models to evaluate anticancer strategies targeting PGE2. PGE2 concentrations were measured by enzyme immunoassay in canine melanoma, soft tissue sarcoma, transitional cell carcinoma, osteosarcoma, and prostatic carcinoma cell lines; in 80 canine tumor tissue samples including oral melanoma (MEL), oral squamous cell carcinoma (SCC), transitional cell carcinoma of the urinary bladder (TCC), lymphoma (LSA), mammary carcinoma (MCA), osteosarcoma (OSA), prostatic carcinoma (PCA); and in corresponding normal organ tissues. High concentrations of PGE2 (range 400^3300 pg/104 cells) were present in cell culture medium from the transitional cell carcinoma, prostatic carcinoma, and osteosarcoma cell lines. PGE2 concentrations in tumor tissues were elevated (tumor PGE2 concentration4meanþ2X sd PGE2 concentration of normal organ tissue) in 21/22 TCC, 5/6 PCA, 7/10 SCC, 5/10 MEL, 3/8 MCA, 4/15 OSA, and 0/9 LSA. Results of this study will help guide future investigations of anticancer therapies that target cyclooxygenase and PGE2. & 2001Harcourt Publishers Ltd
INTRODUCTION Several studies published in the past two decades have demonstrated an important link between cancer development and progression, and arachidonic acid metabolites (reviewed by Lupulescu1 and Ara2). One of these eicosanoids, prostaglandin E2 (PGE2), has received considerable attention. PGE2, a product of the enzyme activity of cyclooxygenase (cox) (including cox-1 and cox-2) on arachidonic acid, has been implicated for its role in carcinogenesis, cancer progression, and metastasis.1–12 High concentrations of PGE2 are produced by tumor cells, tumor-associated macrophages, and peripheral blood monocytes in people with breast, lung, colon, and head and neck cancer, and in experimentallyinduced animal tumors.1–5,13
PGE2 and cox are being evaluated as targets for cancer prevention and treatment. One of the animal models that is being used for such studies consists of pet dogs with naturally-occurring cancer.14 Specific histopathologic types of naturally-occurring cancer in pet dogs closely mimic their human counterparts.14,15 The purpose of this study was to determine the PGE2 concentration in naturally-occurring cancer in pet dogs and in canine cancer cell lines in order to identify specific types of canine cancer with high PGE2 production which could serve as preclinical models to evaluate anticancer strategies targeting PGE2.
METHODS
Cell lines Received 27 July 2000 Accepted 5 September 2000 Correspondence to: Deborah W. Knapp, Department of Veterinary Clinical Sciences, Purdue University,West Lafayette, IN 47907-1248, USA.Tel.: þ1 765 4941107; Fax: þ1 765 496 1108; E-mail: [email protected]
& 2001Harcourt Publishers Ltd
PGE2 concentrations were measured in the culture medium of 10 canine cancer cell lines including transitional cell carcinoma, soft tissue sarcoma, melanoma, osteosarcoma, and prostatic carcinoma cell lines16,17 (Table 1). All cell lines were cultured as adherent Prostaglandins, Leukotrienes and Essential FattyAcids (2001) 64(1), 1^ 4
2
Mohammed et al.
Table 1 PGE2 concentration measured in conditioned medium from canine cancer cell lines (after 72 h incubation at 378C, 5% CO2). Mean PGE2 concentration from four experiments Cell line Origin of cell line MEL STS TCC BC-1 TR5P TR6LM CF-3 GN4 BF-2 LOSA
PGE2 (pg/104 cells)
Melonoma 4.1 Soft tissue sarcoma 13.7 Transitional cell carcinoma 1934.8 Prostatic carcinoma 760.0 Prostatic carcinoma 874.8 Prostatic carcinoma lung metastasis 859.8 Prostatic carcinoma lung metastasis 398.7 Prostatic carcinoma lung metastasis 694.4 Prostatic carcinoma bone metastasis 3299.8 Osteosarcoma lung metastasis 1918.2
monolayers. Cells were harvested, counted with an automated counter (Coulter Counter, Hiaelah, FL, USA) or a hemocytometer, and plated in triplicate wells in a 24 well tissue culture plates at a density of 10 000 to 15 000 cells per ml per well in RPMI, DME, or MEM medium (depending on the cell line) with 10% fetal bovine serum and 1% L glutamine (Sigma Chimcal Co., St Louis, MO, USA). After 72 h, the culture medium (‘conditioned medium’) was collected, centrifuged to remove cells or cell debris, and stored at 7808C until assayed for PGE2 concentration. The cell count from each well was determined and recorded. Culture medium not exposed to the cancer cells (‘unconditioned medium’) served as a negative control. Each experiment was repeated a minimum of four times on each cell line. PGE2 concentrations were normalized for tumor cell number at 72 h and expressed as pg PGE2/104 tumor cells.
Tumor tissues Following approval by the Purdue Animal Care and Use Committee, tumor tissue samples were obtained by trucut or excisional biopsy from pet dogs with naturallyoccurring cancer who were presented to the Purdue Comparative Oncology Program for evaluation and treatment. A complete medical history was reviewed to assure the dogs were not receiving corticosteroids or nonsteroidal antiinflammatory drugs. Tumor tissue samples were collected at the time of biopsy, surgical intervention, or at the time of euthanasia and post mortem examination. Histopathologic diagnosis was made on all tumor samples. Tumor samples collected at post mortem examination included only those collected immediately after the death of the dog. Tumors included: lymphoma (LSA), transitional cell carcinoma of the urinary bladder (TCC), prostatic carcinoma (PCA), osteosarcoma (OSA), oral melanoma (MEL), squamous cell carcinoma (SCC), and mammary carcinoma (MCA). For comparison purposes, normal lymph node, urinary bladder mucosa, prostate gland, bone, oral mucosa, and Prostaglandins, Leukotrienes and Essential FattyAcids (2001) 64(1), 1^ 4
mammary gland were obtained from dogs undergoing euthanasia and post mortem examination. Tumor tissue and normal tissue samples were snap frozen in liquid nitrogen and stored at 7808C until assayed.
Measurement of PGE2 concentrations Conditioned and unconditioned culture medium was added directly to a PGE2 enzyme immunoassay plate (Biotrak PGE2 EIA kit, Amersham Pharmacia Biotech, Piscataway, NI, USA) with each sample tested in duplicate wells. The intra- and inter-assay coefficient of variation has been reported to be 8–11 and 9–18% respectively for this assay system. Tumor tissue samples were weighted and homogenized in ice cold phosphate buffered saline containing 5 mg/ml indomethacin. After homogenization and extraction of PGE2 with methanol, the samples were passed through activated C18 reverse phase columns. PGE2 was eluted using ethylacetate. Samples were dried under nitrogen and resuspended in assay buffer (0.1 M phosphate buffer, pH 7.5, containing 0.9% w/v bovine serum albumin and 0.5% w/v Kathon). PGE2 concentrations were determined by enzyme immunoassay (Biotrak PGE2 EIA kit) following manufacturer’s instructions. PGE2 concentrations in tumor and normal tissues were measured in duplicate wells, and were then normalized for tumor weight and expressed as ng PGE2/g tissue.
Statistical analyses The Wilcoxon two-sided test was used to assess differences between the mean PGE2 concentration in tumor tissues and the mean PGE2 concentration in the corresponding normal organ tissues. A P value of 50.05 was considered statistically significant for all analyses. The PGE2 concentration in tumor tissue sample was considered ‘elevated’ if the tumor PGE2 concentration was greater than the mean þ2X SD of the PGE2 concentration in the corresponding normal tissues. RESULTS The PGE2 concentrations measured in culture medium conditioned by canine tumor cell lines are summarized in Table 1. Concentrations of PGE2 ranging from 400–3300 pg/104 cells were detected in the transitional cell carcinoma, prostatic carcinoma, and osteosarcoma lung metastasis cell lines. The PGE2 concentrations measured in canine tumor tissue samples from 80 dogs and in corresponding normal tissues are summarized in Table 2. PGE2 concentrations were elevated in tumor tissue compared to normal tissues in the majority of cases of TCC (21/22), PCA (5/6), and & 2001Harcourt Publishers Ltd
Prostaglandin E2 concentrations in canine cancer 3
Table 2
PGE2 concentration in tumor tissue samples from naturally-occurring canine cancer and in normal canine tissues
Tissue
n=
Mean PGE2 conc (ng/g)
SD
# elevated*
Transitional cell carcinoma Normal bladder mucosa
22 10
927.6** 46.5
1129.1 32.3
21/22
6 5
195.5** 14.8
315.5 13.8
5/6
10 10 9
209.8 201.5** 72.2
258.1 131.4 33.8
5/10 7/10
8 9
72.2 28.4
48.2 23.25
3/8
Osteosarcoma Normal bone
15 6
57.8 21.4
60.4 31.7
4/15
Lymphoma Normal lymph node
9 11
23.2 93.4
25.8 40.8
0/9
Prostatic carcinoma Normal prostate gland Oral melanoma Oral squamous cell carcinoma Normal oral mucosa Mammary carcinoma Normal mammary gland
*PGE2 concentration in tumor tissue 4meanþ2X sd PGE2 concentration in normal tissue. **Mean PGE2 concentration in tumor tissue was significantly higher (P50.05) than the mean PGE2 concentration in corresponding normal tissue.
SCC (7/10). PGE2 concentrations were elevated in some cases of MEL (5/10), MCA (3/8), and OSA (4/15). In contrast, none of nine LSA samples had elevated PGE2 concentration compared to normal lymph node tissue. DISCUSSION High concentrations of PGE2 were found in naturally occurring canine TCC, PCA, SCC and in some cases of MEL, MCA, and OSA. PGE2 production in cell lines corresponded with the findings in tumor tissue samples. The melanoma cell line did not produce PGE2, and only five of 10 melanoma tissue samples in this study produced PGE2. In contrast to other tumor types, none of the canine LSA samples produced PGE2. The work has been useful in identifying canine tumors (TCC, SCC, PCA) for further study of treatment strategies aimed at PGE2 or at cox. Considerable interest has been generated concerning the possible role of cox and cox metabolites like PGE2 in cancer development and progression. Although the exact mechanisms by which PGE2 and other arachidonic acid metabolites augment tumor development and progression are not known, possible mechanisms include: 1) a role in mutagenesis (ex. malondialdehyde, a direct mutagen produced from breakdown of PGH2); 2) direct and indirect effects of PGE2 and other cox products on proliferation and apoptosis in cancer cells; 3) PGE2 induced immunosuppression which renders the host immune system unable to kill tumor cells; 4) effects of PGE2 and other cox products on the metastatic cascade (enhanced collagenase activity, increased angiogenesis, promotion of a hypercoagulable state allowing tumor cells to arrest in the circulation and invade new organs); and 5) PGE2 induced inflammation with recruitment of & 2001Harcourt Publishers Ltd
multiple growth factors that may contribute to the cancer process.1,2,4,10–12 Eicosanoid production in dogs with naturally-occurring cancer has not been previously studied to any extent. Our laboratory previously reported high PGE2 plasma concentrations and increased PGE2 production by peripheral blood monocytes from dogs with naturally-occurring transitional cell carcinoma (TCC) of the urinary bladder.18 In that study, the monocyte production of PGE2 inversely correlated with tumor remission with a cox inhibiting drug in canine TCC.18 Although studies of the expression of the cox and analyses of cox products have been limited in canine cancer, cox inhibiting drugs have been evaluated for antitumor activity in pet dogs with naturally occurring cancer.18,19 The cox inhibitor, piroxicam, has induced remission of TCC, SCC and other cancers in dogs in phase I and II clinical trials.14,18,19 Considerable variability was noted in the tumor tissue PGE2 concentration within a given tumor type in this study. This is considered most likely to be a biologic effect. Sample collection, storage, and assay techniques were consistent across the samples. In addition, previous studies showed considerable variation within canine and human tumors in the percentages of cells that express cox-2, a major source of PGE2 production in tumors.20,21 The PGE2 concentration would be expected to vary since the cox expression varies from tumor to tumor and within a specific tumor type and within different regions of the same tumor. In conclusion, this work has identified specific histopathologic types of spontaneous canine cancer which may be useful for the study of pharmacological and dietary strategies aimed at controlling or eliminating cancer through effects on PGE2. These tumors and cell Prostaglandins, Leukotrienes and Essential FattyAcids (2001) 64(1), 1^ 4
4
Mohammed et al.
lines may also be useful for mechanistic studies aimed at defining the role of cox and cox products, such as PGE2, in cancer growth and progression.
ACKNOWLEDGEMENTS This work was supported by The Iams Company, Dayton, Ohio, USA.
REFERENCES 1. Lupulescu A. Prostaglandins, their inhibitors and cancer. Prostaglandins, Leukot Essent Fatty Acids 1996; 54: 83–94. 2. Ara G, Teicher B. A. Cyclooxygenase and lipoxygenase inhibitors in cancer therapy. Prostaglandins, Leukot Essent Fatty Acids 1996; 54: 3–16. 3. Furuta Y., Hall E. R., Sanduja S., et al. Prostaglandin production by murine tumors as a predictor for therapeutic response to indomethacin. Cancer Res 1988; 48: 3002–3007. 4. Marnett L. J. Aspirin and the potential role of prostaglandins in colon cancer. Cancer Res 1992; 52: 5575–5589. 5. Earnest D. L., Hixson L. J., Alberts D. S. Piroxicam and other cyclooxygenase inhibitors: potential for cancer chemoprevention. J Cell Biochem 1992; 161: 156–166. 6. Herschman H. R. Prostaglanding synthase2. Biochim Biophys Acta 1996; 1299: 125–140. 7. Rose D. P, Connolly J. M. Effects of fatty acid and inhibitors of eicosanoid synthesis on the growth of a human breast cancer cell line in culture. Cancer Res 1990; 50: 7139–7144. 8. Fulton A. M. The role of eicosanoids in tumor metastasis. Prostaglandins Leukot Essent Fatty Acids 1988; 34: 299–237. 9. Hubbard N. E., Erickson K. L. Enhancement of metastasis from a transplantable mouse mammary tumor by dietary linoleic acid. Cancer Res 1987; 47: 6171–6175.
Prostaglandins, Leukotrienes and Essential FattyAcids (2001) 64(1), 1^ 4
10. Whiteside T. L. Role of NK cells in human desease. Clin Immunol Immunopath 1989; 53: 1–23. 11. Folkman J., Klagsbrun M. Angiogenic factors. Science 1997; 235: 442–447. 12. Tsujii M., Kawano S., Tsuji, et al. Cyclooxygenase regulates angiogenesis induced by colon cancer cells. Cell 1998; 93: 705–716. 13. Hubbard W. C., Alley M. C., McLemore T. L., et al. Profiles of prostaglandin biosynthesis in sixteen established cell lines derived from human lung, colon, prostate, and ovarian tumors. Cancer Res 1988; 48: 4770–4775. 14. Knapp D. W., Glickman N. W., DeNicola D. B., et al. Naturallyoccurring canine transitional cell carcinoma of the urinary bladder. A relevant model of human invasive bladder cancer. Urol Oncol 2000; 5: 47–59. 15. Knapp D. W., Waters D. J. Naturally occurring cancer in pet dogs: important models for developing improved cancer therapy for humans. Mol Med Today 1997; 3: 8–11. 16. Knapp D. W., Chan T. C. K., Kuczek T., et al. Evaluation of in vitro cytotoxicity of nonsteroidal anti-inflammatory drugs against canine tumor cells. Am J Vet Res 1995; 56: 801–805. 17. Walker-Daniels J., Coffman K., Azimi M., et al. Overexpression of EphA2 tyrosine kinase in prostate cancer. Prostate 1999; 41: 275–280. 18. Knapp D. W., Richardson R. C., Chan T. C. K., et al. Piroxicam therapy in 34 dogs with TCC of the urinary bladder. J Vet Intern Med 1994; 8: 273–278. 19. Knapp D. W., Richardson R. C., Bottoms D. G., et al. Phase I trial of piroxicam in 62 dogs bearing naturally-occurring tumors. Cancer Chemother Pharmacol 1992; 29: 214–218. 20. Khan K. N. M., Knapp D W., DeNicola D. B., et al. Expression of cyclooxygenase-2 in transitional cell carcinoma of the urinary bladder in dogs. Am J Vet Res 2000; 61: 478–481. 21. Mohammed S. I., Knapp D. W., Bostwick D. G., et al. Expression of cyclooxygenase-2 (COX-2) in human invasive transitional cell carcinoma (TCC) of the urinary bladder. Cancer Res 1999; 59: 148–152.
& 2001Harcourt Publishers Ltd
Prostaglandin E2 concentrations in naturally occurring canine cancer S. I. Mohammed,1 K. Coffman,1 N.W. Glickman,2 M. G. Hayek,3 D. J.Waters,1 D. Schlittler,1 D. B. DeNicola,4 D.W. Knapp1 1
Department of Veterinary Clinical Sciences, Purdue University,West Lafayette, IN, USA Research Programs, Purdue University,West Lafayette, IN, USA 3 The Iams Company, Dayton, OH, USA 4 Department of Veterinary Pathobiology, Purdue University,West Lafayette, IN, USA 2
Summary The purpose of this study was to determine the PGE2 concentration in naturally-occurring cancer in pet dogs and in canine cancer cell lines in order to identify specific types of canine cancer with high PGE2 production which could serve as preclinical models to evaluate anticancer strategies targeting PGE2. PGE2 concentrations were measured by enzyme immunoassay in canine melanoma, soft tissue sarcoma, transitional cell carcinoma, osteosarcoma, and prostatic carcinoma cell lines; in 80 canine tumor tissue samples including oral melanoma (MEL), oral squamous cell carcinoma (SCC), transitional cell carcinoma of the urinary bladder (TCC), lymphoma (LSA), mammary carcinoma (MCA), osteosarcoma (OSA), prostatic carcinoma (PCA); and in corresponding normal organ tissues. High concentrations of PGE2 (range 400^3300 pg/104 cells) were present in cell culture medium from the transitional cell carcinoma, prostatic carcinoma, and osteosarcoma cell lines. PGE2 concentrations in tumor tissues were elevated (tumor PGE2 concentration4meanþ2X sd PGE2 concentration of normal organ tissue) in 21/22 TCC, 5/6 PCA, 7/10 SCC, 5/10 MEL, 3/8 MCA, 4/15 OSA, and 0/9 LSA. Results of this study will help guide future investigations of anticancer therapies that target cyclooxygenase and PGE2. & 2001Harcourt Publishers Ltd
INTRODUCTION Several studies published in the past two decades have demonstrated an important link between cancer development and progression, and arachidonic acid metabolites (reviewed by Lupulescu1 and Ara2). One of these eicosanoids, prostaglandin E2 (PGE2), has received considerable attention. PGE2, a product of the enzyme activity of cyclooxygenase (cox) (including cox-1 and cox-2) on arachidonic acid, has been implicated for its role in carcinogenesis, cancer progression, and metastasis.1–12 High concentrations of PGE2 are produced by tumor cells, tumor-associated macrophages, and peripheral blood monocytes in people with breast, lung, colon, and head and neck cancer, and in experimentallyinduced animal tumors.1–5,13
PGE2 and cox are being evaluated as targets for cancer prevention and treatment. One of the animal models that is being used for such studies consists of pet dogs with naturally-occurring cancer.14 Specific histopathologic types of naturally-occurring cancer in pet dogs closely mimic their human counterparts.14,15 The purpose of this study was to determine the PGE2 concentration in naturally-occurring cancer in pet dogs and in canine cancer cell lines in order to identify specific types of canine cancer with high PGE2 production which could serve as preclinical models to evaluate anticancer strategies targeting PGE2.
METHODS
Cell lines Received 27 July 2000 Accepted 5 September 2000 Correspondence to: Deborah W. Knapp, Department of Veterinary Clinical Sciences, Purdue University,West Lafayette, IN 47907-1248, USA.Tel.: þ1 765 4941107; Fax: þ1 765 496 1108; E-mail: [email protected]
& 2001Harcourt Publishers Ltd
PGE2 concentrations were measured in the culture medium of 10 canine cancer cell lines including transitional cell carcinoma, soft tissue sarcoma, melanoma, osteosarcoma, and prostatic carcinoma cell lines16,17 (Table 1). All cell lines were cultured as adherent Prostaglandins, Leukotrienes and Essential FattyAcids (2001) 64(1), 1^ 4
2
Mohammed et al.
Table 1 PGE2 concentration measured in conditioned medium from canine cancer cell lines (after 72 h incubation at 378C, 5% CO2). Mean PGE2 concentration from four experiments Cell line Origin of cell line MEL STS TCC BC-1 TR5P TR6LM CF-3 GN4 BF-2 LOSA
PGE2 (pg/104 cells)
Melonoma 4.1 Soft tissue sarcoma 13.7 Transitional cell carcinoma 1934.8 Prostatic carcinoma 760.0 Prostatic carcinoma 874.8 Prostatic carcinoma lung metastasis 859.8 Prostatic carcinoma lung metastasis 398.7 Prostatic carcinoma lung metastasis 694.4 Prostatic carcinoma bone metastasis 3299.8 Osteosarcoma lung metastasis 1918.2
monolayers. Cells were harvested, counted with an automated counter (Coulter Counter, Hiaelah, FL, USA) or a hemocytometer, and plated in triplicate wells in a 24 well tissue culture plates at a density of 10 000 to 15 000 cells per ml per well in RPMI, DME, or MEM medium (depending on the cell line) with 10% fetal bovine serum and 1% L glutamine (Sigma Chimcal Co., St Louis, MO, USA). After 72 h, the culture medium (‘conditioned medium’) was collected, centrifuged to remove cells or cell debris, and stored at 7808C until assayed for PGE2 concentration. The cell count from each well was determined and recorded. Culture medium not exposed to the cancer cells (‘unconditioned medium’) served as a negative control. Each experiment was repeated a minimum of four times on each cell line. PGE2 concentrations were normalized for tumor cell number at 72 h and expressed as pg PGE2/104 tumor cells.
Tumor tissues Following approval by the Purdue Animal Care and Use Committee, tumor tissue samples were obtained by trucut or excisional biopsy from pet dogs with naturallyoccurring cancer who were presented to the Purdue Comparative Oncology Program for evaluation and treatment. A complete medical history was reviewed to assure the dogs were not receiving corticosteroids or nonsteroidal antiinflammatory drugs. Tumor tissue samples were collected at the time of biopsy, surgical intervention, or at the time of euthanasia and post mortem examination. Histopathologic diagnosis was made on all tumor samples. Tumor samples collected at post mortem examination included only those collected immediately after the death of the dog. Tumors included: lymphoma (LSA), transitional cell carcinoma of the urinary bladder (TCC), prostatic carcinoma (PCA), osteosarcoma (OSA), oral melanoma (MEL), squamous cell carcinoma (SCC), and mammary carcinoma (MCA). For comparison purposes, normal lymph node, urinary bladder mucosa, prostate gland, bone, oral mucosa, and Prostaglandins, Leukotrienes and Essential FattyAcids (2001) 64(1), 1^ 4
mammary gland were obtained from dogs undergoing euthanasia and post mortem examination. Tumor tissue and normal tissue samples were snap frozen in liquid nitrogen and stored at 7808C until assayed.
Measurement of PGE2 concentrations Conditioned and unconditioned culture medium was added directly to a PGE2 enzyme immunoassay plate (Biotrak PGE2 EIA kit, Amersham Pharmacia Biotech, Piscataway, NI, USA) with each sample tested in duplicate wells. The intra- and inter-assay coefficient of variation has been reported to be 8–11 and 9–18% respectively for this assay system. Tumor tissue samples were weighted and homogenized in ice cold phosphate buffered saline containing 5 mg/ml indomethacin. After homogenization and extraction of PGE2 with methanol, the samples were passed through activated C18 reverse phase columns. PGE2 was eluted using ethylacetate. Samples were dried under nitrogen and resuspended in assay buffer (0.1 M phosphate buffer, pH 7.5, containing 0.9% w/v bovine serum albumin and 0.5% w/v Kathon). PGE2 concentrations were determined by enzyme immunoassay (Biotrak PGE2 EIA kit) following manufacturer’s instructions. PGE2 concentrations in tumor and normal tissues were measured in duplicate wells, and were then normalized for tumor weight and expressed as ng PGE2/g tissue.
Statistical analyses The Wilcoxon two-sided test was used to assess differences between the mean PGE2 concentration in tumor tissues and the mean PGE2 concentration in the corresponding normal organ tissues. A P value of 50.05 was considered statistically significant for all analyses. The PGE2 concentration in tumor tissue sample was considered ‘elevated’ if the tumor PGE2 concentration was greater than the mean þ2X SD of the PGE2 concentration in the corresponding normal tissues. RESULTS The PGE2 concentrations measured in culture medium conditioned by canine tumor cell lines are summarized in Table 1. Concentrations of PGE2 ranging from 400–3300 pg/104 cells were detected in the transitional cell carcinoma, prostatic carcinoma, and osteosarcoma lung metastasis cell lines. The PGE2 concentrations measured in canine tumor tissue samples from 80 dogs and in corresponding normal tissues are summarized in Table 2. PGE2 concentrations were elevated in tumor tissue compared to normal tissues in the majority of cases of TCC (21/22), PCA (5/6), and & 2001Harcourt Publishers Ltd
Prostaglandin E2 concentrations in canine cancer 3
Table 2
PGE2 concentration in tumor tissue samples from naturally-occurring canine cancer and in normal canine tissues
Tissue
n=
Mean PGE2 conc (ng/g)
SD
# elevated*
Transitional cell carcinoma Normal bladder mucosa
22 10
927.6** 46.5
1129.1 32.3
21/22
6 5
195.5** 14.8
315.5 13.8
5/6
10 10 9
209.8 201.5** 72.2
258.1 131.4 33.8
5/10 7/10
8 9
72.2 28.4
48.2 23.25
3/8
Osteosarcoma Normal bone
15 6
57.8 21.4
60.4 31.7
4/15
Lymphoma Normal lymph node
9 11
23.2 93.4
25.8 40.8
0/9
Prostatic carcinoma Normal prostate gland Oral melanoma Oral squamous cell carcinoma Normal oral mucosa Mammary carcinoma Normal mammary gland
*PGE2 concentration in tumor tissue 4meanþ2X sd PGE2 concentration in normal tissue. **Mean PGE2 concentration in tumor tissue was significantly higher (P50.05) than the mean PGE2 concentration in corresponding normal tissue.
SCC (7/10). PGE2 concentrations were elevated in some cases of MEL (5/10), MCA (3/8), and OSA (4/15). In contrast, none of nine LSA samples had elevated PGE2 concentration compared to normal lymph node tissue. DISCUSSION High concentrations of PGE2 were found in naturally occurring canine TCC, PCA, SCC and in some cases of MEL, MCA, and OSA. PGE2 production in cell lines corresponded with the findings in tumor tissue samples. The melanoma cell line did not produce PGE2, and only five of 10 melanoma tissue samples in this study produced PGE2. In contrast to other tumor types, none of the canine LSA samples produced PGE2. The work has been useful in identifying canine tumors (TCC, SCC, PCA) for further study of treatment strategies aimed at PGE2 or at cox. Considerable interest has been generated concerning the possible role of cox and cox metabolites like PGE2 in cancer development and progression. Although the exact mechanisms by which PGE2 and other arachidonic acid metabolites augment tumor development and progression are not known, possible mechanisms include: 1) a role in mutagenesis (ex. malondialdehyde, a direct mutagen produced from breakdown of PGH2); 2) direct and indirect effects of PGE2 and other cox products on proliferation and apoptosis in cancer cells; 3) PGE2 induced immunosuppression which renders the host immune system unable to kill tumor cells; 4) effects of PGE2 and other cox products on the metastatic cascade (enhanced collagenase activity, increased angiogenesis, promotion of a hypercoagulable state allowing tumor cells to arrest in the circulation and invade new organs); and 5) PGE2 induced inflammation with recruitment of & 2001Harcourt Publishers Ltd
multiple growth factors that may contribute to the cancer process.1,2,4,10–12 Eicosanoid production in dogs with naturally-occurring cancer has not been previously studied to any extent. Our laboratory previously reported high PGE2 plasma concentrations and increased PGE2 production by peripheral blood monocytes from dogs with naturally-occurring transitional cell carcinoma (TCC) of the urinary bladder.18 In that study, the monocyte production of PGE2 inversely correlated with tumor remission with a cox inhibiting drug in canine TCC.18 Although studies of the expression of the cox and analyses of cox products have been limited in canine cancer, cox inhibiting drugs have been evaluated for antitumor activity in pet dogs with naturally occurring cancer.18,19 The cox inhibitor, piroxicam, has induced remission of TCC, SCC and other cancers in dogs in phase I and II clinical trials.14,18,19 Considerable variability was noted in the tumor tissue PGE2 concentration within a given tumor type in this study. This is considered most likely to be a biologic effect. Sample collection, storage, and assay techniques were consistent across the samples. In addition, previous studies showed considerable variation within canine and human tumors in the percentages of cells that express cox-2, a major source of PGE2 production in tumors.20,21 The PGE2 concentration would be expected to vary since the cox expression varies from tumor to tumor and within a specific tumor type and within different regions of the same tumor. In conclusion, this work has identified specific histopathologic types of spontaneous canine cancer which may be useful for the study of pharmacological and dietary strategies aimed at controlling or eliminating cancer through effects on PGE2. These tumors and cell Prostaglandins, Leukotrienes and Essential FattyAcids (2001) 64(1), 1^ 4
4
Mohammed et al.
lines may also be useful for mechanistic studies aimed at defining the role of cox and cox products, such as PGE2, in cancer growth and progression.
ACKNOWLEDGEMENTS This work was supported by The Iams Company, Dayton, Ohio, USA.
REFERENCES 1. Lupulescu A. Prostaglandins, their inhibitors and cancer. Prostaglandins, Leukot Essent Fatty Acids 1996; 54: 83–94. 2. Ara G, Teicher B. A. Cyclooxygenase and lipoxygenase inhibitors in cancer therapy. Prostaglandins, Leukot Essent Fatty Acids 1996; 54: 3–16. 3. Furuta Y., Hall E. R., Sanduja S., et al. Prostaglandin production by murine tumors as a predictor for therapeutic response to indomethacin. Cancer Res 1988; 48: 3002–3007. 4. Marnett L. J. Aspirin and the potential role of prostaglandins in colon cancer. Cancer Res 1992; 52: 5575–5589. 5. Earnest D. L., Hixson L. J., Alberts D. S. Piroxicam and other cyclooxygenase inhibitors: potential for cancer chemoprevention. J Cell Biochem 1992; 161: 156–166. 6. Herschman H. R. Prostaglanding synthase2. Biochim Biophys Acta 1996; 1299: 125–140. 7. Rose D. P, Connolly J. M. Effects of fatty acid and inhibitors of eicosanoid synthesis on the growth of a human breast cancer cell line in culture. Cancer Res 1990; 50: 7139–7144. 8. Fulton A. M. The role of eicosanoids in tumor metastasis. Prostaglandins Leukot Essent Fatty Acids 1988; 34: 299–237. 9. Hubbard N. E., Erickson K. L. Enhancement of metastasis from a transplantable mouse mammary tumor by dietary linoleic acid. Cancer Res 1987; 47: 6171–6175.
Prostaglandins, Leukotrienes and Essential FattyAcids (2001) 64(1), 1^ 4
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