in vitro effects of prostaglandin e2 or indomethacin on the proliferation of lymphokine-activated...

In Vitro Effects of ProstaglandinE2 orIndomethacin on the ProliferationofLymphokine-ActivatedKiller Cells andtheir Cytotoxicity against BladderTumorCells in Patients with BladderCancer

WangZhiping”f,Chen Yirongf,ZhengRongliang’,QinDashanf,ChenXuehon@, WangYiqiu*,andLiu Guodong’

*Department of Biology, Lanzhou University, Lanzhou 730000, P.R.China; ‘Institute of Urology, 2nd Affiliated Hospital of LanzhouMedical College, Lanzhou 730030, P.R. China; *Department of IsotopicAssay, 2nd Affiliated Hospital of Lanzhou Medical College, Lanzhou730030, P.R. China

Purpose: To investigate the combined effects of interleukin-2 (IL-2)with either prostaglandin E2 (PGE2) or indomethacin (IM) on the pro-liferation and cytolysis of bladder tumor cells by lymphokine-acti-vated killer (LAK) cells in patients with bladder cancer.

Methods: LAK cell proliferation was assayed in the presence of vari-ous concentrations of either PGE2 or IM by cell counting. Bladder can-cer cell lines BIU-87, EJ and bladder tumo~ cells (BTC) from the pa-tients were cultured as target cells, and the cytotoxicity of LAK cellswas determined by 3-(4,5 -dimethylthiazol-2 -yl)-2,5-diphenyltetrazo -lium bromide (MTT) assay. In addition, PGE2 in samples of condi-tioned medium from bladder cancer cells or peripheral blood mononu-clear cells (PBMC) as well as plasma from 21 patients with bladdercancer and 20 healthy donors were determined by radioimmunoassay(RIA).

Results: The proliferation of LAK cells induced by IL-2 was inhib-ited by PGEP (0.05 to 5 ng/mL) in concentration-dependent manner.

Correspondenceto: Chen Yirong,Institute of Urology,2nd AffiliatedHospitalofLanzhouMedical College, Lanzhou730030, P.R. ChinaSupportedby Natural Science Foundationof Gansu Province and Scientific Re-search Project from Gansu EducationCommission

Prostaglandins54:769-779, 199701997 by ElsevierScience Inc. 0090-6980/97/$17.00655 Avenueof the Americas,NewYork, NY 10010 PII S0090-6980(97)00161-5

—. —.. -.— ——

PGE2, NK-Cells and Bladder Cancer: Wang et al.

The enhanced growth of LAK cells was observed at certain concentra-tions of IM (100–400 ng/mL) from 48 to 96 h. Pretreatment of LAKcells with IM (200 ng/mL) significantly enhanced cytotoxicity againstBIU-87, El cells, or BTC. More PGEZ was present in conditioned me-dium from BIU-87 cells than in the conditioned medium from PBMC.

Conclusions: These studies indicate that LAK cell proliferation in-duced by IL-2 in patients with bladder cancer is inhibited by PGE2produced by PBMC and bladder cancer cells. This inhibition can beovercome by IM, which may be of use in immunotherapy of bladdercancer.

Keywords:Lymphokine-activatedkiller cells; bladder cancer; cytotoxicityjprostaglandin E2; indomethacin

Introduction

The stimulation of peripheral blood lymphocytes with interleukin-2(IL-2) results in the generation of lymphokine-activated killer (LAK) cells.These cells can lyse tumor cell targets that are resistant to natural killer(NK) cells and do not affect normal cells (l). Wang (2) reported thatalthough bladder cancer cell lines BT-A and BT-B have low sensitivity tothe cytotoxic activity of mononuclear and NK cells, they are greatlyaffected by LAK cells. Although LAK cells are potently cytotoxic to manyhistologically different tumors, the use of LAK cells in clinical practice isnot very satisfactory. It is therefore important to understand their anti-tumor mechanism.

Prostaglandin Ez (PGE2) can play a significant role in immunosuppres-sion (3). It can be generated by immunocytes such as macrophages (3–7),and in addition high PGE2 levels have been found in many types oftumors (8,9). Alleva reported that PGE2 production by macrophages wasblocked by indomethacin (IM) (5). Constantin (7) demonstrated that ele-vated PGEZ production by monocytes was responsible for the depressedlevels of NK and LAK cell function in patients with breast cancer.Tumor-derived PGE2 promotes the in vivo growth of tumor cells bysuppressing the host antitumor immunity (6,9). Depressed functions ofLAK and NK cells can be overcomed by IM (7).

This study was undertaken to investigate the effects of PGE2 or IM onthe generation of LAK cells in patients with bladder cancer and thecytotoxicity of LAK cells against bladder cancer cell lines.

Materialsand Methods

Drugs and Reagents

PGE2 and IM were purchased from Chongqing 3rd Pharmaceutical Fac-tory (Chongqin, China). RPMI 1640 medium was obtained from GIBCO.

770 Prostaglandins 1997:54, November


3-(4,5 -dimethylthiazol-2 -yl)-2,5-diphenyltetrazolium bromide (MTT),DNase, collagenase hyaluronidase, and L-glutamine were obtained fromSigma. Recombinant interleukin-2 (IL-2) was provided by the Institue ofChangchong Bioproducts (China). Fetal calf serum (FCS), insulin, anddexamethasone were purchased from The Sine-American BiotechnologyCompany.

Patients

Between March 1995 and July 1996, 21 patients with diagnosed transi-tional cell carcinoma of the bladder entered this study. Blood samplingwas performed before surgical procedures or other therapy.

Cultivation of LAK Cells

Peripheral blood mononuclear cells (PBMC) were isolated by Ficoll-paque(Shanghai 3rd Chemical Reagents Ltd.) and density-gradient centrifuga-tion. The interface cells were aspirated, washed three times with Hank’ssolution and suspended PBMC (106 cells/mL) in complete medium (CM)which consists of RPMI 1640, 100 U/mL penicillin, 100 U/mL strepto-mycin, 50 U/mL gentamycin, 2 mM L-glutamine, 1 mM sodium pyruvate,and 15 YO heat-inactivated FCS. The cells were allowed to settle for 2 h in25 cm3 tissue culture flasks at 37°C in 5’7. C02 in air. The nonadherentPBMC (2 x 105 cells/mL) were moved into another 25 cm3 flask andfurther cultivated for 96 h in CM supplemented with 1000 U/mL IL-2.

LAK Cell Proliferation Assay

The nonadherent PBMC (3.0 X 104 cells per well in 300 p,L CM + IL-2]were plated in 96-well plates in the presence of various concentrations ofeither PGE2 (O–5 ng/mL) or IM (0–400 ng/mL). Each concentration wasadded to 6 wells. Cell counts were made in a hemocytometer chamberevery 24 h. Five separate tests were made.

Target Cells

Tumor cells were prepared as described elsewhere (10). Specimens oftransitional bladder tumor tissues were mechanically minced and treatedwith DNase, collagenase, and hyaluronidase. The cells were then washedand centrifuged at 40 x g for 10 min on a three-step discontinuous Percollgradient. Tumor cells were collected from the bottom, washed threetimes in Hank’s solution, and then resuspended in CM for cytotoxicityassay.

Human bladder transitional cell carcinoma cell lines BIU-87 and EJwere kindly provided by the Institute of Urology, Beijing Medical Uni-versity. The cells were maintained in CM at 37°C in 5Y0 C02 in air. Theywere harvested by overlaying the monolayer with a solution of 0.05’Yotrypsin (Sigma) and 0.53 mM EDTA, resuspended in CM, and plated in

Prostaglandins 1997:54, November 771


96-well plates at 8 X 103 cells per well in 150 IALCM for cytotoxicityassay.

Cytotoxicity Assay

The cytotoxicity of LAK cells was determined by the 3-(4,5 -dimethylthia-zol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (11), which Hus-sain developed to replace the conventional radioactive techniques. Re-sults of MTT and Cr51 release assays showed a good correlation over awide range of cell numbers and types (r value 0.96, p < 0.03) [1 1].

BIU87 or EJ cells cultured in wells for 24 h were used as the target cells.After removing the medium, the LAK cells which had been pretreatedwith PGE2 (0.5 ng/mL)or IM (200 ng/mL)for 48 h were added to the targetcells in effecter/target (E/T) ratio of 30:1 in 250 I-LLCM. Four separatecytotoxicity assays with each for the four wells were performed. The cellmixtures were then incubated at 37°C for 4 h before washing the platewith RPMI 1640 and 2Y0 FCS. RPMI 1640 medium, which contained0.5Y0 MTT and 0.5Y0 FCS, was then added to each well. After incubationfor 4 h, the medium was replaced by 100 ~L DMSO [Sigma). The absor-bance at 570 nm in each well was determined using a microplate auto-reader (Nanjing, China).

The LAK killing of nonadherent bladder tumor cells from specimentissues was carried out in a similar manner. At the termination of the 4 hculture the cells were pelleted by centrifugation (25 x g for 5 rein) and,after discarding the supernatant, the cell mixtures were resuspended inRPMI 1640 medium, which contained 0.5Y0 MTT and 0.5Y0 FCS, andcultured for another 4 h. The cells were again pelleted by centrifugation(25 x g for 5 rein) and, after discarding the supernatant, 100 IJ,LDMSOwere added. After solubilization, the absorbance at 570 nm in each wellwas measured. The percentage of lysis was calculated by the formula:

OD of targetcells – (ODoftargetcellsplusLAKcells – OD of LAKcells)% killing= OD of targetcells

x 100

Preparation of Conditioned Medium

Near-confluent BIU-87 cells and PBMC (2 X 105 cells/mL) were culturedin 25 cmz flasks in serum-free medium (SFM), which consists of RPMI1640 supplemented with 100 U/mL penicillin, 100 U/mL streptomycin,50 U/mL gentamycin, 2 mM L-glutamine, 1 mM sodium pyruvate, 0.5mg/mL insulin, and 1 PM dexamethasone. The medium was changedonce, discarded, and the cells were cultured for a further 48 h in 10 mL ofSFM. The samples of conditioned medium were clarified by centrifuga-tion (25 x g for 10 rein), filtered through a 0.2 pm filter, and stored at–200c until further use within 3 months.


PGE2, NK-Celh and Bladder Cancer: Wang et al.

RIA for PGE2

The amounts of immunoreactive PGEZ in samples of conditioned me-dium or plasma from 21 patients with bladder cancer, and in SFM weredetermined by RIA using a commercially available RIA kit (Institute ofBlood, Suzhou Medical University, China) according to the manufactur-er’s instructions. Briefly, to each polypropylene RIA tube were added 100

12.51.PGE2, and PGE2 or the sample. Immune com-WLeach of anti-PGE2,plexes were precipitated 24 h later with 1 mL of 167. polyethylene glycolsolution, and the radioactivity in the precipitate was determined by agamma counter. There was no nonspecific interference of the assay by thecomponents of the sample. Determinations were carried out in triplicateand the means and standard deviations were obtained.

Statistical Analysis

Statistical analysis of all LAK cell proliferation experiments was per-formed by using analysis of variance. The amounts of PGE2 in the plasmaand samples of conditioned medium were compared using Student’st-test for unpaired data (2-tailed). The statistical analysis of all LAK cellcytotoxicity determinations against tumor cells was carried out by usingthe unpaired Wilcoxon’s u test.

Results

Effect of PGEP on the Growth of LAK Cells

Increasing concentrations (O–5 ng/mL) of PGE2 were added to LAK cellsfor up to 96 h. PGE2 0.05 to 5 ng/mL inhibited LAK cell proliferation ina concentration-dependent manner (p < 0.01) (Fig. 1) starting from 24 h(Fig. 2).

Stimulation of IM on LAK Cell Growth

The proliferation of LAK cells was stimulated by IM 100–400 ng/mLfrom 48 to 96 h. The greatest stimulation was achieved with 200 ng/mLIM (p <0.01, Fig. 3).

Effect of PGE2 or IM on the Cytotoxicity of LAK Cells against TumorCells

The LAK cells from patients with transitional cell cancer of bladder werepretreated with IL-2 plus 0.5 ng/mL PGE2 or IM 200 ng/mL. IM enhancedthe cytotoxicity of LAK cells against BIU-87, EJ cells or bladder tumorcells (BTC) from the patients, and the BTC cultures were more responsivethan the bladder cell lines to IM-treated LAK cells (Table 1).


i

\I,I

I

!I

t


250

o

J-

-Ctr

-I-

A

-L

●☛

II_●☛

✍

B c t) E F

COitcentratiOn of PGE2 ( ng/ml)

FIGURE1. Concentration-dependentinhibitionby PGE2 on IAK cell proliferation.Peripheralblood mononuclearcells (3.0 x 104cells per well in 300 WLcomplete medium)culturedfor 96 hwith 1000 U/mL IL-2 (Ct~,or with added PGE20.5 pg/mL (A),5 pg/ml (B),0.05 ng/mL (C), 0.5ng/mL (D),5 ng/mL (E),and 50 ng/mL (F).Eachconcentrationgroupcontained6 wells,withfiveindependentexperiments.**p<0.01 compared with Ctr.

PGEPin Conditioned Medium or Plasma /rem Patients with BladderCancer

More PGE2 was present in conditioned medium from BIU-87 cells (0.75 *0.11 pg/mL)than from PBMC (0.48 * 0.10 pg/mL).SFM that contained noPGEZ was used as a control.

Discussion

PGE2 is generally accepted to be an immunosuppressant produced bycancer cells and their associated macrophages (12). Tumor growth en-hances the suppressor activity of macrophages by increasing their syn-thesis of PGEZ (5,7). In tumor-bearing animals, PGE2 production by peri-toneal and splenic macrophages was raised (13). Alleva et al. (5) reportedthat macrophages that do not express major histocompatibility complexclass II molecules are the major PGE2-producing cells. Ikemoto et al. (3)demonstrated PGEZ production by monocytes from 48 patients withbladder cancer using enzyme immunoassay and RIA with anti-humanPGE2 antibody. In addition, many types of carcinoma produce immuno-suppressive PGE2 (8,14). Malignant human breast tumors can producehigh of levels PGE2 (9,15). The mouse cultured fibrosarcoma cell line


PGE2, NK-Cells and Wadder Cancer: Wang et al.

T

o ‘c 24 4t3 “72 96

Incubationtime(h)

FIGURE2. Growthof IAK cellstreated with PGE2.The peripheralblood mononuclearcells (3.0 x104 cells per well in 300 I.LLcomplete medium)were treated with 1000 U/mL IL-2 alone (U),1000 U/mL IL-2 plus0.05 ng/mL PGE2(0), 1000 U/mL IL-2 plus0.5 ng/mL PGE. (A), and 1000U/mL IL-2 plus 5 ng/mL PGE2 (A). Each concentrationgroup contained 6 wells, with fiveindependentexperiments.‘p <0.05 and ‘“p <0.01, compared with 1000 U/mL IL-2 alone.

QR-32 produces large amounts of PGEZwhen co-cultured with immuno-cytes, consistent with the possibility that enhanced PGE2 production bytumor cells results in their progression (16). Henderson et al. (17) foundthat photodynamic treatment in vitro caused release of large amounts ofPGEZ from mouse radiation-induced fibrosarcoma cells and peritonealmacrophages. Nevertheless, PGE2 levels produced by head and necktumor cells demonstrated no correlation to the tumor site, stage, orhistopathologic differentiation (18). Our results showed that althoughthere was no significant difference in the levels of PGE2 in plasma frompatients with bladder cancer or healthy donors, there was more PGE2 inconditioned medium from BIU-87 cells than from PBMC.

The mechanisms controlling PGE2 production in cancer cells are notunderstood fully (9). The protein kinase C agonist 12-O-tetradecanoyl-phorbol-13-acetate, and inflammatory mediators such as interleukin-1 orarachidonic acid, induced PGE2 production in breast fibroblasts or thebreast cancer cell line MDA-MB-231 (9,15). In addition, Okada (19]showed that enhanced PGE2 production by tumor cells was inhibited inthe presence of radical scavengers such as superoxide dismutase (SOD)and catalase, suggesting that oxygen radicals are involved in PGE2 pro-duction by tumor cells. Free radicals, singlet oxygen, peroxide molecules


PGE2, NK--Cel]s and

0 24 48 72 96

Incubationtime(h)

FIGURE3. Effectof IM on LAKcell proliferationinducedby 1000 U/mL IL-2. The peripheralbloodmononuclearcells (3.0 x 104cells per well in 300 I.LLcomplete medium)were treated with 1000U/mL IL-2 alone (El),1000 U/mL IL-2 plus 100 ng/mL IM (0), 1000 U/mL IL-2 plus 200 ng/mLIM (A), and 1000 U/mL IL-2 plus400 ng/mL (A). Each concentrationgroup had 6 wells, in fiveindependentexperiments.“p<0.05 and “-p<0.01, compared with 1000 U/mL IL-2 alone.

and others are known to result in increased peroxidation of membranelipids (20). Endoperoxide synthase activity appears to depend on thecellular peroxide tone—being inactivated by excess peroxides but requir-ing a certain peroxide level to function (21). In addition, phospholipase A2activity might be affected by the generation of lipid peroxides (21).

Further evidence that PGE2 is implicated in tumor-associated subver-sion of immune function (22) was produced by Okuno et al. (12), whofound that continuous administration of PGE2 in the portal vein dramat-

TABLE1. Effectof PGE2or IM on the CytolyticEffectof LAKCellsAgainstBladderTumor Cells

BladderCytolysis(%)

Tumor PGE,Cells Control (0.5 ng/mL) (200 ~g/mL)

BIU-87 47.6 ? 0.9 45.8 z 1.4 54.7 ? 0.2’EJ 48.3 ? 0.2 44.3 ? 0.1 56.8 t 0.5’BTC 38.4 ~ 0.7 33.2 t 1.1 73.8 Y 0.4”

Each concentrationhad4 wells, in four independentexperiments.*p<0.05 and ‘*p <0.01, comparedwith controls.



ically suppressed the cytotoxic activity of hepatic sinusoidal lympho-cytes against NK-sensitive YAC-1 tumor cells in a rat model. Flowcytometric analysis showed that the large granular lymphocyte fraction,hepatic NK cells, and CD4–8+ killer cells were mainly reduced innumber in the hepatic sinusoidal lymphocytes following PGE2 infusion(12). The data presented here demonstrate that PGE2 inhibited prolifera-tion of LAK cells in a concentration-dependent manner. PGE2 inhibitsT-cell proliferative responses to mitogens (19). Kokudo and Chun (23) alsodemonstrated that LAK cells retain their responsiveness to inhibition byPGEZ and that the inhibition can be partially suppressed by additionalIL-2. The antitumor activities of macrophages collected from renal pa-tients, or NK and LAK cells from patients with breast cancer, are in-versely correlated to PGE2 production (7,24). Enhanced PGE2 productionby tumor cells and immunoytes is considered to be an important mech-anism for facilitating tumor cell escape from host immune surveillance(19). PGE2 may act as a suppressor signal for many immune functionssuch as a proliferative response to antigens and alloantigens, lymphokineproduction, natural and antibody-dependent cellular cytotoxicity, andcell-mediated cytolysis (3].

PGE2 may modulate the proliferation and cytotoxicity of LAK cells bymultiple pathways. Elkashab et al. (4) revealed that splenocytes in normaland tumor-bearing mice have specific receptors for PGE2. Reduction ofcellular cAMP may also be involved in the inhibition of LAK cells (24). Itis assumed that the effect of PGE2 on human lymphocytes is mediated viaother means, including downregulation of MHC class II gene productsand IL-2-specific receptors on activated lymphocytes (25–27). Constantin(7) showed that PGE, suppressed the production of IL-2 in culturedPBMC, and the reduced LAK cell activity by PGE2 correlates with down-regulation of IL-2 receptor expression on CD56+ cells in patients withbreast cancer. In addition, decreased production of superoxide or nitricoxide by immunocytes may also be involved in the depression of cell-mediated immunity (13).

Since IM can inhibit synthesis PGE2 by tumor cells or macrophages(5,15,17,28), IM may prevent this PGE,-mediated inhibition of immuno-cytes (22). IM increased the activity of NK and LAK cells in patients withbreast cancer up to the levels of healthy donors (7). Chao (29) demon-strated that IM accelerated the cell proliferation of IL-2-activated tumor-infiltrating lymphocytes (TIL), resulting in a fast-acting and long-lastingenhancement of cytotoxicity. In agreement with the above data, thecurrent results demonstrate that IM enhanced LAK cell proliferation andcytotoxicity against the bladder tumor cells BIU-87, EJ, and BTC. Theseresults may help to provide a basis for the clinical use of immunotherapycombined with IM for the management of bladder cancer.



References1.

2.

3.

4.

5.

6.

7.

8.

9.

10.

11.

12.

13.

14.

15.

778

Jackon, A.M., Hawkyard, S.J., Prescott, S., Ritchie, A.W.S., James, K., andChisholm, G.D. An investigation of factors influencing the in vitro inductionof LAK activity against a variety of human bladder cancer cell lines. J. Urol.147:207-21 1, 1992.Wang, M. H., Flad, H., and Bohle, A. Cellular cytotoxicity of human naturalkiller cells and lymphokine activated killer cells against bladder carcinomacell lines. Immunol. Lett. 27:191–198, 1991.[kemoto, S., Kishimoto, T., Nishio, S., Wada, S., and Maekawa, M. Correla-tion of tumor necrosis factor and prostaglandin Ez production of monocyte inbladder cancer patients. Cancer 64:2076-2080, 1989.Elkashab, M. and Lala, P. PGE2 receptors on murine splenic lymphocytes:Effects of tumor bearing. Immunol. Lett. 30:7-15, 1991.Alleva, D. G., Burger, C.J., and Elgert, K.D. Interferon-y reduces tumor in-duced Ia- microphage mediated suppression: Role of prostaglandin Ez, Ia andtumor necrosis factor-a. Immunopharmology 25:215–227, 1993.Alleva, D.G., Burger, C.J., and Elgert, K.D. Tumor growth increases Ia-macrophages synthesis of tumor necrosis factor-a and prostaglandin E2:changes in macrophages suppressor activity. J. Leukocyte Biol. 53:550–558,1993.Baxevanis, C.N., Reclos, G.J., Gritzapis, A.D., Dedousis, G.V.Z., Missitzis, I.,and Papamichail, M. Elevated prostaglandin Ez production by monocytes isresponsible for the depressed levels of natural killer and lymphokine acti-vated killer cell function in patients with breast cancer. Cancer 72:491–401,1993.Burrai, I., Petti, R., and Grille, R.L. Prostagladine (PGE2]: Production andbiologic significance in cancer. J. Exp. Clin. Cancer Res. 14:185-188, 1995.Schrey, M.P. and Patel, K.V. Prostagladin Ez production and metabolism inhuman breast cancer and breast fibrolasts. Regulation by inflammatory me-diators. Br. J. Cancer 72:1412-1419, 1995.Kariya, Y., Okamoto, N., Fujimoto, T., Inoue, N., Kihara, T., and Sugie, K.Lysis of fresh human tumor cells by autologous peripheral blood lymphocytesand tumor-infiltrating lymphocytes activated by PSK. Jpn. J. Cancer Res.82:1044-1050, 1991.Hussain, R.F., Nouri, A.M. E., and Oliver, R.T.D.A. New approach for mea-surement of cytotoxicity using calorimetric assay. J. Immunol. Methods160:89-96, 1993.okuno, K., Jinnai, H., and Yung, Sun Lee. A high level of prostagladine (PGE2)in the portal vein suppresses liver-associated immunity and promotes livermetastasis. Surg. Today 25:954–958, 1995.Gardner, T. E., Naama, H., and Daly, J.M. Peritoneal and splenic microphagefunction in the tumor-bearing host. J. Surg. Res. 59:305-310, 1995.Jimbo, T., Akimoto, T., and Tohgo, A. Effect of combined administration ofa synthetic low toxicity lipid A derivative, DT-5461a, and indomethacin invarious experimental tumor models of colon 26 carcinoma in mice. CancerImmunol. Immunother. 40:10-16, 1995.Hughes, R., Timmermans, P., and Schrey, M.P. Regulation of arachidonicacid metabolism, aromatase activity and growth in human breast cancer cells

Prostaglandins 1997:54, November


16.

17.

18.

19.

20.

21.

22.

23.

24.

25.

26.

27.

28,

29.

by interleukin-1 and phorbol ester: Dissociation of a mediatory role forprostagladine Ez in the autocrine control of cell function. Int. J. Cancer67:684-689, 1996.Okada, F., Hosokawa, M., Hamada, J.I., Hasegawa, J., Kate, M., Mizutani, M.,Ren, J., Takeichi, N., and Kobayashi, H. Malignant progression of a mousefibrosarcoma by host cells reactive to a foreign body (gelatin sponge). Br. J.Cancer 66:635-639, 1992.Henderson, B.W. and Donovan, J.M. Release of prostagladin Ez from cells byphotodynamic treatment in vitro. Cancer Res. 49:6896-6900, 1989.Snyderman, C.H., Klapan, L, and Milanovich, M. Comparison of in vivo andin vitro prostagladin Ez production by squamous cell carcinoma of the headand neck. Otolaryngol. Head Neck Surg. 111:189–196, 1994.Okada, F., Hosokawa, M., Hasegawa, J., Kuramitsu, Y., Nakai, K., Yuan, L.,Lao, H., Kobayashi, H., and Takeichi, N. Enhancement of in vitro prostagla-din Ez production by mouse fibrosarcoma cells after co-cultured with variousanti-tumour effecter cells. Br. J. Cancer 70:233–238, 1994.Girotti, A.W. Mechanisms of photosensitization. Photochem. Photobiol. 38:745-751, 1983.Menconi, M. and Polgar, P. Radiation, lipid peroxidation and the role ofoxygen radicals in eicosanoid metabolism. In: P. Polgar (cd.), Eicosanoids andRadiation, pp. 119-131. Boston: Kluwer Academic, 1988.Young, M.R.I. Eicosanoids and the immunology of cancer. Cancer MetastasisRev. 13:337-348, 1994.Kokudo, S. and Chu, T.M. Responsiveness of murine lymphokine activatedkiller activity to prostagladin Ez at late phase of interleukin-2 induction.Tumor Biol. 14:144-154, 1993.Ben-Efaim, S., Tak, C., and Fieren, M.J.W.A. Activity of human peritonealmacrophages against a human tumor: Role of tumor necrosis factor-cr PGE2and nitrite, in vitro studies. Immunol. Lett. 37:27–33, 1993.Chouaib, S., Welter, K., Mertelsman, R., and Dupont, B. Prostagladin E2 actsat two distinct pathways of T lymphocyte activation: inhibition of interleu-kin-2 production and down regulation of transferring receptor expression.J. Immunol. 135:1172-1179, 1985.Nelson, J.A.S., Karhar, R.S., Scodras, J.M., and Lala, P.K. Characterization ofmacrophages subsets regulating murine natural killer cell activity. J. Leuko-cyte Biol. 48:382–393, 1990.Parhar, R.S., Yagel, S., and Lala, P.K. PGE2-mediated immunosuppression byfirst trimester human decidua cells blocks activation of material leukocytesin the decidua with potential anti-trophoblast activity. Cell Immunol. 120:61-74, 1984.Lonnroth, C., Svaninger, G., and Gelin, J. Effects related to indomethacinprolonged survival and decreased tumor growth in a mouse tumor model withcytokine dependent cancer cachexia. Int. J. Oncol. 76:1405–1413, 1995.Chao, T.Y. and Chu, T.M. Effect of indomethacin on tumor infiltratinglymphocytes of a spontaneously developed murine mammary adenocarci-noma. Cancer Immunol. Immunother. 30:158–164, 1989.

Editor: Dr. A. Bennett Received: 02-27-97 Accepted: 09-25-97


—. ———.

in vitro effects of prostaglandin e2 or indomethacin on the proliferation of lymphokine-activated...

Documents