(CANCER RESEARCH 49. 3853-3856, July 15. 1989]

Modulation of Prostaglandin Biosynthesis in Hypoxie Murine MammaryAdenocarcinoma Cells by Misonida/ole'

David R. Shalinsky,2 Dennis B. McNamara, and Krishna C. Agrawal1

Department of Pharmacology; Tulane University School of Medicine, New Orleans, Louisiana 70112

ABSTRACT

Resistance of hypoxic cells to radiation and chemotherapy remains amajor limitation to effective therapy of solid tumors. Misonidazole, a 2-nitroimidazole analogue, has been studied extensively as a radiosensitizerof hypoxic cells and has been shown to undergo bioreductive metabolismto exert preferential cytotoxicity against hypoxic cells. We have investigated the effects of misonidazole on the biosynthesis of prostaglandins(PCs) in a murine mammary adenocarcinoma cell line (No. 4526) underaerobic and hypoxic conditions in attempts to exploit modulation of PGlevels under hypoxia as a means of improving therapeutic approaches forthe treatment of solid tumors. We report a time-dependent inhibition ofPG biosynthesis by the suspended cells under hypoxia induced by flushingsealed vials with \. (1.5 liters/min). After 30 min of hypoxia, PGformation was inhibited by 50%. Indomethacin was able to further inhibitthe PG formation in a concentration-dependent manner under hypoxia.Misonidazole, however, selectively increased the PGE2 biosynthesis under hypoxia by 49% at 100 /¿M.This increase was concentration dependent over the range of 25 to 100 fiM and was blocked by indomethacin(0.1 MM).Imidazole, the heterocyclic moiety in misonidazole without thenitro function, had no effect on PG biosynthesis at these concentrations.These data suggest that arachidonic acid metabolism is sensitive to thedifferential oxygen levels which exist within solid tumors and that PGlevels may be modulated by electron-affinic agents in hypoxic tumorcells.

INTRODUCTION

Metabolism of AA4 has been associated with modulation ofcell growth. Several reports have demonstrated growth-inhibitory activity for PCs of the E, A, D, and J series (1, 2).Administration of nonsteroidal antiinflammatory agents suchas indomethacin to mice bearing tumors has been shown toinhibit tumor growth (3) or to potentiate the effects of chemo-therapeutic agents such as melphalan (4), further suggestingthat modulation of PG levels may be beneficial in the treatmentof cancer. The antiproliferative activity of these prostanoidsseems to depend on the chemical transformation to the cyclo-pentenone ring containing an a,/3-unsaturated ketone (5, 6). Ithas been suggested that the mechanism of growth inhibition bythese prostanoids does not seem to involve either the activationof adenylate cyclase or an increase in the levels of cyclic AMP(7). Recent reports have suggested that the uptake and intra-cellular accumulation of cyclopentenone PCs in the nuclei maybe responsible for their growth-inhibitory effects (2, 8). Inhibition of the synthesis of macromolecules such as DNA, RNA,and proteins by PCs has also been demonstrated (1, 9). However, inhibition of macromolecular synthesis by PGD: in a B16melanoma cell line required higher concentrations than theconcentrations needed to inhibit the replication of these cellsin vitro (10). In contrast, elevated levels of PCs, notably PGE2,

Received 8/22/88; revised 4/6/89; accepted 4/14/89.The costs of publication of this article were defrayed in part by the payment

of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1Supported in part by USPHS Grant CA 21050 awarded by the NationalCancer Institute, Department of Health and Human Services.

1 Portions of these investigations were submitted as a dissertation in partialfulfillment of the requirements for the degree of Doctor of Philosophy.

3To whom requests for reprints should be addressed.4The abbreviations used are: AA. arachidonic acid; PG, prostaglandin; DO.

dissolved oxygen; HBSS, Hanks' buffered salt solution.

have been associated with solid neoplasms in humans andanimals, but it is not known whether these elevated levels reflectderegulated neoplastic biosynthesis or whether the excessiveformation represents an endogenous mechanism for aidingtumor growth (11).

Since one of the major limitations to the effective treatmentof solid tumors, e.g., of the colon, breast, or lung, is the presenceof resistant hypoxic cells, it is of fundamental importance toinvestigate means of increasing the sensitivity of hypoxic cellsto radiation or chemotherapy. These cells exist within theoxygen-deficient central core of the proliferating tumor (12)and have become hypoxic due to decreased oxygen diffusionfrom the peripheral blood supply. The hypoxic cells are generally not in the cell cycle and therefore are resistant to cell cycle-active chemotherapeutic agents (13) as well as to radiation,which requires the presence of oxygen for its full lethal effect.After radiation and/or chemotherapy of solid tumors, revascu-larization may induce the surviving hypoxic cells into cyclingcells, thus enabling tumor regrowth.

Previous reports have suggested that synthesis of prostanoidsin various tissues is stimulated by hypoxic conditions such aslow arterial Po2 or ischemia (14). Recently, hypoxia has beenshown to stimulate PGE2 synthesis in renal mesangial cells incultures maintained under chronic exposure to 2% oxygen (15,16). However, PG biosynthesis in neoplastic cells under hypoxiahas not been reported. Hence, we have investigated the influenceof hypoxia on in vitro production of PGE2 in murine mammaryadenocarcinoma cells (line 4526) which have been shown tohave a high level of activity of the cyclooxygenase pathway ofarachidonic acid metabolism (17). Furthermore, it is not knownwhether the PG biosynthesis in neoplastic cells under hypoxiacan be modulated by pharmacological agents. Since a class ofelectron-affinic compounds, the nitroimidazoles, such as misonidazole, has been shown to sensitize hypoxic cells to theeffects of ionizing radiation and has been suggested to haveoxygen-mimicking effects, it was anticipated that this class ofcompounds may modulate PG biosynthesis under hypoxia.

MATERIALS AND METHODS

Reagents. [l-'4C]Arachidonic acid (40 to 60 mCi/mmol) was pur

chased from New England Nuclear Corp. (Boston, MA). Prostaglandinstandards and ibuprofen were obtained from The Upjohn Co. (Kala-mazoo, MI). AA and indomethacin were purchased from Sigma Chemical Co. (St. Louis, MO). All ingredients needed for cell culture werepurchased from GIBCO (Grand Island, NY). Double-distilled waterwhich had been filtered through a 0.22-^m filter was used in all studies.

Cell Culture. The murine mammary adenocarcinoma cell line (No.4526) was derived from a lung metastasis of a BALB/c mouse bearinga spontaneously arising mammary adenocarcinoma tumor ( 18) and wasobtained from Dr. Amy Fulton of the Michigan Cancer Foundation(Detroit, MI). The cells were grown in monolayer in 150-cm2 tissueculture flasks (Costar, Cambridge, MA) in 10% heat-inactivated fetalbovine serum-supplemented Waymouth's (MB 752/1) growth medium

containing 250 units of penicillin/250 ng of streptomycin per ml, 0.025HIML-glutamine and adjusted to pH 7.4 with a solution of NaHCO.,(2.6 g/liter). The cells were kept in an air:CO2 (95:5) incubator at 37'C

and transferred (1/4) at confluency using standard tissue culture procedures. Cells from exponentially growing cultures were detached from

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PROSTAGLANDIN BIOSYNTHESIS IN HYPOXIC CELLS

the flasks by using trypsin (2%)-EDTA (0.5%) and resuspended ingrowth medium to neutralize the action of trypsin. The cells werecentrifugea (1100 x g for 5 min), and the pellet was washed in HBSS(pH 7.4), recentrifuged, and resuspended in HBSS to produce a cellsuspension containing approximately IO7 cells/ml. The cell number

was obtained using a Coulter Counter. Cellular viability was assessedby using 0.4% trypan blue.

Assay of Arachidonic Acid Metabolism. The radiolabeled AA poolwas prepared by mixing 0.9 nmol of [I4C]AA with 1.1 nmol of AA inethanol. The [I4C]AA pool was then diluted with HBSS to provide 2

nmol of AA per 100 ^1 aliquot added to each incubation vial (120,000dpm). Ethanol concentration did not exceed 0.1%.

One million cells suspended in 1.0 ml of HBSS in 10 ml glass vialswere prewarmed to 37'C for 5 min and incubated for 20 min (or asnoted) with 2.0 ¿AI['''CjAA (2 nmol). In experiments performed under

hypoxic conditions, vials containing cells were sealed using rubberstoppers. A flow of N; (1.5 liters/min) was maintained over the cellsuspension through tubing connected to the stoppers via 22-gauge inletand outlet needles. After 25 min when the cells have become hypoxic,the drug solution or HBSS was injected into the treated or controlvials, respectively, for 5-min preincubation. Then, [14C]AA(in 100 n\)

was injected through the stopper into each vial to initiate AA metabolism. Control vials containing cells for aerobic biosynthesis were alsokept in the water bath for 30 min prior to incubation with [14C]AA.The reaction was stopped at appropriate time intervals, and the eicos-anoids were extracted, separated, identified, and quantified as previously reported (19). A typical radiochromatographic scan was similarto those previously published (20).

Measurement of Dissolved Oxygen. DO level of HBSS in the vialswas initially determined under experimental conditions without cellsusing a Rank Brothers oxygen electrode (Cambridge, England). Theoxygen electrode was calibrated at the beginning of each experiment byadjusting to 100% DO level in HBSS (37°C)which was calculated to

be 283 ¿«Munder basal conditions (21) and for 0% of oxygen afteraddition of dithionite, a compound which avidly reduces dissolvedoxygen in solution. The dithionite was then completely washed outbefore starting the experiment. Nitrogen gas was passed through theair space over HBSS in the incubation vials via an inlet and outletneedle connection set up while monitoring the DO content. The electrode baseline was stable at the 100% DO level before monitoring theeffect of the nitrogen gas using 1 x 10*cells suspended in the electrode

chamber.Statistical Analysis. The data are expressed as the group mean ±

SEM of duplicate determinations from each "n". Student's t test for

grouped data was used. The 50% inhibitory concentration values weredetermined by probit analysis. In all cases, significance was at the levelof P < 0.05.

RESULTS

Formation of PCs in Murine Mammary AdenocarcinomaCells. The intactness of the plasma membrane as a marker forviability of the prepared cellular suspension was measured byexclusion of 0.4% trypan blue. Exclusion always exceeded 96%prior to and during incubation with arachidonic acid. The tumorcells were incubated with [I4C]AA for varying time intervals

(Fig. 1). A rapid rise in PGE2 formation was observed for up to20 min at the level of approximately 15% conversion of AA toPGE2, after which there was no further significant increase inPGE2 formation. The increases in the levels of PGF2., andPGD2 were relatively smaller. It is unlikely that the PG metabolites were reincorporated into the cells since the outer cellmembrane has been generally reported to be impermeable totheir inward diffusion (22). However, a relatively smaller decrease in the formation of all three prostanoids at 60 min mayrepresent catabolism by 15-hydroxyprostaglandin dehydro-genase. Formation of 6-keto-PGFi,, or thromboxane B2was notdetected in extracts of the cells. Subsequent studies used the20-min reaction period by which time the formation of PGE2had reached a plateau.

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Fig. 1. Formation of prostaglandins in mammary adenocarcinoma cells as afunction of time. Suspended cells (1 x 10") in HBSS were incubated with 2.0 JIM[I4C]AA for varying time intervals. Columns, mean; bars, SEM (n = 20).

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Fig. 2. Effect of indomethacin on prostaglandin formation in mammaryadenocarcinoma cells. Suspended cells (I x 10') were prcincubated with drug for30 min prior to incubation with 2.0 JIM[MC]AA for 20 min. Points, mean; bars,SEM (n = 6).

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Fig. 3. Measurement of DO levels under experimental conditions. At Arrow1, dithionite was added into the electrode chamber to deplete the DO. At Arrow2, an \ flush (1.5 liters/min) was initiated through the sealed vials, and the DOhad dropped by 87% after 30 min (Arrow 3). At Arrow 4, the DO baseline wasmonitored under aerobic conditions. A representative chromatogratn is shown (n= 3).

Effect of Inhibitors of Cyclooxygenase. The cells were prein-cubated for 30 min with either indomethacin (10~9 to IO"6 M)or ibuprofen (10~5to IO'1 M)prior to metabolism of arachidonic

acid. Both cyclooxygenase inhibitors decreased the formationof PGE2, PGD2, and PGF2„in a concentration-dependent manner as shown for indomethacin in Fig. 2. The data for ibuprofenare not shown. The 50% inhibitory concentration value forPGE2 inhibition was 6.3 x 10~s M and 9.6 x IO'5 M for

indomethacin and ibuprofen, respectively.Effects of Hypoxia on PC Biosynthesis. The dissolved oxygen

level in the vials was determined under experimental hypoxicand aerobic conditions (Fig. 3). The baseline for 0% dissolvedoxygen was determined by addition of dithionite, which avidlyreduces dissolved oxygen and immediately decreased its levelin the chamber to 0%. After the dithionite was washed out anda 10-min period to monitor baseline stability, nitrogen flow (1.5

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PROSTAGLANDIN BIOSYNTHESIS IN HYPOXIC CELLS

liters/min) was begun. A rapid decrease in dissolved oxygenlevel was observed as it dropped to 13% of control (37 /¿Moxygen) after 30 min. The decreasing dissolved oxygen levelapproached the 0% level after 60 min of N2 flow.

The cellular PG biosynthesis was decreased in a time-dependent manner with increasing periods of hypoxia prior toincubation with AA (Fig. 4). By 15 min, the biosynthesis wassignificantly decreased for PGF2„,PGE2, and PGD2. After 30min, the PG levels were decreased by approximately 50%. Thistime interval was chosen for subsequent studies. The synthesisof PGE2 and PGF2„was further lowered significantly in aconcentration-dependent manner to 3% of control by 1 /ÕMindomethacin under hypoxia, whereas alteration in PGD2 formation was not significant (Fig. 5). In this set of studies,hypoxia reduced PGE2 formation to 38% of aerobic control.

Effect of Misonidazole on PGE2 Biosynthesis. The effect ofmisonidazole on PG biosynthesis under hypoxic conditions isshown in Fig. 6. Under aerobic conditions, misonidazole didnot alter PG biosynthesis (data not shown), whereas underhypoxia, misonidazole increased the formation of PGE2 by 49%in a concentration-dependent manner over the range of 25 to100 UM. However, at 500 fiM and higher concentrations ofmisonidazole, PGE2 formation was attenuated. Hypoxic PGF2„production was also significantly increased in the presence of25 UMmisonidazole, but was not increased consistently. Underhypoxic conditions, indomethacin (0.1 J/M)completely blockedthe enhanced PG biosynthesis produced by misonidazole (100UM). At this concentration of indomethacin, PG biosynthesis was inhibited by approximately 40% of hypoxic control(Fig. 7).

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Fig. 4. Effect of hypoxia on prostaglandin biosynthesis in mammary' adenocarcinoma cells. Cells (1 x 10*) were exposed to increasing periods of hypoxiaprior to 20-min incubation with [14C]AA. Columns, mean; bars, SEM (n —¿�6).*, P < 0.05 as compared with control at zero time.

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Fig. 7. Effect of indomethacin on the formation of prostaglandins in thepresenceof misonidazole under hypoxic conditions. Cells were preincubated withindomethacin (INDO) and misonidazole (MISO) for 30 and 5 min, respectively.Hypoxia was induced by 30-min N2 flush, and the metabolism of [""CIAA wascarried out for 20 min. Columns, mean; bars, SEM (n = 3). *,f< 0.05 ascompared with hypoxic control.

At the maximum concentration studied, misonidazole neitheraffected cellular viability as determined by the intactness ofplasma membrane to trypan blue exclusion nor was the growthrate of these cells altered (data not shown). Imidazole at equi-molar concentrations did not change prostaglandin formationunder aerobic or hypoxic conditions. Imidazole also did notaffect cellular viability as measured by trypan blue staining(data not shown).

DISCUSSION

The 4526 murine mammary adenocarcinoma cell line wasshown to biosynthesize primarily PGE2, and this formation wasinhibited in a concentration-dependent manner by cyclooxygen-ase inhibitors such as indomethacin and ibuprofen, suggestingthat PGE2 formation in this model system was enzymatic.However, as PGE2, PGF2„,and PGD2 can be formed spontaneously by hydrolysis of their precursor, PGH2, demonstrationof the presence of the isomerase that forms PGE2 is requiredin order to determine whether or not the PGE2 formation inthis model was enzymatic. A time-dependent inhibition of PGE2formation was observed under hypoxia and was reversed selectively by misonidazole (25 to 100 UM) in a concentration-dependent manner. The enhanced PGE2 biosynthesis by misonidazole under hypoxia was completely blocked by indomethacin, suggesting that misonidazole does not act independently ofcyclooxygenase. The conditions used for the induction of hypoxia in these experiments were similar to those used previously

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PROSTAGLANDIN BIOSYNTHESIS IN HYPOXIC CELLS

for hypoxic cytotoxicity and radiosensitization studies (23).Incubation of the tumor cells with exogenously added AA

resulted in the formation of PGE2, PGF2„,and PGD2 similarto other studies (17). No evidence of prostacyclin or thrombox-ane formation was observed as indicated by the absence offormation of the stable hydrolytic products 6-keto-PGFi,, orthromboxane B2, respectively. Additional studies using sonicated cells to metabolize the endoperoxide substrate, PGH2,have demonstrated the presence of the reduced glutathione-dependent PGE2 isomerase in these cells (24). These studiesfailed to demonstrate the presence of reduced glutathione-dependent or -independent PGH2 to PGD2 isomerase or theenzymatic conversion of PGH2 to PGF2„;therefore, it seemslikely that the formation of PGF2„and PGD2 in this model issecondary to hydrolysis of PGH2.

After 30-min flushing with nitrogen through the sealed vials,the DO level decreased to 13% of control (37 MM)and continuedto drop over time. The Km of oxygen for cyclooxygenase hasbeen reported to be 5 MM(25), suggesting the observed inhibition of PGE2 biosynthesis under hypoxia was due to the decreasing DO level over the incubation period. Prostaglandinformation was inhibited by 50% under these conditions. After120 min of N2 flushing when the oxygen content decreased tothe 0% level similar to that produced by dithionite, the PGE2biosynthesis still remained higher than expected at approximately 20% of control values, suggesting that amounts of airsufficient to support cyclooxygenase activity might have beenintroduced with injection of substrate into the sealed vials.Since the controls for hypoxic vials received the same protocolas the drug-treated groups, any extra oxygen inserted duringthe injection of drug into the vial would have similar effects foreach group.

Misonidazole selectively augmented the hypoxic PGE2 formation without affecting aerobic PGE: biosynthesis up to aconcentration of 100 MM. In contrast, the concentrations ofmisonidazole required for radiosensitization of hypoxic cellsare in the range of 0.25 to 1 mM or higher. Moreover, thehypoxic cytotoxicity due to misonidazole is observed only atconcentrations of 1 mivi or higher and requires a longer incubation time of more than 4 h (23). It may be postulated thatreduction of the nitro group was coupled to the oxidation ofarachidonic acid, since the nitro function has been observed toundergo rapid reduction under these conditions (26). Alternatively, misonidazole may have acted as an oxygen-mimickingagent as a radical anión(27) to enhance the conversion of AAto PGH2 with subsequent metabolism to PGE2. When the cellswere incubated under hypoxia, with indomethacin (0.1 MM)¡nthe presence of misonidazole (100 MM),the resulting inhibitionof enhanced PGE? biosynthesis indicated that the increase insynthesis under hypoxia was due to the cyclooxygenase activity.These data also suggest that misonidazole is not able to protectthe cyclooxygenase activity from the inhibitory effect of indomethacin. The parent molecule, imidazole, which lacks the nitrofunction, did not elevate hypoxic PGE2 formation, indicatingthe necessity of the electron-affinic group. These data demonstrate that AA metabolism is sensitive to the differential oxygenlevels which exist within solid tumors and that PG levels maybe modulated by electron-affinic agents in hypoxic tumor cells.

ACKNOWLEDGMENTS

Great appreciation is expressed to Dr. Amy Fulton of the MichiganCancer Foundation for kindly supplying the tumor cell line and to

Suzanne M. Knoop and Barbara J. Rider for assistance in the analysisof these studies.

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1989;49:3853-3856. Cancer Res   David R. Shalinsky, Dennis B. McNamara and Krishna C. Agrawal  Mammary Adenocarcinoma Cells by MisonidazoleModulation of Prostaglandin Biosynthesis in Hypoxic Murine

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