Vol. 3, 433-438, July/August 1994 Cancer Epidemiology, Biomarkers & Prevention 433

Antiproliferative Effect of Nonsteroidal Antiinflammatory Drugsagainst Human Colon Cancer Cells1

Lee J. Hixson, David S. Alberts,2 Mary Krutzsch, JanineEinsphar, Klaus Brendel, Paul H. Gross, Nancy ShippParanka, Monika Baier, Scott Emerson, Rifat Pamukcu,and Randall W. Burt

Departments of Medicine IL. J. H., D. S. Al, Pharmacology ID. S. A.,

K. B.I, and Biometry IM. B., S. El, and the Arizona Cancer Center IM. K.,I. El, University of Arizona, Tucson, Arizona 85724; Department of

Chemistry, University of the Pacific, Stockton, California 9521 1 IP. H. Cl;Cell Pathways, Inc., Denver, Colorado 80290 IN. S. P.1; Department ofMedicine, University of Cincinnati, Cincinnati, Ohio 45221 IR. P.1; andDepartment of Medicine, University of Utah, Salt Lake City, Utah 841 12

IR.W.B.I

Abstrad

Several lines of evidence suggest that nonsteroidalantiinflammatory drugs may be effedive in preventingcoloredal cancer. These include animal experiments,case-control studies, and clinical experience withsulindac in promoting the regression of adenomatouscolon polyps in adenomatous polyposis coli. Wedetermined the antiproliferative activity of variousnonsteroidal antiinflammatory drugs, including twosulindac derivatives, against human colon cancer cells invitro. Ht-29, SW480, and DID-i cells were continuouslyincubated with serial drug dilutions for 6 days prior tofixation. Cell number was determined using thesulforhodamine B assay, and drug concentrations whichinhibited cell growth by 50% were estimated for eachagent by interpolation. All drugs exhibitedantiproliferative adivity against Ht-29 and DID-i cells,and most inhibited SW480 cells. For Ht-29 cells, the50% inhibitory concentration varied from 55 �M fordiclofenac to 21 00 �M for 5-aminosalicylic acid, withthree drug groups of high, intermediate, and lowpotency evident. Inhibition of cell growth by sulindacsulfide was reversible following drug removal.Nonsteroidal antiinflammatory drugs exert anantiproliferative effed against human colon cancer cellswith a wide range of potencies. A cytostatic responsewas demonstrated with sulindac sulfide. These datafurther support the potential role of these agents forchemoprevention of coloredal neoplasia.

Introduction

NSAIDs3 have considerable potential as chemopreventiveagents for colorectal cancer. Two recent case-control drug

Received 7/29/93; revised 4/18/94; accepted 4/18/94.1 This work was supported by NIH Grants CA-41 108 and CA-23074, and a

grant from Cell Pathways, Inc., Denver, Colorado.2 To whom requests for reprints should be addressed, at Arizona Cancer

Center, 1515 N. Campbell Avenue, Tucson, AZ 85724.3 The abbreviations used are: NSAID, nonsteroidal antimnflammatory drug;SRB, sulforhodamine B; DMSO, dimethyl sulfoxide; IC50, 50% inhibitory

concentration.

surveillance studies and one large cohort study found thatpatients with regular aspirin use had a reduced incidence ordecreased death rate from colonectal cancer (1 -3). Severaldifferent NSAIDs have been shown to reduce the formationof both colon adenomatous polyps (the precursor lesion ofcolon cancer) and cancers in experimental animals givenknown carcinogens (4-10). NSAIDs have also been dem-onstrated in animal models to inhibit the growth and din-cal expression of transplanted tumors and metastatic

spread (11-15), and to potentiate the antitumor effects ofimmunotherapy, radiotherapy, and cytotoxic drug therapy(1 6-21). Perhaps most intriguing are the reports that theNSAID sulindac promotes regression and inhibits recur-rence of adenomatous colon polyps in patients with adeno-matous polyposis coli (22-26).

NSAIDs inhibit cell growth and [3Hlthymidine incor-poration into cellular DNA in various cell culture models(e.g., rat hepatoma and colon carcinoma and human fibro-blast) and in human rectal mucosa explants (27, 28). Indo-methacin exerts a cytostatic effect by reversibly freezingcells in the C1 phase in vitro (29). We now extend these

earlier observations in assessing the relative inhibitory ef-fects of a series of 1 6 NSAIDs against 3 well characterizedhuman colon carcinoma cell lines. This assay is used as aprimary screening tool by the National Cancer Institute’sDivision of Cancer Treatment (Developmental TherapeuticsProgram) to identify candidate chemopreventive drugs (30).

Materials and Methods

Cell Culture Technique. The human colon carcinoma celllines Ht-29, DLD-l and SW480 were obtained from ATCC,Bethesda, MD and have been previously characterized (31).Cells were maintained in monolayer culture in RPMI 1640supplemented with 5% fetal bovine serum (Gemini Bio-products, Inc., Calabases, CA) and 2 m�i glutamine in ahumidified atmosphere of 95% air and 5% CO2 at 37#{176}C.Cell lines were monitored for the absence of Mycop!asmausing the Gen-Probe Mycoplasma Detection System (Gen-Probe, Inc., San Diego, CA). Subcultuning was done atsubconfluent densities. The cells were dispersed with asolution of 0.05% trypsin and 0.02% ethylenediamine-tetraacetic acid.

Measurement of Cell Proliferation. Cell number was de-termined using the SRB colonimetnic protein stain assay(30). Cells (500 cells/well) were plated in nonpenimeterwells in 96-well microtiter plates and incubated for 24 h at37#{176}Cprior to addition of drugs. All NSAIDs tested exceptsulindac sulfide and sulindac sulfone were purchased fromSigma Chemical Co. (St. Louis, MO). Sulindac sulfide andsulindac sulfone were synthesized as outlined below. Thesulindac compounds were solubilized in 1 00% ethanol andall other agents in 100% DMSO. Concentrations were di-luted with media so that final concentrations within wells

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434 NSAIDs Inhibit Human Colon Cancer Cells

contained a maximum of 0.5% DMSO or 1 0/ ethanol. Forinitial screening, each agent was tested at 5-6 10-fold

dilutions, generally starting with a maximum concentration

of 100-300 �M as solubility permitted. Smaller serial dilu-

tions were tested subsequently with selected agents to fur-then clarify a drug concentration which achieved an IC50.

For each plate, 1 2 wells were designated as media controls,6 wells were designated as DMSO or ethanol controls, and

the remainder of the wells were designated for drug dilu-tions with 6 wells per drug concentration. After 6 days of

culture, cells were fixed by the addition of cold tnichlono-acetic acid to a final concentration of 10%. Plates were

incubated at 4#{176}Cfor 60 mm; then the supernatant was

aspirated and the plates were washed with deionized water.

SRB (Aldrich Chemical Co., Milwaukee, WI) solution was

formulated to 0.4% w/v in 1% acetic acid; 100 p1 was

added to each well and the plates were incubated for 10

mm at room temperature. Unbound SRB was removed by

washing with 1 % acetic acid followed by air drying. Bound

stain was solubilized with 50 mt’�i unbuffered Tnis and ab-sorbance was read by an automated spectrophotometer at a

single wavelength of 540 nm.

To assess whether inhibition of Ht-29 cell growth isreversible, microtiter plates were prepared as described,

and after 24 h, drug was added at a single concentration to

designated wells. Cultures were incubated with drug orcontrol media continuously until assayed (after 1-7 days of

drug exposure) on drug was removed after 3 days and

replaced with control media after a single wash for the

remainder of the experiment (from 1 to 4 days after drug

removal).

Synthesis of Sulindac Sulfide and Sulindac Sulfone. (Z)-5-Fluoro-2-methyl-1 -(p-methylthiobenzylidene)-3-mndenylac-

etic acid (sulfide) and (Z)-5-fluono-2-methyl-1 -(p-methyl-sulfmnylbenzylidene)-3-mndenylacetic acid (sulfoxide) were

prepared according to the procedures described by Shuman

et a!. (32) and were found to be 99.7% pure. (Z)-5-Fluoro-2-methyl-i -(p-methylsulfonylbenzylidene)-3-mndenylacetic

acid (sulfone) was prepared as follows; to a cooled, stirred

mixture of sulfoxide (1 00 g, 0.281 mol) in MeOH (250 ml)

and CH3CN (500 ml), CH3ONa (4.4 M in MeOH, 68.5 ml,

0.30 mol) is added dropwise. After the mixture clears,NaHCO3 (47 g, 0.56 mol) is added, followed by the drop-

wise addition of H2O2 (30% in H20, 3.63 ml, 0.56 mol)keeping the reaction temperature below 5#{176}C.After sitting at-10#{176}Cfor 18 h, NaHCO3 is filtered off and washed withMeOH. The cooled (0#{176}C)and stirred filtrate is neutralized topH 7 by the dropwise addition of HCI (1 M, 350 ml). The

crystalline mass is stirred at 0#{176}Cfor i h, filtered off, andwashed with MeOH to give 93 g (89% yield); melting point,

thin layer chromatography, infrared spectroscopy, � H nu-clear magnetic resonance, 13C nuclear magnetic neso-nance, and elemental analysis confirm purity to be above

99. 7%�

Analysis of Data. For each drug, a minimum of three ex-

peniments measuring the growth inhibition of Ht-29 cellswas conducted. One or two experiments with each drugwere conducted with DLD-i and SW480 cells. For these

experiments, the mean ± SD of absorbance data from atleast 6 replicated wells were calculated for each drug con-centration tested. From these means, the inhibitory effect ofthe drug at each concentration was expressed as:

Table 1 IC50 (pm ) for NSAIDs again 51 colon cancer cells

DrugCell Type

Ht�29a DLD-1 b SW480”

5-Aminosalicylic acid 2100 ± 424

Acetylsalicylic acid 1867 ± 58

Diclofenac 55 ± 14 37 170

Fenoprofen 240 ± 66 300 360

Flurbiprofen 280 ± 85 210 330

Ibuprofen 520 ± 125 320 420

Indomethacin 66 ± 23 65 190

Ketoprofen 590 ± 204 280 NAC

Mefenamic acid 61 ± 1 9 45 NAC

Naproxen 623 ± 155 450 2200

Oxyphenbutazone 114 ± 41 16 41

Phenylbutazone 247 ± 67 84 105Piroxicam 410 ± 128 230 330

Salicylic acid 1 733 ± 666 520 NAC

Sulindac sulfide 64 ± 24 35 80

Sulindac sulfoxide 318 ± 85 175 280

Sulindacsulfone 119± 18 49 130

Tolmetin 630 ± 118 600 2500

.,Mean of �3 assays.5, Single assay or average of 2 assays.

C IC50 not achievable with maximum concentration of soluble drug in cell

media.

absorbance of treated cells% inhibition = x 100

absorbance of control cells

The IC50, the drug concentration causing a SO% reductionin the absorbance relative to the control wells, was thenestimated by interpolation from the inhibitory effects meas-ured at each concentration tested. The IC50 values fromeach experiment were then compared in an analysis ofvariance to test for differences among the drugs. The statis-tical significance between all pairs of drugs was assessedusing Tukey’s multiple comparisons test (33).

ResultsThe mean estimated IC50 values of 1 8 different NSAIDs(including 3 sulindac compounds) for Ht-29 cells are listedin Table 1 . The pairwise comparisons of the antiprolifera-tive potency of these agents in Ht-29 cells is demonstratedin Fig. 1 . Grouping ofthe drugs into high, intermediate, and

low potency categories is apparent. No statistically signifi-cant differences were noted among the estimated IC50 val-ues for salicylic acid, acetylsalicylic acid, and 5-aminosal-icylic acid. However, the estimated IC50 values of thesethree low potency drugs were judged to be significantlyhigher than for all other drugs tested. On the other end ofthe potency spectrum, the IC50 values for diclofenac, mefen-amic acid, sulindac sulfide, and indomethacin could not bestatistically distinguished from each other using this data,nor could they be statistically distinguished from oxyphen-butazone or sulindac sulfone on this basis. The 4 highpotency drugs were, however, judged to have significantlylower IC50 values than the other 1 2 drugs. Fenoprofen,phenylbutazone, flurbiprofen, sulindac sulfoxide, piroxi-cam, ibuprofen, ketoprofen, naproxen, and tolmetin, there-fore, appear to constitute a class of intermediate potencydrugs.

Representative drug concentration time curves forthree NSAIDs of various potencies in Ht-29 cells are dem-

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120

100

� 80>

U) 60

C

Q� 40

20

..--f

..f,.,

-I

,�

Tolmetin

uuululul � I I T�JUUU9�

-5 -4

Log 10 Molar Concentration-3

A

Sulindac Sulfide

C0

.cC

Ca0

a0.

(b)

2.4

2.0

� 1.6Ca

� 1.2

0.8

0.4

0.0

2.4

2.0

aC) 1.6a

� 1.2

0.8

0.4

0.0

1 2 3 4 5

Days

B

5-Fluorouracil

-4Log 10 Molar Concentration

(b)

6 7 8

onstrated in Fig. 2. Each curve represents the mean of atleast 3 experiments with the estimated IC50 for diclofenac,fenoprofen, and salicylic acid equivalent to 55, 240, and1 735 pM, respectively. Concentration time curves for thethree sulindac compounds are shown in Fig. 3. EstimatedIC50 values for these agents are sulindac sulfide (40 pM),

sulindac sulfone (120 pM), and sulindac sulfoxide (280 pM).

Inhibition of tumor cell growth by sulindac sulfide wasreversible after the removal of drug, suggesting a cytostatic

effect (Fig. 4). Following a 3-day drug exposure, cells re-sumed growth at a rate similar to control cultures after adelay of 24-48 h. Cell growth remained markedly sup-pressed as long as cultures were exposed to drug (up to 7days). In contrast, a cytotoxic response was evident with

5-fluorouracil in which cell growth did not recover despitedrug removal (Fig. 4).

The antiproliferative activity of most ofthe NSAIDs wasalso assessed in two additional human colon cancer cell

Cancer Epidemiology, Biomarkers & Prevention 435

Diclofenac

Mefenamic Acid

Sulindac Sulfide

Indomethacin

Oxyphenbutazone

Sulindac Sulfone

Fenoprofen

Phenylbutazone

Flurbiprofen

Sulindac Sulfozide

. . Fig. 3. Drug concentration time curves for sulindac sulfide )-), sulindacPiroxicam sulfone I. . . .) and sulindac sulfoxide (- - -I in Ht-29 cells. The IC5,, values

Ibuprofen are sulindac sulfide (40 pM), sulindac sulfone (1 20 pM), and sulindac sulfox-ide (280 pM). Each curve represents the mean ± SEM from �3 experiments.

Ketoprofen

Naproxen �

Salicylic Acid

Acetylsalicylic Acid

5- Aminosalicylic Acid

Fig. 1. Relative antiproliferative activity of NSAIDs against Ht-29 cells as

compared using Tukey’s multiple comparisons test. Vertical lines, groups ofdrugs which cannot be statistically distinguished on the basis of their IC50

values from the drug marked by the open circle.

(c)

Fig. 2. Drug concentration time curves for diclofenac (. . . .), fenoprofen

(- - -), and salicylic acid (-) in Ht-29 cells. The estimated IC50 values are (c)

diclofenac (55 pM), fenoprofen (240 pM), and salicylic acid (1 733 pM). Eachcurve represents the mean ± SEM from �3 experiments. 1 2 3 4 5

Days

Fig. 4. A and B, reversibility ofthe antiproliferative effect of sulindac sulfide

and 5-fluorouracil on Ht-29 colon cancer cell line growth. Drug sulindacsulfide (100 pM) or 5-fluorouracil (10 pM)l or control media was added to cellcultures at 24 h. Control wells (a) were assayed at timed intervals out to 8

days; wells with drug were exposed between days 1 -4, then washed free ofdrug and incubated in control media until assayed )b), or continually ex-posed to drug (c). A cytostatic response to sulindac sulfide is demonstrated

in which inhibition of cell growth is reversible (values for b at days 7 and 8are significantly higher than corresponding values for c). The response to5-fluorouracil is cytotoxic in that there was no reversal of 5-fluorouracil

growth inhibitory effects after its removal. Horizontal solid bars, drug expo-

sure duration.

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436 NSAIDs Inhibit Human Colon Cancer Cells

lines, DLD-1 and SW480 (Table 1 ). Although fewer nepli-

cative experiments were performed as compared to the datacollected with the Ht-29 cell line, the IC50 values observedwith each of the tested NSAIDs against the DLD-i andSW480 cells tend to sort these agents into similar categoriesof high, intermediate, and low potency. For example, tol-metin and salicylic acid tend to fall into the low potencygroup and sulindac sulfide and oxyphenbutazone fall intothe high potency group of NSAIDs with respect to the IC50values determined for each ofthe drugs in the three differentcell lines.

Discussion

This study demonstrates that a wide variety of NSAIDs exertantiproliferative activity against human colon cancer cellsin vitro. As reported previously with indomethacin (27, 29),we found that sulindac sulfide exerts a cytostatic rather thancytotoxic effect in that a normal rate of cell growth resumesafter removal of drug. This response in vitro concurs withreported clinical experience that sulindac prevents re-growth of macroscopic colorectal polyps in patients withadenomatous polyposis coli only with continuous drug dos-ing (22-25).

The best characterized pharmacological effect of theNSAIDs is to diminish prostaglandmn synthesis by inhibitingcyclooxygenase, which catalyzes the formation of prosta-glandmn precursors from arachidonic acid. Prostaglandmns

are potent mediators of numerous biological responses andprobably play a role in maintaining cellular viability and in

modulating both normal and neoplastic cell proliferation(34-37).

The mechanism by which the NSAIDs exert their cy-tostatic effects against Ht-29 colon cancer cells is unclean,but inhibition of prostaglandin synthesis would appear to bea plausible hypothesis because a variety of experimentalanimal and human tumors contain and/or synthesize highlevels of prostaglandins (7, 34, 38, 39). In addition, pros-taglandin synthesis has been associated with tumor promo-tion and increased metastatic potential (34, 40-42), and itpredicts which tumors will demonstrate slowing of growthin response to indomethacin (43).

Other lines ofevidence are contrary to the concept thatinhibition of prostaglandin synthesis is central to the NSAIDantiproliferative effect. Relatively high levels of prostaglan-dins have been reported to inhibit tumor cell growth both invivo and in vitro, and to inhibit differentiation in sometumor cell lines (34, 36, 44-49). Exogenous prostagland inshave also been demonstrated to inhibit basal mucosal DNAsynthesis in colon explants from animals (50, Si ). DeMelloet a!. (52) reported that NSAID concentrations which inhibitcell growth in rat hepatoma and human fibroblast cell linesin vitro correlate poorly with concentrations reported toinhibit cyclooxygenase in other studies. In addition, pros-taglandin synthetic activity was not detected in the rathepatoma cell line, and exogenous prostaglandmns did notreverse the antiproliferative effects of indomethacin.

Our results concur with those of DeMello et a!. (52) inthat the antiproliferative effect of NSAIDs does not appear tocorrelate with cyclooxygenase inhibitory activity. Piroxi-cam, the most potent cyclooxygenase inhibitor assessed inthis study, demonstrated intermediate activity against thehuman colon cancer Ht-29 cell line, whereas sulindac sul-foxide and sulindac sulfone which are reported to haveminimal or no prostaglandmn inhibitory activity (53, 54) arein the high potency range. Hypothetically, sulindac sulfox-

ide might have to some extent been reduced intracellulanlyto sulindac sulfide, a known potent inhibitor of cyclooxy-genase; however, sulindac sulfone is a metabolite resultingfrom irreversible oxidation of sulindac sulfoxide (53).

Mechanisms postulated to explain the antiproliferative/antitumor effects of NSAIDs other than through prosta-glandmn modulation include: (a) NSAID interference with aspectrum of membrane-associated processes including Cprotein signal transduction, tnansmembrane calcium flux,and cell-to-cell binding (55-57); (b) in addition to cyclooxy-genase, NSAID inhibition of the activity of other enzymesincluding phosphodiesterase, cyclic AMP-dependent pro-tein kinase, and some folate-dependent enzymes (58-60);(C) NSAID inhibition of cyclooxygenase cooxidation of non-lipid substrates to carcinogens during prostaglandmn synthe-sis (61-63); and (d) NSAID enhancement of a multitude ofimmunological responses which may have an importantrole in restoring host antitumor immunity (64, 65).

In conclusion, all of the tested NSAIDs demonstrateantiproliferative activity in two human colon cancer celllines (Ht-29, DLD-i ) and most agents inhibit proliferation ina third line (SW480). In additional studies, we have ob-served similar results in human lung and breast carcinomaand melanoma cell lines (A-427, MCF7/S, UACC375) (datanot shown). Although the effects of NSAIDs on colon can-cer cells may not be generalizable to all predysplastic ordysplastic cells, the ability of sulindac to promote regres-sion and prevent recurrence of adenomatous polyps in thesetting of adenomatous polyposis coli suggests that thesedrugs have a potential role for the chemoprevention ofcolorectal neoplasia. Which, if any, of the NSAIDs mightprevent the development/growth of sporadic adenomatouscolonic polyps in humans is currently unknown. Becausemost toxicity associated with chronic NSAID therapy isrelated to antiprostaglandmn activity, low-dose therapyand/or utilization of agents with less potent prostaglandininhibition conceptually would be more attractive as a po-tential ch non ic chemoprevention treatment.

AcknowledgmentsWe acknowledge Dr. Ken Drew for the chemical synthesis work and LindaLaFleur for preparation of the manuscript.

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