Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
(CANCER RESEARCH 55, 537-543, February 1. 1995) Evaluation of Chemopreventive Agents in Different Mechanistic Classes Using a Rat TrachéalEpithelial Cell Culture Transformation Assay1 Julia T. Arnold,2 Betty P. Wilkinson, Sheela Sharma,3 and Vernon E. Steele Cellular and Molecular Toxicology Program, ManTech Environmental Technology. Research Triangle Park. North Carolina 27709 ¡J. T. A., B. P. W.. S. S.I. and Chemoprevention Branch, Division of Cancer Prevention and Control, National Cancer Institute, NIH, Bethesda, Maryland 20892 ¡V.E. S.] ABSTRACT progress to human clinical trials (1). Chemopreventive agents already undergoing clinical trials include retinoic acid, ß-carotene, yV-(4-hydroxyphenyl) retinamide, vitamins C and E, piroxicam, cal cium, ibuprofen, oltipraz, difluoromethylornithine, 18-ß-glycyrrhetinic acid and /V-acetyl-L-cysteine (2, 3). The rat trachea! epithelial (RTE) cell focus inhibition assay was used to identify potential Chemopreventive agents. Ninety-nine agents were eval uated for their ability to inhibit benzo[a]pyrene-induced transformation of RTE cells. Freshly isolated RTE cells were exposed to benzo[a]pyrene alone or in combination with a Chemopreventive agent. After 30 days in culture, transformed foci were scored and inhibition was quantitated. In these studies, foci formation was inhibited mainly by agents which mod ulate the initiation of carcinogenesis by altering drug-metabolizing en zymes, inhibiting the binding of benzo[a]pyrene to DNA, enhancing de toxification of activated carcinogens, or by inducing epithelial cell differentiation. Such agents include antioxidants, free radical scavengers, glutathione S-transferase enhancers, vitamins, retinoids, and sulfhydryl compounds. Agents which inhibit ornithine decarboxylase and arachi- The multistage nature of the process of cancer development in cludes perturbations in the normal functioning within cells and the genome of the organism over a period of many years. It is a cyclical process of DNA damage, proliferation, clonal selection, and progres sion. This process could potentially be modulated by chemicals that effect cellular enzyme systems, gene expression, signal transduction pathways, differentiation, or interactions with surrounding cells and extracellular matrices. Many chemical compounds may have the abil ity to inhibit, retard, or reverse one or more stages of carcinogenesis and thus could affect the overall cancer incidence (4). A wide range of compounds has shown the ability to inhibit carcinogenesis in vivo (5). The most extensively studied suppressing agents are the retinoids (6, 7). Saffiotti et al. (8) found inhibition of hamster respiratory tract tumors with vitamin A, and Mass el al. (9) were able to inhibit the transformation of carcinogen-treated primary donic acid metabolism were not as effective. The RTE assay provides important data for agent selection prior to whole animal-screening assays in the development of chemoprevention drugs. INTRODUCTION Chemoprevention is an important defense strategy against human cancer since it is highly unlikely for one to avoid all carcinogenic insults. The objective of chemoprevention is to administer one or more chemical agents, naturally occurring or synthetic, which may have multiple biological mechanisms to inhibit various stages of carcinogenesis. Target populations for chemoprevention include those who by genetic background or previous occupational exposure are at higher risk for developing cancer, those who have had a primary cancer and seek to reduce recurrence, and the general population with unknown risks. Candidate Chemopreventive agents are identified by epidemio- trachéalepithelial cells with retinoid exposure. Other inhibitory agents occur naturally in allium and cruciferous vegetables (10-12). There is a large variety of chemical classes that can protect against cancer, including phenols, Õndoles,aromatic isothiocyanates, methylated flavones, coumarins, terpenes, dithiolthiones, plant sterols, protease in hibitors, selenium salts, ascorbic acid, tocopherols, and retinol (13). These inhibitors could be classified as blockers or suppressors by the stage in the carcinogenic process at which they exert their inhibitory effects: (a) preventing metabolic activation of carcinogen; (b) block ing reactive metabolites from cellular target sites; or (c) suppressing logical surveys, experimental research findings, clinical observations, promotion or progression of neoplasia. Antineoplastic agents such as antioxidants, modifiers of mixed-function oxidases, free radical scav or structural homology with known Chemopreventive agents. engers, or inducers of glutathione S-transferase could inhibit the A rigorous and systematic evaluation of the efficiency of these natural and synthetic agents is necessary before their usefulness in metabolism and binding of the carcinogen in the initiation phase, while anti-inflammatory agents, protease inhibitors, or inhibitors of cancer prevention can be evaluated in clinical trials. Because of the large number of potential agents, rapid and cost-efficient means of prostaglandin synthesis, ornithine decarboxylase, or protein kinases screening them are needed. In vitro assays such as the rat trachéal could suppress the promotion phase of carcinogenesis. Many of the epithelial cell focus inhibition assay (referred to as the RTE4 assay) agents tested in this study are multimechanistic and may have several are relatively inexpensive and have been developed to evaluate the of the above mentioned activities. effects of various agents on inhibiting cell transformation. These In this RTE assay, primary trachea! epithelial cells are treated with short-term in vitro systems provide data for the selection and ranking B[a]P for 24 h in the presence of the test Chemopreventive agent. The of potential Chemopreventive chemicals for whole animal tests, ac carcinogen is removed and the cells are cultured for 30 days with the celerate the rate of chemical evaluation, and provide data on possible test agent. This allows the agent to be present in the early stages of initiating events. Normal cells differentiate after 2-3 weeks, whereas mechanisms of action. Agents found positive in the whole animal tests B[Ã-Ã-]P-induced cells continue to grow to form colonies or foci. At 30 Received 8/8/94; accepted 12/1/94. days, foci of morphologically transformed, preneoplastic cells are The costs of publication of this article were defrayed in part by the payment of page identified, and inhibition is scored as a decrease in the number of these charges. This article must therefore be hereby marked advertisement in accordance with foci compared to the B[a]P alone-treated cultures. 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported by the National Cancer Institute Contracts N01-CN-55503-05, N01The RTE assay has been shown to be sensitive to several classes of CN-95172-02, and NOI-CN-95172-06. Chemopreventive agents (14-19). Of the 99 compounds tested in this 2 Present address: Department of Pathology, CB #7525, University of North Carolina, Chapel Hill, NC 27599-7525. study, it was generally found that morphological transformation was 3 To whom requests for reprints should be addressed, at ManTech Environmental inhibited or reduced by agents that are more effective in protecting Technology, P.O. Box 12313, Research Triangle Park, NC 27709. against DNA damage than those that may be antiproliferative. The 4 The abbreviations used are: RTE, rat trachéalepithelial; B[a]P, benzo|«]pyrene; ODC, ornithine decarboxylase; CFE, colony-forming efficiency. compounds that tend to be positive in the assay are those that prevent 537 Downloaded from cancerres.aacrjournals.org on July 28, 2017. © 1995 American Association for Cancer Research. CHEMOPREVENTIVE MECHANISMS or block DNA damage by: (a) altering drug-metabolizing enzymes (antioxidants, modifiers of mixed-function oxidases); (b) inhibiting binding of B[a]P to DNA (inducers of glutathione 5-transferase and free radical scavengers); (c) detoxifying B[a]P by increasing gluta thione levels; or (d) inducing differentiation (retinoids). Other posi tive classes of compounds include antihistamines, immunomodula- CELLS Compound Solubility. For each chemopreventive test agent, the highest soluble concentration up to 1 mM was tested for solubility in RTE cell culture media (see below). If insoluble in media, one of the following solvents was used: DMSO; ethanol; acetone; or tetrahydrofuran, with the final concentration on the cells not exceeding 0.2, 0.5, 0.3, or 1.0% respectively. Assay for Inhibition of Transformation. Trachea! cell isolation methods, culture media requirements, assay protocols, and data analysis have been described previously (16), and are summarized briefly here. An initial rangefinding assay was performed for each chemopreventive test agent over a wide range of concentrations to determine nontoxic concentrations for the focus inhibition assay. Concentrations were considered nontoxic if the mean CFE was within 20% of control values, since such values were statistically com parable to control. The highest nontoxic dose plus four half-log dilutions were tors, vitamins, flavonoids, and sulfhydryl compounds. The RTE assay has been found to be a good predictor of in vivo activity of chemopreventive agents. It has a predictive value for the hamster lung model, correctly identifying 15 of 23 agents, and was especially helpful in identifying the negative agents (20). Compounds that affect the later stages of cell proliferation and progression are not as frequently positive in the RTE assay. The short duration of the RTE assay may not be adequate to identify agents with mechanisms associated with progression, such as inhibitors of orni- used for the focus inhibition assay, which included the following nine exper imental groups: medium control, solvent control, B[a]P alone at 10 fig/ml, B[o]P plus all-/rans-retinoic acid at 30 nM (positive control), and B[a]P plus test chemopreventive agent (five groups at half-log concentrations). thine decarboxylase activity, prostaglandin synthesis, protein kinase activity, or anti-inflammatory agents. This assay, in conjunction with Primary rat trachéalcells were isolated. All experimental groups except media and solvent controls were exposed to 10 /xg/ml B[a]P and the test agent or retinoic acid on day 1 for 24 h. On day 2 the carcinogen was rinsed from the cells which were then cultured until day 30, refeeding twice/week. The test groups received fresh chemopreventive agent, while the B[a]P and solvent controls received culture media with appropriate solvent concentrations. Par allel dishes were set up for testing of cytotoxicity under actual assay conditions and were scored on day 6 for CFE. On day 15, the media were reduced in serum and growth factors to increase selection for transformed foci. On day 30, the cultures were fixed, stained, and scored for morphologically transformed colonies or foci. Previous studies have shown that Class III transformed colonies or foci that have greater than 2500 cells/mm2 can progress to form a battery of other in vitro assays that are able to detect inhibition at later stages of cell transformation, should provide an effective screen to identify cancer-preventing compounds. MATERIALS IN EPITHELIAL AND METHODS Chemicals. The following test agents were obtained from Aldrich Chem ical Co. (Milwaukee, WI): /V-(6-Aminohexyl)-5-chloro-l-naphthalene sulfonamide; apigenin; arginine HC1; benzyl isothiocyanate; carnosine; chlorogenic acid; curcumin; dehydroepiandrosterone; dimethyl fumerate; ethylvanillin; fu marie acid; sodium molybdate; propyl gallate; purpurin; simethicone; sodium thiosulfate; ursolic acid; vanillin; and vitamin Kv Folie acid, quinacrine HC1, and uric acid were purchased from Chemical Dynamics Corp. (South Plainfield, NY). The garlic-derived compounds allyl tumors when injected into nude mice (21). A very high percentage of Class II foci (1300-2500 cells/mm2) also became tumorigenic. Class I foci (<1300 cells/mm2) usually do not progress to tumorigenicity. Therefore, in this assay only Class II and III foci were considered as morphological evidence of early transformation events. Numbers of foci from experimental groups were aver aged, solvent control background was subtracted, and the results were com pared to the group treated with B[a)P alone to calculate the percentage of inhibition. The data was analyzed and considered positive if the agent inhibited carcinogen-induced foci formation by 20% or more over the control of B[o]P methyl disulfide, diallyl sulfide, and diallyl trisulfide were supplied by Co lumbia Organic Chemical, Inc. (Camden, SC). Anethole trithione was obtained from Solvay Pharma LTM, Suresnes, France. The following agents were obtained through the National Cancer Institute Division of Cancer Prevention and Control Repository, (Bethesda, MD): BASF 47848; BASF 47850; BASF 47851; BASF 51328; carbenoxolone; etoperidone; ß-glycyrrhetinic acid; oltipraz; retinoyl-D,L-leucine; RO16-9100; and RO19-2968. alone at nontoxic concentrations. All culture materials and actual test cultures were tested routinely for contamination by bacteria, fungus, yeast, and Mycoplasma. If contamination was found, the media, media components, or cultures were not used in the experiments. Other agent suppliers include: allyl methyl trisulfide, Oxford Chemicals, Ltd. (Bloomfield, NJ); p-aminobenzoic acid, Spectrum Chemical Manufactur ing (Gardner, CA); p-bromophenacyl bromide and DMSO, Pierce (Rockford, IL); cromolyn sodium, Interchem Corp. (Paramus, NJ); 5,8,11,14-eicosatet- RESULTS AND DISCUSSION In this study, 99 natural or synthetic compounds were screened for chemopreventive activity using the RTE assay. Out of the 99 potential chemopreventive agents tested, 58 were found to be positive and 41 were negative for inhibiting B[a]P-induced morphological transfor mation. The concentration ranges for testing the agents in the RTE assay were chosen to be the highest nontoxic concentrations as de termined by the initial range-finding assay. A cytotoxicity assay was raynoic acid, Biomolecular Research Labs, Inc., (Plymouth Meeting, PA); lovastatin, Merck, Sharp and Dohme (West Point, PA); Maharishi Amrit Kalash 4 and 5, Maharishi Ayurveda Products (Lancaster, MA); myricetin, Fluka Biochemika (Ronkonkoma, NY); phenethylisothiocyanate, Eastman Kodak Co. (Hartford, CT); phloretin, Transworld Chemical, Inc. (Rockville, MD); sodium suramin, FBA Pharmaceuticals (West Haven, CT); thiolutin, Pfizer (Doreville, GA); a-tocopherol succinate-polyethylene glycol-1000, Eastman Chemical Products Company (Kingsport, TN). Ajoene was obtained from Dr. Eric Block of Albany State University. Chlorophyll was obtained from American Tokyo Kasei, Inc. (Atlanta, GA). Ascorbyl palmitate, glycerol monooleate, lanosterol, propylene glycol, and riboflavin-5'-phosphate were performed concurrently with the transformation inhibition assay to assess the cytotoxicity at the actual test concentrations. If cytotoxicity was noted during the actual test, the data for that concentration was not considered. Compounds were scored positive if they reduced the transformation frequency by >20% compared to control (B[a]P exposure alone). Results of all agents tested are given in Table 1 where the 99 compounds are listed in alphabetical order in column 1. In column 2, the concentration that induced maximum inhibition of transformation is given in (UM.If transformation was not inhibited, then the highest concentration tested is shown. The third column indicates the maxi mum percentage of inhibition of B[a]P-induced foci, compared to B[a]P alone controls. If some of the test concentrations were cytotoxic (with less than 80% of control colony-forming efficiency) those obtained from Pfaltz and Bauer, Inc. (Waterbury, CT). The remaining compounds were purchased from Sigma Chemical Co. (St. Louis, MO): acetylsalicylic acid; amiloride HC1; antineoplaston AIO; benzo[a]pyrene; ß-carotene; caffeic acid; cysteamine HC1; diphenhydramine; ferulic acid; glycine; a-glycyrrhetinic acid; hydrochlorothiazide; inositol hexaphosphate sodium; indomethacin; D-mannitol; meclizine; meclofenamate sodium; melatonin; D-L-methionine; /»-methoxyphenol; méthylène blue; miconazole; morin; nicotinic acid; D-L-palmitoylcarnitine Cl; polyvinylpyrrolidone; praziquantel; promethazine; all-/rans-retinoic acid; rhapontin; sodium selenite; ß-sistosterol; steviol; sulfasalazine; sulindac; tetracycline; transforming growth factor /3; thioctic acid; 2-thioxo4-thiazolidone; triprolidine; and verapamil. 538 Downloaded from cancerres.aacrjournals.org on July 28, 2017. © 1995 American Association for Cancer Research. CHEMOPREVENTIVE MECHANISMS [N EPITHELIAL CELLS Table 1 In vitro screening of potential chemopreventive agents using the rat trachéalepithelial cell focus inhibition assav Primary rat trachea! epithelial cells were treated with B[fl]P for 24 h in the presence of the test chemopreventive agent. The carcinogen was removed and the cells were cultured for 30 days with the tesi agent. Foci of morphologically transformed, preneoplastic cells were identified, and inhibition was scored as a decrease in the number of these foci compared to the B[a]P-alone treated cultures. AgentAcetylsalicylic (fAM)"5550.30.00251.9740.3752.1880.124121.811.1043.77.2290.2640.3132.760.20113216.65.2513265.58828.2156558.60.27126.410.40.263 concentration inhibition (%)ME*NE25NENE963923NENE98NE1007610058NENENE4954NENE70911 acidAjoeneAllyl disulfideAllyl methyl trisulfideAmiloride/j-Aminobenzoic methyl acidAnethole tritinoneAntineoplaston AIOApigeninArginine HC1Ascorbyl palmitateBASF 47848BASF 47850BASF-47851BASF 51328Benzyl isothiocyanate/7-Bromophenacyl BrCaffeic acidCarbenoxoloneCarnosineß-CaroteneChlorogenic acidChlorophyllCromolyn, NaCurcuminCysteamine HC1DHEADiallyl sulfideDiallyl trisulfideDimethyl fumerateDiphenhydramineEthylvanillinEtoperidoneETYAFerulic acidFolie acidFumarie acidGlycerol monooleateGlycinea-Glycyrrhetinic acidß-Glycyrrhetinic acidHydrochlorothiazideIMPIndomethacinLanosterolLovastatinMAK-4MAK-5D-MannitolMeclizineMeclofenamate, tig/ml10 fig/ml54890.65218842.620.124.10.0802.0914579.91.08.121330.00690.0180.10941000.320.09414.1131420.1170.00062.382382092.620.0030. NaMelatoninD.L-Methionine/7-MethoxyphenolMéthylène blueMiconazoleMolybdate, NaMorinMyricetinNicotinic acidOltiprazD.L-Palmitoylcarnitine HC1Phenethylisothiocy anatePhloretinPolyvinylpyrrolidonePraziquantelPromethazinePropyl gallatePropylene glycolPurpurinQuinacrine HC1Retinoyl-D,L-leucineRhapontinRiboflavin-5'-phosphateRO16-9100RO 19-2968Selenite, NaSimethiconeß-SistosterolSteviolInhibitory 539 Downloaded from cancerres.aacrjournals.org on July 28, 2017. © 1995 American Association for Cancer Research. CHEMOPREVENTIVE MECHANISMS IN EPITHELIAL CELLS Table 1 Cimunued inhibition Result'NENE100 (%)* AgentSulfasalazineSulindacSuramin, concentration (/¿M)"75.328.1216.80.4 NaTetracyclineTGF-ßThioctic +64 +90 nM48.50.0001319.022.50.0660.31817.90.66065.70.610.100.2220.080Maximum +NE69 acidThiolutinThiosulfate, +NE85 Na2-Thioxo-4-thiazolidonea-Tocopherol +52 +33 +23 +NENE57 1000TriprolidineUric succinate PEGacidUrsolic acidVanillinVerapamilVitamin +100 +55 +NE AVitamin K,W-7Inhibitory " The concentration that induced maximum inhibition of transformation is given in /IM. If transformation was not inhibited, then the highest concentration tested is shown. '' Maximum percentage of inhibition of B[«]P-induced foci, compared to B[u]P alone controls. Agents with less than 20% inhibition are listed as not effective. ' If the inhibitory activity at a nontoxic dose was greater than 20%, a positive sign (+) is shown in the result column. If less than 20% then the result is negative (-). '' NE, not effective; DHEA, dehydroepiandrosterone; ETYA. 5.8,11,14-eicosatetraynoic acid; IHP, inositol hexaphosphate, sodium; MAK-4 and MAK-5. Maharishi Amrit Kalash, formula 4 and 5, aqueous extract used; TGF-ß,transforming growth factor ß;a-tocopherol succinate PEG, ot-tocopherol succinate polyethylene glycol 1000; W-7, W-(6-aminohexyl)5-chloro-l-naphthalene sulfonamide. inhibition results were discarded and only nontoxic results are shown. Agents with less than 20% inhibition are listed as not effective. The last column indicates a positive or negative response in the RTE assay. If the inhibitory activity at a nontoxic dose was greater than 20%, a positive sign (+) is shown in the result column. If the inhibitory activity was less than 20% then the result is negative (-). All results shown are from single trials due to the screening nature of the study. Tests are considered valid if: (a) the retinoic acid-positive control inhibits agents found to have these activities in other systems, 22 were found to be positive in the RTE assay. Table 2 lists these agents starting with the most effective at inhibiting B[a]P-induced transformation. Anti oxidants block initiation by scavenging free radicals that are involved in the activation of carcinogens. Antioxidants may produce changes in the metabolite profile of B[a]P, contributing to increased detoxifica tion or decreased activation of B[a]P. They can also act by altering carcinogen-metabolizing enzymes in microsomes (uridine 5'-diphos- transformation by 20% or more; (b) B[a]P induces transformation at least twice that of background levels; and (c) there are at least 8 dishes scored in critical control or treatment groups. Transformation frequencies of B[a]P-treated cultures averaged 3-6 times over background. These chemopreventive test agents have been reported to exhibit various biological activities and mechanisms of chemoprevention. An analysis of the mechanisms of the positive agents in this assay gives insight into what mechanisms of chemoprevention may be involved in the inhibition of cell transformation in the RTE assay. The most common mechanistic categories of the test agents are listed: (a) antioxidants and free radical scavengers; (b) retinoid derivatives; (c) enhancers of glutathione 5-transferase; (d) antihistamines; (e) anti-inflammatory agents; (/) phoglucuronyltransferase, aniline hydroxylase) or in the cytosol (glucose-6-phosphate dehydrogenase, UDP-glucose dehydrogenase, glutathione 5-transferase, and epoxide hydratase) (22). Beyond initiation, free radical scavengers may be effective by preventing the formation of tumor-promoting reactive electrophiles, by preventing lipid peroxidation, or by gene or enzyme activation or deactivation. Compounds with these activities could block the neoplastic process in various stages. One of the effective free radical scavengers was sodium selenite. Selenium is a necessary cofactor for the enzyme glutathione peroxidase, which catalyzes the reduction of hydrogen peroxide and hydroperoxides within the cell. Selenium-dependent glutathione inhibitors of arachidonic acid metabolism and prostaglandin synthesis; (g) inhibitors of ODC; and (h) inhibitors of protein kinase C. These mechanisms can be grouped as anti-initiating or antipromoting. A potential chemopreventive agent may have an inhibitory effect anywhere in the cancer process. Some agents act to block metabolism of the carcinogen or increase detoxification pathways in the cell. These mechanisms would be considered anti-initators since they would inhibit carcinogen-DNA adduci formation. Other agents may inhibit signal transduction pathways, ornithine decarboxylase, or pros taglandin synthesis that are regulators of cellular proliferation. These agents are called antiproliferators. Many agents have been found to have multiple biological activities, and may be represented in several mechanistic classes. While it was beyond the scope of this study to determine the exact mechanisms involved in inhibiting B[o]P-induced transformation in the RTE assay, it is helpful to gain insight into those classes of agents that are detected by the RTE assay to aid in future selection of test agents. Fig. 1 indicates the percentage of positive agents in each mechanistic class and gives the number of positive agents out of the total number in that category. Two mechanisms strongly represented by the agents positive in the RTE assay are the antioxidants and free radical scavengers. Out of 32 Antioxidants/FRS |326 Retinoids |85|93 GSH Enhancers AntihistaminesAntiinflammatory ¡31 |79 188|16lj4 InhibitorsODC AA | Inhibitors PKC Inhibitors22 O 20 40 60 80 100 120 Percent Positive Agents Fig. 1. Response of various mechanistic classes in the RTE Assay. Most common mechanistic classes are represented, expressing the percentage of positive agents. For each class, actual numbers of positive agents (expressed within ham) is given out of total numbers (expressed outside bars). FRS, free radical scavengers; GSH, glutathione; A4, arachidonic acid; PKC, protein kinase C. 540 Downloaded from cancerres.aacrjournals.org on July 28, 2017. © 1995 American Association for Cancer Research. CÕIEMOPREVENTIVE MECHANISMS peroxidase may lower the level of potentially damaging peroxide radicals generated from B[a]P metabolism. Selenium affects the me tabolism and DNA binding of polcyclic hydrocarbons such as 7,12- IN EPITHELIAL CELLS Table 3 Mechanistic classes of agents weakly detected hy the RTE assay (%)"10091787364Ml412929NENENI AgentArachidonic inhibitorsMeclofenamatc, acid NaCurcuminIMPa-Glycyrrhetinic dimethylbenz(a)anthracene (23). Selenium was positive in the RTE assay, completely suppressing transformation (100%) at 0.578 ¿IM. Agents that induce glutathione 5-transferase are found to be posi acidTelracyclineMorinß-Glycyrrhetinic tive in the RTE assay. Five of nine agents with this mechanism tested positive (Table 2). These agents also contain a sulfhydryl group. Increasing cellular levels of glutathione can protect against initiation events by directly scavenging or trapping electrophilic carcinogenic metabolites such as B[o]P-7,8-diol-9,10 epoxide, thus detoxifying the carcinogen (24). The most effective agent in this group was oltipraz, a synthetic dithiolthione which provided 98% inhibition at 133 /AM. acidETYAOuinacrine HC1Acetylsalicylic acidAjoeneApigenin/>Bromophenacyl NI;NENENENENE917360 BrCaffeic acidHydrochlorothiazideMyricetinPropyl gallateW-7Ornithinc Table 2 Mechanistic Classes of agents effectively detected by the RTE assay inhibitorsCurucumina-Glycyrrhetinic decarboxylase AgentAntioxidants/frec scavengersFerulic radicai acidSelenite, NaMeclofenamate, NaAscorbyl palmitatePromethazine/j-Aminobenzoic acidMorinVerapamilPurpurinSteviolß-Glycyrrhetinic acidD.L-Palmitoylcarnitine HC1ApigeninArginine acidCurcuminEthylvanillinIHF'MAK-5TetracyclineMAK-4PhloretinMorinFolie HC1/»-Bromophenacyl BrGlycineIndomcthacinMyricetinPropyl HNL;NI;NE29NENENE100NI;NENENENI;NE gallateW-7Prolein acidVitamin K,ß-Carotenea-Tocopherol inhibitorsD,L-Palmitoylcarnitine kinase C HCIAmilorideRiboflavin-5 1000CarnosineETYAUric succinate-PEG '-PhosphateThiosulfate, NaAnti-inflammatory agentsMeclofenamate. acidDimethyl (umeraleApigeninChlorophyllFumarie NaAcetylsalicylic acidCarbenoxoloneIndomethacinu-MannitolSulfasalazineUrsolic acidCaffeic acidMéthylène blue/)-MethoxyphenolMyricetinPropylene " Maximum AcidInhibition percentage of inhibition of B[n]P-induced foci, compared to B[fl]P alone controls. Agents with less than 20% inhibition arc listed as noi effective. Abbreviations: 1HP, inositol hexaphosphate, sodium: ETYA, 5,8,11,14-eicosaletraynoic acid: NE, not effective (<20% inhibition): W-7, ¿V-(6-aminohexyl)-5-chloro-l -naphthalene sulfonamide. glycolPropyl gallaleChlorogenic acidGlutathione enhanchersOltiprazBenzyl isothiocyanateDiallyl trisulfideAnethole trithioneAllyl disulfideDiallyl methyl sulfideAjoeneAllyl Oltipraz is an antischistosomal drug which causes induction of the glutathione 5-transferase liver enzymes, and has been found recently to induce another protein, alfatoxin B, aldehyde reducÃ-ase,in livers of oltipraz-treated animals and is used as a biomarker for oltipraz effect.5 trisulfidePraziquantclRetinoid methyl The retinoids are highly effective as chemopreventive agents and some, such as 4-hydroxyphenol retinamide (tested previously, Ref. 16), are currently in use in clinical trials to prevent recurrence of breast cancers (25). The retinoid analogues tested in this study were synthesized to provide chemoprotection with less toxicity. In the RTE ABASF 47850BASF-47851RO assay 6 of 9 retinoids were positive at nontoxic doses, providing complete inhibition in nearly all assays (Table 2). The retinoids have 19-2968BASF anti-initiating mechanisms as well as the ability to induce differenti 47848AntihistaminesDiphenhydramineCromolyn. ation (26). All of the three agents known to be antihistamines gave positive NaTriprolidineInhibition(%)"100UHI10098"7%4187787h64f>4h260565554524')292323NENENENENENENENENENEW58533925NENENENEUH)10010010010076NENE1007033 results in this assay (Table 2). How they may function to inhibit Maximum percentage of inhibition of B[a)P-induced foci, compared to B[a]P alone B[a]P- induced transformation in an in vitro system is unclear. derivativesBASF 1328Retinoyl-D-L-leucineR016-9100Vilamin 5 " controls. Agents with less than 20% inhibition are listed as not effective. * NE, not effective (<20% inhibition); IHP, Inositol hexaphosphate, sodium; ETYA, 5 S. Sharma, G. P. Wyatt, L. N. Anderson, V. E. Steele, and G. J. Kelloff, Character 5,8,11,14-eicosatetraynoic acid; MAK-4 and MAK-5, Maharishi Amrit Kalash, formula 4 and 5, aqueous extract used; a-tocopherol succinate PEG 1000, a-tocopherol succinate polyethylene glycol 1000. ization of a 38 Kdalton prolein that is highly induced by oltipraz, preparation. manuscript 541 Downloaded from cancerres.aacrjournals.org on July 28, 2017. © 1995 American Association for Cancer Research. in CHEMOPREVENTIVE MECHANISMS Diphenhydramine, the active agent in nonprescription antihistamines, was the most effective agent in this group with 100% inhibition at 34 /UM. Agents classified as antiproliferators are those that inhibit the progression of an initiated, preneoplastic cell. This group would include agents that inhibit ODC activity, inhibit the metabolism of arachidonic acid to prostaglandins, and inhibit protein kinase activity. These activities are characteristics of proliferating cells that when modulated, appear to inhibit cancer progression. ODC is the ratelimiting enzyme in the synthesis of polyamines, which are involved in proliferation and differentiation (27). Arachidonic acid metabolites serve as second messengers in proliferating cells (28). Protein kinases are involved in signal transduction, and protein kinase C is the putative receptor for the phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (29). Agents which act to inhibit these mechanisms did not show as strong a positive response in the RTE assay as did the agents with anti-initiating mechanisms (Fig. 1 and Table 3). Some of the positive agents are listed both within antiproliferating and anti-initi ating mechanistic classes. For instance, curcumin (found in tumeric) can act as an antioxidant and an ODC inhibitor. Another agent, meclofenamate sodium, is a pharmaceutical anti-inflammatory agent, and also acts as a free radical scavenger and arachidonic acid inhibitor. These agents and others had been shown to be effective chemopreventive agents by multiple mechanisms. While the exact mechanisms in the RTE assay are not known, these compounds may prove to be valuable candidates for cancer pre vention due to their multiple activities. Individual biomarker stud ies using these cells would aid in defining the exact mechanism used by these agents in the RTE assay (30). In examining the mechanistic nature of the 58 agents which were positive in this assay, several chemopreventive mechanisms appear to be consistently prominent. These were agents that: (a) induce differ entiation such as the retinoids and analogues; (b) increase glutathione levels or enhance conjugation of carcinogenic metabolites; (c) display antioxidant activity or sequester free radicals; and (d) act as antihis tamines. These results confirm our previous findings of 28 chemo preventive agents (16). There were 41 agents that were negative in the RTE Assay. The assay does not readily detect compounds which are: (a) antiprolifer ating in nature; (b) anti-inflammatory agents; (c) inhibitors of arachi donic acid metabolism and prostaglandin synthesis; (d) inhibitors of ornithine decarboxylase; or (e) inhibitors of protein kinase C. Since many agents have multiple mechanisms, it is difficult to tell which mechanism is key in this assay. Some antipromoter mechanisms may also be important in anti-initiation, for instance, inhibitors of prosta glandin synthetase may also inhibit carcinogen-activating enzymes. It is felt that due to the treatment protocol and the relatively short 30-day term, the RTE assay is not as sensitive to inhibition by antipromoters. The data compiled and analyzed from 99 chemopreventive agents indicate that the RTE assay was most effective in identifying chemo preventive compounds which act to block DNA damage during the carcinogenic process. Any effort to fit the agents into categories in order to better understand the mechanisms of cancer prevention could be criticized as oversimplifying the complex interaction of the mul tiple mechanisms of these agents within the complex etiology of cancer. One can at best look for trends in the data to validate the ability of the RTE assay to predict potential chemopreventive agents and to try to understand which mechanisms are most effective in inhibiting B[a]P-induced carcinogenesis in RTE cells. These results help identify other chemicals that have similar mechanistic or struc tural classes, which may provide greater potential chemopreventive activities or less toxicity. As part of a screening program this assay [N EPITHELIAL CELLS helps to quickly define agents with chemopreventive potential for further development in animal studies and clinical trials. ACKNOWLEDGMENTS We acknowledge CCS Associates, Inc. (Mountain View, CA) for informa tion on agent mechanisms. This information was provided to National Cancer Institute under contract No. NO1-CN-25417. We are also grateful to Gail P. Wyatt and Kyle Garris for technical support. REFERENCES 1. Boone, C. W., Steele, V. E., and Kelloff, G. J. Screening for chemopreventive (anticarcinogenic) compounds in rodents. Mutât.Res., 267: 251-255, 1992. 2. Boone, C. W., Kelloff, G. J., and Malone, W. E. Identification of candidate cancer chemopreventive agents and their evaluation in animal models and human clinical trials: a review. Cancer Res., 50: 2-9, 1990. 3. Kelloff, G. J., Boone, C. W., Malone, W. F., and Steele, V. E. Chemoprevention clinical trials. Mutât.Res., 267: 291-295, 1992. 4. Wattenberg, L., Lipkin, M., Boone, C. W., and Kelloff, G. J., (eds.) Cancer Chemo prevention, Boca Raton, FL: CRC Press, 1992. 5. Bagheri, D.. Doeltz, M. K., Fay J. R., Helmes, C. T., Monasmith, L. A., and Sigman, C. C. Database of inhibitors of carcinogenesis. J. Environ. Sci. Health C6: vii-xviii, 262-403, 1988-1989. 6. Sporn, M. B., Roberts, A. B., and Goodman, D. S. (eds.). The Retinoids, New York: Academic Press, 1984. 7. Bertram, J. S., Kolonel, L. N., and Meyskens, F. L. Rationale and strategies for chemoprevention of cancer in humans. Cancer Res., 47: 3012-3031, 1987. 8. Saffiotti, U., Montesano, R., Sellakumar, A. R., and Borg, S. A. Experimental cancer of the lung: inhibition by vitamin A of tracheobronchial squamous metaplasia and squamous cell tumors. Cancer (Phila.), 20: 857-864, 1976. 9. Mass, M. J., Nettesheim, P., Beeman, D. K., and Barrett, J. C. Inhibition of trans formation of rat trachéalepithelial cells by retinoic acid. Cancer Res., 44: 5688—5691, 1984. 10. You, W. C., Blot, W. J., Chang, Y. S., Ershow, A., Yang, Z. T., An, Q., Henderson, B. E., Fraumeni, J. F., Jr., and Wang, T. G. Alunni vegetables and reduced risk of stomach cancer. J. Nail. Cancer Inst., 81: 162-164, 1989. 11. Wattenberg, L. W. Chemoprevention of cancer. Cancer Res., 48: 1-8, 1985. 12. Wattenberg, L. W., Sparnins, V. L., and Barany, G. Inhibition of /V-nitrosodiethylamine carcinogenesis in mice by naturally occurring organosulfur compounds and monoterpenes. Cancer Res., 49: 2689-2692, 1989. 13. Dragsted, L. O., Strube, M., and Larsen, J. C. Cancer-protective factors in fruits and vegetables: biochemical and biological background. Pharmacol. Toxicol., 72 (Suppl. 1): 116-135, 1993. 14. Arnold, J. T., Wilkinson, B. P., Sharma, S., and Steele, V. E. Identification of chemopreventive agents using a rat trachea! epithelial cell assay. Proc. Am. Assoc. Cancer Res., 33: 161, 1992. 15. Wilkinson, B. P., Arnold, J. T., Sheela, S., and Steele, V. E. Biological activity and chemical class of 125 chemopreventive agents in relation to efficacy in the rat trachéal epithelial cell foci inhibition assay. In Vitro Cell. Dev. Biol., 28: 167, 1992. 16. Steele, V. E., Kelloff, G. J., Wilkinson, B. P., and Arnold, J. T. Inhibition of transformation in cultured rat trachéalepithelial cells by potential chemopreventive agents. Cancer Res., 50: 2068-2074, 1990. 17. Arnold, J. T., Wilkinson, B. P., and Steele, V. E. Inhibition of benzo[a]pyrene induced transformation in cultured rat trachéalepithelial cells by various classes of chemopreventive agents. Proc. Am. Assoc. Cancer Res., 31: 121, 1990. 18. Arnold, J. T., Wilkinson, B. P., and Steele, V. E. Evaluation of chemopreventive agents of several chemical classes using an epithelial cell culture foci inhibition assay. In Vitro Cell Dev. Biol., 26: 57, 1990. 19. Wilkinson, B. P., Arnold, J. T., and Steele, V. E. The use of cultured respiratory epithelial cells to screen chemicals for potential chemopreventive activity. In Vitro Cell Dev. Biol., 25: 56, 1989. 20. Steele, V. E., Boone, C. W., and Kelloff, G. J. Use of four in vitro assays in predicting the efficacy of cancer chemopreventive agents in whole animals. Proc. Am. Assoc. Cancer Res., 32: 127, 1991. 21. Steele, V. E., Arnold, J. T., and Mass, M. J. In vivo and in vitro characteristics of early carcinogen-induced premalignant phenotypes in cultured rat trachea] epithelial cells. Carcinogenesis (Lond.), 9: 1121-1127, 1988. 22. Slaga, T. J., DiGiovanni, J. Inhibition of chemical carcinogenesis. In: C. E. Searle (ed.), Chemical Carcinogens, Vol. 2, pp. 1279-1321. Washington DC: American Chemical Society, 1984. 23. Milner, J. A., Pigoli, M. A., Dipple, A. Selective effects of selenium selenite on 7,12-dimethylbenz(ij)anthracene-DNA binding in fetal mouse cell cultures. Cancer Res., 45: 6347-6354, 1985. 24. Hesse, S., and Jernstrom, B. Role of glutathine S-transferases: detoxification of reactive metabolites of benzo(u)pyrene-7,8-dihydrodiol by conjugation with glutathi one. In: H. Greim, R. Jung, M. Kramer, H. Marquardt, and F. Oesch (eds.), Bio chemical Basis of Chemical Carcinogenesis, pp. 5-12. New York: Raven Press, 1984. 25. Costa, A., Veronesi, U., De Palo, G., Chiesa, F., Permeili, F., Marubini, E., Del Vecchio, M., and Nava. M. Chemoprevention of cancer with the synthetic retinoid fenretinide: clinical trials in progress at the Milan Cancer Institute. In: L. Wattenberg, M. Lipkin, C. W. Boone, and G. J. Kelloff (eds.), Cancer Chemoprevention, pp. 95-112. Boca Raton, FL: CRC Press, 1992. 542 Downloaded from cancerres.aacrjournals.org on July 28, 2017. © 1995 American Association for Cancer Research. CHEMOPREVENTIVE MECHANISMS 26. Lotan, R. Suppression of squamous cell carcinoma growth and differentiation by retinoids. Cancer Res., 54 (Suppl.): 1987s-1990s, 1994. 27. Verma, A. K. Ornithine decarboxylase, a possible target for human cancer prevention. In: V. E. Steele, G. D. Sloner, C. W. Boone, and G. J. Kelloff (eds.), Cellular and Molecular Targets for Chemoprevention, pp. 207-224. Boca Raton, FL: CRC Press, 1992. 28. Zenser, T. V. and Davis, B. B. Arachidonic acid metabolism. In: V. E. Steele, G. D. Stoner, C. W. Boone, and G. J. Kelloff (eds.), Cellular and Molecular Targets for Chemoprevention, pp. 225-245. Boca Raton, FL: CRC Press, 1992. IN EPITHELIAL CELLS 29. O'Brian, C. A., Ward, N. E., loannides, C. G., and Dong, Z. Potential strategies of chemoprevention through modulation of protein kinase C activiiy. In: V. E. Steele, G. D. Stoner, C. W. Boone, and G. J. Kelloff (eds.). Cellular and Molecular Targets for Chemoprevention, pp. 161-172. Boca Raion, FL: CRC Press, 1992. 30. Sharma, S., Stutzman, J. D., Kelloff, G. J., and Steele, V. E. Screening of potential chemopreventive agents using biochemical markers of carcinogenesis. Cancer Res., 54: 5848-5855, 1994. 543 Downloaded from cancerres.aacrjournals.org on July 28, 2017. © 1995 American Association for Cancer Research. Evaluation of Chemopreventive Agents in Different Mechanistic Classes Using a Rat Tracheal Epithelial Cell Culture Transformation Assay Julia T. Arnold, Betty P. Wilkinson, Sheela Sharma, et al. Cancer Res 1995;55:537-543. Updated version E-mail alerts Reprints and Subscriptions Permissions Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/55/3/537 Sign up to receive free email-alerts related to this article or journal. To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at [email protected]. To request permission to re-use all or part of this article, contact the AACR Publications Department at [email protected]. Downloaded from cancerres.aacrjournals.org on July 28, 2017. © 1995 American Association for Cancer Research.