Download Evaluation of Chemopreventive Agents in

(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.