Download Inhibition of Anaerobic Bacteria as a Screen for

Inhibition of Anaerobic Bacteria as a Screen
for Anti-Tumor Agents*
WILLIAMT. BRADNER
ANDDONALDA. CLARKE
WITH THETECHNICAL
ASSISTANCE
or ANAROTHANDBARBARAWEISS
(Division of Experimental Chemotherapy, Sloan-Kettering Institute for Cancer Research, Sloan-Kettering
Division, Cornell University Medical College,New York, N.Y.)
Results of tests with bacteria are often used
as a preliminary indicator of biological activity
in the choice of material for anti-tumor screen
tests. However, anaerobic organisms per se have
been largely overlooked in this type of investiga
tion because of the inherent inconvenience in their
cultivation. The fact that an anaerobic type of
metabolism is a general property of tumors raised
the question whether or not anaerobic bacteria
might have sensitive systems in common with
tumors which either would not be widely found
or would be less exposed among aerobic organisms.
Two observations germane to this question can
be cited. Malmgren and Flanigan (14) noted the
selective localization of Clostridium tetani in tumor
tissue following parenteral administration of spores
to the host. This suggested that the tumor tissue
Eh (oxidation-reduction potential) was sufficiently
negative to meet the stringent requirements of
this bacterium. Two potent inhibitors of animal
tumors, azaserine and diazooxonorleucine have
been shown by Coffey et al. (6) and Ehrlich et al.
(8) to inhibit six out of six and four of five clostridia tested, respectively. In the experiments to
be described, 75 selected compounds of theoretical
interest in cancer research were tested for inhibi
tion of ten anaerobic or microaerophilic bacteria
(4). The most pertinent results are presented
below and their implications discussed.
MATERIALS AND METHODS
From a group of organisms that was obtained
from various sources, ten strains were selected
which would grow consistently and without diffi
culty in common media.1 The agar-plate pen-assay
disc procedure was used to test for inhibition of
the organisms. Routinely, the bacteria were first
grown in broth medium, usually thioglycollate,
* This investigation was supported by a research grant,
CY-3192, from the National Cancer Institute of the National
Institutes of Health, Public Health Service.
Received for publication October 25, 1957.
and the cells were harvested by centrifugation and
resuspended in saline. Five ml. of this material
was inoculated into 100 ml. of melted heart in
fusion agar, from which plates were poured and
allowed to harden. The compounds were dissolved
in sterile distilled water. If solubility problems
were encountered, a final concentration of up to
0.05 N HC1 or 0.1 N NaOH could be used without
inhibiting any of the organisms. Acetone could
also be used if the discs were dried before placing
them on the agar. All compounds were tested
initially at three levels: 10, 100, and 1000 /ig/disc.
The effects of control discs bearing any solvents
used and of a disc with 2 units of penicillin (posi
tive control) were always determined with each
strain in every experiment. The plates were incu
bated in Brewer anaerobic jars under a nitrogencity gas atmosphere for 3 days at 37°C.
RESULTS
To assess the inhibition noted from the stand
point of a screening procedure for anti-tumor
agents, it was necessary to compare these results
with those obtained from trials of the same com
pounds against animal neoplasms. The only de
tailed study available for comparison was that
edited by Gellhorn and Hirschberg (10). Therein
were reported the results of tests of 27 selected
compounds against fifteen rodent tumor systems,
21 microbiological, seventeen mutation, differen
tiation, developmental, and 21 biochemical systems.
These were further combined into the two
categories: tumor and nontumor systems. A re
sponse by one third or more of the units in
a system to a given compound was arbitrarily
considered positive. Thus, each compound could
1The following organisms were obtained from the American
Type Culture Collection, Washington, D.C.: Bacteroides
limosità 8436, B. pseudoramosus 8489, B. tumidus 8496,
Butyribacterium rettgeri 10835, Clostridium cylindrosporum
7905, C. feseri 1009%, Micrococcus aerogenes 9321, Propionibacterium freudenreichii 6207, and P. shermanii 9617.
Clostridium acidi-urici 9A was received through the
courtesy of Dr. H. A. Barker.
299
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300
Cancer Research
be rated as "active or inactive" in the tumor
and nontumor tests and appropriate comparisons
made. So that advantage could be taken of this
available information, these 27 compounds were in
cluded in the tests with the anaerobes. The results
are presented in Table 1. Since in nearly all cases
two or more tests were made, an average is pre
sented for each compound. If a compound was
slightly inhibitory on one test and not in another,
it was scored as showing activity. This was done
on the premise that any such observation in a
screen test would be the subject of further investi
gation. It can be seen that at least one organism
responded to every compound. In determining the
scoring methods to be used for assigning a rating
of "active" or "inactive," cognizance was taken
of the major criticism of the "1/3" method used
for the Diverse Systems investigation. The objec
tion has been raised that such an arbitrary division
would tend to minimize the importance of com
pounds which were strongly active in just one or
a few systems. In consideration of the limited
knowledge available regarding the validity of antirodent tumor screening data for the predicting
of therapeutic efficacy in humans, these agents
might best be given a rating equivalent to that
assigned to compounds which exhibit weak ac
tivity in many systems. To assess this situation
with respect to the anaerobes, a value of 5 for ±,
10 for +, and 20 for + + was assigned, and the
total activity of each compound determined. A
baseline sum of 20 was chosen as minimum for
"active," since this would acknowledge any com
pound causing maximum inhibition of just one
anaerobic species. With this method only one
compound, D-glucosamine, was included in addi
tion to those obtained by counting any response
of one-third or more of the organisms as indicating
an "active" compound. Using the \ procedure, the
total anaerobe results were compared with the
Diverse Systems tumor responses for mismatched
positives, i.e., cases in which anaerobes were inhibit
ed and the tumors were not; mismatched negatives ;
and total matching positive and negative results
(Table 1). Dividing the latter figure by 27 yielded
the so-called "matching score." This was calcu
lated for the anaerobes as a group, for each indivi
dual species used (Table 2), and for various com
binations of species. The anaerobes as a group
were no better in this respect than the other micro
biological systems reported in the Diverse Systems
Investigation. However, Clostridium feseri by it
self had a superior score, with respect not only
to the other nine anaerobes, but also to that of
the 21 individual microorganisms previously de
scribed (10). Moreover, there were only two mis
Vol. 18, April, 1958
matched negatives; these concerned two tumoractive compounds which did not inhibit C. feseri
and which, therefore, could be overlooked in a
screening program. This result was comparable to
that of the mouse mammary Adenocarcinoma 755
and superior to all other tumors tested for the
Diverse Systems Investigation. The matching
score could not be improved by any combination
of additional anaerobes. Table 3 shows the results
with a selection of fifteen additional compounds
known to exhibit activity as inhibitors of various ex
perimental neoplasms. Here again, C.feseri had the
minimum of mismatched negative results (shared
with C. cylindrosporum). Four of the five tumor
inhibitors missed by C. feseri in both groups of
compounds were purine derivatives. Obviously, C.
feseri could not be used if this were the type of
agent sought. Nevertheless, the organism appears
worthy of inclusion in some of the primary micro
biological screening programs in which a critical
evaluation of its usefulness could be made.
One important application of microbiology in
cancer research is the study of special classes of
compounds. In this respect several examples of
responses by the anaerobes will be presented. C.
feseri was inhibited by all the alkylating agents
tested that have reached clinical trial. However,
in view of the limited number of compounds, ap
praisal of this observation must await further
screening results. The inhibition of B. limosus by
2-deoxy-D-glucose (2DG) was found to be specific
(glucose, mannose, and 2-D-glucosamine did not
inhibit). Tube dilution tests revealed that 2DG
inhibition could be blocked competitively by glu
cose with an I/S ratio of 1 (7, 16).
Diethylstilbestrol was of interest, because it was
the most potent inhibitor of all the compounds
tested. On repeated tests all ten species were in
hibited by 10 ng/disc. Clostridium acidi-urici was
inhibited strongly by diethylstilbestrol but little
or not at all by para- or metahydroxypropiophenone. The parahydroxypropiophenone might be a
metabolite of diethylstilbestrol, and its low ac
tivity suggests that this is not the inhibitor. In
addition, the lack of response of the meta com
pound rules out possible inhibition by the phenolic
function. C. acidi-urici was also inhibited by 4,4'dimethylstilbene and a,a'-dihydroxy-a,a'-diethylp,p'-dihydroxy-bibenzyl.
The glutamine antagonist 6-diazo-5-oxo-L-norleucine (DON) was inhibitory to all ten species,
as was its close relative, azaserine. Results with
DON, L-glutamic acid-5-hydrazide, and glutamine
vs. B. rettgeri and M. aerogenes are shown because
of their similar pattern of responses (Table 4). The
compounds are very closely related if considered
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302
Cancer Research
as a common glutamyl moiety with a diazomethane, a hydrazino, or an amino group attached
to the 5-carbonyl, as shown in the structural dia
gram. Though the hydrazide has been shown by
others (13) to be a glutamine antagonist, there
is a striking difference between it and DON with
respect to inhibition of these organisms. Various
agents have been found to antagonize inhibitions
caused by DON or azaserine, depending upon the
biological system studied. Therefore, experiments
were carried out with these inhibitors and with
several of the reported antagonizing agents. By
incorporating either DON (2 /ug/ml) or azaserine
(50 ¿ug/ml)into the agar with B. rettgeri, it was
found that the inhibitions could be blocked by
glutamine at 10 /ig/disc (as evidenced by a zone
of growth), but not by adenine, guanine, or hy-
Vol. 18, April, 1958
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Mismatchednegatives.positives.7467
poxanthine (2,15), nor by glutamic acid or a
mixture of the aromatic amino acids—phenylalanine, tyrosine, and tryptophan—at concentrations
up to 1000 /ig/disc (12). Inhibition of M. aerogenes
was blocked only by hypoxanthine at 100 ¿ig/disc.
These responses cannot be examined in detail until
the organisms are grown in defined media.
Of the various purine derivatives tested, purinyl-6-thiocyanate (P-6-T) was the strongest in
hibitor of the anaerobes. It differs from 6-MP
and hypoxanthine in the functions on the 6position of the purine ring (Table 5). From a
structure-activity point of view some results with
C. cylindrosporum were of interest, since the or
ganism was not inhibited by 6-MP, hypoxanthine,
cyanide, or thiocyanate—compounds which might
be split products of P-6-T. This suggests that it
is necessary for the thiocyanate function to remain
intact on the 6-position for either the actual
inhibitory process or for entry of the compound
into the cell.
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Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1958 American Association for Cancer Research.
BRADXERANDCLARKE—AnaerobicBacteria and Anti-Tumor Agents
DISCUSSION
In the experiments described, a series of com
pounds that have been studied for anti-neoplastic
activity were tested for inhibition of ten anaerobic
bacteria. Though the responses noted indicate that
the organisms as a group offer no advantage as
a screening tool, the results obtained with one
strain, C. feseri, were correlated closely enough
with results obtained with tumors to warrant more
detailed study. It is difficult to ascertain whether
the fact of anaerobic requirements was responsible
TABLE 4
INHIBITIONTESTSWITHTwo
GLUTAMINE
ANTAGONISTS
Compound
B. rettgcrì
1. 6-Diazo-5-oxo-L-norleucine
2. L-Glutamic acid-5-hydrazide
3 L-Glutamine
Af. aerogene»
+
±
—
O
NH2
CCH2CH2CHCOOH
O
NH2
II
TABLE 5
FURINEDERIVATIVES
INHIBITIONOF
CLOSTKIDIUM
CYLINDROSPORUM
Purinyl-6-thiocy++
anate
6-Mercaptopurine
—
Hypoxanthine
—
Potassium cyanide —
(KCN)
Potassium thio—
cyanate (KSCN)
SUMMARY
1. Various compounds of theoretical and prac
tical interest in cancer research were tested for
their ability to inhibit ten species of anaerobic
bacteria.
2. The results obtained with at least one or
ganism (Clostridium feseri) correlated sufficiently
well with the anti-tumor activities of certain com
pounds to warrant further investigation as a
screening tool.
3. The results seen with special classes of com
pounds suggest that some of the anaerobes may
prove to be independently useful in the examina
tion of these materials for inhibitory properties.
REFERENCES
CCH2CH2CHCOOH
Initial
test
since this has not been the usual observation with
other bacteria inhibited by these agents. This
effect and the activity of purinyl-6-thiocyanate,
however, can only be considered as responses of
certain microorganisms rather than as unique prop
erties of anaerobes.
I
CCH2CH2CHCOOH
O
NH2
Compound
303
id
3d
4th
(Position 6)
+
+
++
-SCN
—
—
—
-SH
-OH
—
—
for some of the results seen or whether this only
represents the normal expectation for a group of
bacteria not previously examined. The striking in
hibition caused by diethylstilbestrol might be a
function of anaerobe sensitivity. On theoretical
grounds anaerobic bacteria should be highly sus
ceptible to glycolytic inhibitors when it is con
sidered that they lack the alternate oxidative
pathways of hexose metabolism and the efficient
respiratory means of pyruvate utilization. Indeed,
all ten organisms were inhibited to some degree
by 2-deoxy-D-glucose. The finding with one strain
that glutamine was the most effective antagonist
of inhibition by azaserine and DON is of interest,
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Inhibition of Anaerobic Bacteria as a Screen for Anti-Tumor
Agents
William T. Bradner, Donald A. Clarke, Ana Roth, et al.
Cancer Res 1958;18:299-304.
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