[CANCER
RESEARCH
37, 4559-4567,
December
1977]
Inhibitory Effect of Abrin and Ricin on the Growth of
Transplantable Murine Tumors and of Abrin on Human
Cancers in Nude Mice1
Oystein Fodstad, Sjur Olsnes, and Alexander Pihl
Norsk Hydro's Institute
for Cancer Research,
Montebello,
Oslo 3, Norway
SUMMARY
21). Each toxin consists of 2 polypeptide chains with differ
ent functions (19, 20). The B-chain, or "haptomer," binds
The toxic lectin abrin increased the survival time of mice
inoculated with Ehrlich ascites cells by 43 to 93% and that
of mice carrying B16 melanoma by 36%. The best results
were obtained when several doses were given. The growth
of Lewis lung carcinoma was retarded in abrin-treated
mice, but the animals did not survive longer than did
untreated animals. Ricin reduced the formation of ascites
but increased survival only by 29%.
HeLa cells, baby hamster kidney cells, and abrin-resistant
variants obtained from these lines all formed solid tumors
after injection into nude mice. When the animals were
treated i.v. with abrin, tumors derived from parent cell
lines sensitive to abrin in vitro were inhibited in their
growth, whereas tumors derived from the abrin-resistant
variants were unaffected. The data indicate (a) that when
the cell lines grow as tumors in nude mice they retain their
characteristic sensitivity to abrin and (b) that the inhibition
of tumor growth is caused by the action of abrin on the
tumor cells as such.
Human cancer tissue from a fibrosarcoma humeri, an
ovarian carcinoma, an ovarian sarcoma, a melanoma, and
an Ewing's sarcoma was transplanted s.c. into nude mice.
the toxin to certain cell surface receptors carrying terminal
galactose residues. After being bound to the cell surface,
the toxin or its active part, the A-chain or "effectomer,"
which is attached to the B-chain by a disulfide bond,
somehow penetrates into the cytoplasm where it inactivates
the 60 S ribosomal subunits, thus inhibiting protein synthe
sis (1,30).
A tumor-inhibiting effect of ricin was reported by Mosinger in 1951 (14). Lin et al. (10) found a strong protective
effect of abrin and ricin against Ehrlich ascites tumor cells
in mice. Others (6, 27) have found a growth-inhibiting
effect of abrin and ricin on Ehrlich ascites tumor and
Yoshida sarcoma, but the effect was much less than that
reported by Lin ef al. A certain protective effect against
experimental leukemia was also reported (26). In prelimi
nary studies, the toxins have also been used in the treat
ment of certain forms of human cancers (7, 32). In the few
cases reported thus far, the results appear promising, and
few side effects have been observed.
In view of the preliminary nature of most of the studies
on the cancerostatic properties of abrin and ricin and the
discrepancies between the observations in different labo
ratories, further studies seemed warranted. Here we started
out to test the effect of abrin and ricin on the growth in
mice of transplantable tumors. The toxins had a clear
inhibitory effect on tumor growth without a depressive
effect on the level of WBC. These findings encouraged us
to explore the effect of abrin on the growth of human
tumors transplanted into nude mice.
When the transplanted tumors had reached an easily meas
urable size, some of the animals were treated with abrin (3
to 5 injections) or with other cytostatics. In all cases abrin
in doses well tolerated by the mice had a strong inhibitory
effect on the tumor growth. Abrin and ricin, even when
given in nearly lethal doses, did not depress the level of
peripheral leukocytes.
The finding that abrin and ricin inhibits growth like a
number of cytostatics in common clinical use suggests
that abrin and ricin may be useful, particularly in combina
tion therapy and under conditions when bone marrow
depression rules out further treatment with other cytostatic
agents. The data also indicate that studies in nude mice
may be of value in assessing the sensitivity of human
tumors to cytotoxic agents.
INTRODUCTION
The toxic plant lectins abrin and ricin are potent cytotoxic
agents that inhibit protein synthesis in eukaryotic cells (9,
1 This work was supported
by the Norwegian
Received March 3, 1977; accepted
DECEMBER
1977
Cancer Society.
August 9. 1977.
MATERIALS
AND METHODS
Tumors. Three different mouse tumors, Ehrlich ascites
tumor, Lewis lung tumor, and B16 melanoma, were used.
The Ehrlich ascites tumor had been passaged in C57BL6 x
DBA/2 (hereafter called B6D2F,) mice for more than 2 years
at this institute. The Lewis lung and B16 tumors were ob
tained from the NIH, Bethesda, Md.
Five different human tumors were used. A fibrosarcoma
of the huméruswas obtained from a 60-year-old woman,
an ovarian sarcoma was obtained from a 70-year-old
woman, and an ovarian carcinoma (serous cystadenocarcinoma) was obtained from a 60-year-old woman. A malignant
melanoma was obtained from a lymph node metastasis
removed from a 20-year-old man. An Ewing's sarcoma was
4559
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1977 American Association for Cancer Research.
0. Fodstad et al.
obtained from a lymph node metastasis from a 9-year-old
boy who had previously been treated with X-irradiation.
Animals. In the study of the transplantable mouse tu
mors, different strains of mice were used. The B16 mela
noma was maintained by s.c. serial transplantation in the
C57BL6 mouse and transplanted into female B6D2F, mice
for the chemotherapy experiments. Female B6D2F, mice,
weighing 19 to 22 g, were also used in the experiments
involving Ehrlich ascites tumor. Male B6D2F, mice weigh
ing 22 to 26 g were used in the experiments with Lewis
lung tumor. The animals were housed 3 to a cage and were
maintained on a standard pellet diet with water ad libitum.
Six- to 8-week-old nude athymic mice of both sexes were
used for the experiments with human tumors. The animals,
purchased from the Laboratory Animals Breeding and Re
search Center, G1. Bomholtgaard, Ry, Denmark, were the
outcome of back-cross cycles in gene transfer to either
BALB/c or NMRI mice. The animals were housed 5 to 10
per cage under conventional conditions.
Toxins. Abrin and ricin were extracted from seeds of
Abrus precatorius and Ricinus communis and were purified
to homogeneity as described earlier (19, 20).
The doses of the toxins lethal to 50% of animals here
used were 14 and 65 ng for abrin and ricin, respectively,
after i.v. administration of a single dose to B6D2F, mice
weighing 20 g (4). The dose-lethality curve was very steep,
and all mice survived treatment with 11 ng abrin or 50 ng
ricin. BALB/c nude and NMRI nude mice showed essentially
the same sensitivity to the toxins as did the B6D2F, strain.
Drugs. 5-Fluorouracil was obtained from Hoffmann La
Roche, Basel, Switzerland. DTIC2 was obtained from Dome
Laboratories, Stoke Poges, Buckinghamshire, England. Actinomycin D (Cosmegen Lyovac) was obtained from E.
Merck AG, Darmstadt, West Germany.
Inoculation of Tumors. One x 106 Ehrlich ascites cells in
0.2 ml of ascites fluid diluted with 0.9% NaCI solution were
injected i.p. Small fragments (30 mg) of the solid mouse
tumors were inoculated s.c. into the left axillary region of
the host.
HeLa cells with different sensitivities to the toxins when
grown in culture were injected s.c. into nude mice. The
cells in all cases grew as solid tumors. The microscopic
pictures of parent and resistant cells in culture and of the
corresponding tumors are shown in Fig. 1.
The human tumors were serially transplanted into nude
mice through 5 to 12 passages before being used in the
experiments. Small pieces of solid tumor tissue (about 2 x
2x2
mm) were transplanted into the s.c. space in the
flanks of the mice. Microscopic and electron-microscopic
examination of the tumors showed that the original struc
ture was retained after several passages. Only mice bearing
a growing tumor were included in the experiments. After
the tumors had been adapted to growth in nude mice, the
take rate upon further transfers was 90 to 95%. The therapy
was initiated when the tumor diameter was 4.5 ±1.5 mm.
Administration of Toxins. In the experiments with Ehrlich
ascites tumor, abrin was injected i.p. on the day of tumor
transplantation, and in most cases the injection was re2 The abbreviations used are: DTIC. 5-(3,3-dimethyl-1-triazeno)imidazole4-carboxamide;
BHK, baby hamster kidney.
4560
peated once or twice within the next 12 days. In general,
there was a weight reduction of about 5% during the first 2
to 4 days after the injection of abrin. When the animals had
regained their weight, after 2 to 3 days, the next injection
was given.
In the experiments with solid tumors, the toxins were
injected i.v. into the tail vein. On the day of tumor transplan
tation, 7 ng of abrin or 25 ng of ricin per 20 g body weight
were injected into each mouse. The injections were re
peated either 2 or 3 times within 14 days, depending on the
weight change of the host. Therapy was discontinued after
2 weeks, in part because i.v. injections were difficult to
perform after 2 or 3 previous injections and in part because
the response to the toxins gradually decreased. In the
tumor-bearing control animals 0.9% NaCI solution was
injected instead of toxins.
Evaluation of Tumor Growth. The growth of the Ehrlich
ascites tumor was evaluated by weighing the animals every
second day.
For the solid mouse tumors, 2 perpendicular tumor di
ameters were measured with vernier calipers 3 times a
week. The results were expressed in sq mm as the product
of the 2 diameters.
The absolute size of the s.c. human tumors proved
difficult to measure, because the difference in the thickness
of the skin and s.c. tissue above the tumor strongly influ
enced the measurement of the tumors, which were rela
tively small by the time the treatment was initiated. Although
the pieces of tumor tissue inoculated in the different mice
were about the same size, the tumor diameters showed
some variation from mouse to mouse when treatment was
initiated.
Subsequently, however, the increase in tumor diameter
was quite similar in each group of mice. Because of the
difficulties mentioned above, the mean diameter difference
was used as a measure of increase or decrease in tumor
size. Two diameters on each tumor were measured 3 times
a week and the corresponding diameters measured on the
day treatment was started were subtracted from these
values. This proved to be a reproducible measure and has
been used in the following studies. The observation time
as given on the charts varies from one experiment to
another, because the experiments were terminated when
more than 10% of the animals had died.
Animals died from the tumor only in those experiments
in which tumors were derived from cultured BHK cell lines.
The animals carrying tumors transplanted from patients
apparently died for reasons unrelated to the implanted
tumors or the abrin treatment. Thus, the death rate was the
same in control animals as in tumor-bearing abrin-treated
and untreated animals.
Cells. HeLa cells and BHK cells and their toxin-resistant
variants were maintained in shaker cultures at 37°in Grand
Island Biological Co. minimum essential medium contain
ing 10% calf serum (GIBCO BlO-Cult) at a concentration of
1 to 4 x 105cells/ml. Cells were harvested by centrifugation
for 5 min at 100 x g and were then suspended to a
concentration of about 5 x 106 cells/ml in Eagle's medium
containing 10% calf serum.
BHK cells and the resistant variants R7, R,a, RM, R,,¡,R17,
and R^j were obtained from Dr. R. C. Hughes, National
CANCER
RESEARCH
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1977 American Association for Cancer Research.
VOL. 37
Effect of Abrin on Human Cancers in Nude Mice
Institute for Medical Research, Mill Hill, London, England
(12,13). Toxin-resistant variants of HeLa cells were isolated
by selection for resistance to ricin. Ricin was used because
we needed ricin-resistant
cells for other purposes and
material was precipitated by adding trichloroacetic
acid to
a final concentration
of 10% (w/v), and the acid-precipitable
because we had reason to expect that such cells would
also be resistant to abrin. The resistant variant R"l was
blood were taken from the tail of the mice and immediately
diluted in 10 ml Isoton II (Coulter Electronics, Harpenden,
Hertfordshire.
England). Then 2 drops Zaponin (Coulter
Electronics)
were added to dissolve the RBC. and the
number of WBC was measured in a Coulter counter.
isolated by adding ricin (20 ng/ml) to a suspension culture
of HeLa cells (100 ml containing 5 x 105 cells) in medium
containing 5% calf serum. Every 3 days 25 ml of a fresh
suspension of HeLa cells were added to provide the re
quired growth factors for surviving cells. The medium
contained 20-ng/ml amounts of ricin throughout the exper
iment.
When dividing cells were observed in the microscope,
the addition of fresh cell suspensions was stopped. HeLa
R"lll was obtained from R"l in the following way: 50 ml (3 x
105 cells/ml) of HeLa R"l was seeded into Falcon tissue
culture flasks (25 sq cm). In this case the medium contained
only 2% calf serum. Ricin (1 /¿g/ml) was added after
incubation for 14 days, surviving cells were cloned and a
single clone was selected. RHII was isolated after x-irradiation (450 to 500 rads) of 10 samples of 5 ml suspension
each (3 x 10s cells/ml) in Carrel flasks. After x-irradiation
the cells were seeded into Falcon tissue culture flasks in
Grand Island Biological
Co. minimum essential medium
with Earle's salt and 10% calf serum. The medium was
changed every 4 days. Under these conditions dead cells
were removed. After 2 weeks the concentration
of calf
serum was reduced to 2%, and ricin (100 ng/ml) was
added. Four days later the medium was changed to a
medium of 10% calf serum and no ricin. The surviving cells
formed colonies, and these cells were later used to select 1
clone. Isoenzyme studies of the ricin-resistant
variants
confirmed that they originated from HeLa cells. The isola
tion and characterization
of the resistant variants will be
described in detail elsewhere.3
Test of Protein Synthesis Inhibition. The assay was
carried out in suspension
cultures or in cells grown as
monolayers. To test cells in suspension, we washed the
cells twice with Hanks' solution and resuspended them to a
concentration
of 3 x 105 cells/ml in serum-free Eagle's
medium containing
one-tenth the usual concentration
leucine and 21 mM /V-2-hydroxyethylpiperazine-/V'-2-eth-
of
anesulfonic acid (pH 7.7). To test cells in monolayer, we
seeded 3 x 10s cells in Eagle's medium containing
10%
calf serum into each well of Linbro tissue culture plates
equipped with 16-mm troughs (FB-16-24-tc; Linbro Chemi
cal Co., Ltd., New Haven, Conn.). After a 20-hr incubation,
radioactivity was measured.
Measurement
of WBC Levels.
Twenty-//!
samples of
RESULTS
Effect of Abrin and Ricin on the Growth of Transplantable Mouse Tumors. The untreated mice given 1 x 106
Ehrlich ascites cells i.p. lived for 14 ±0.2 days. Table 1
shows that the abrin-treated mice all survived longer than
the untreated controls. The prolongation
in survival time
varied from 43 to 93%. Tumor-free control animals given
the same amounts of abrin remained in good condition
throughout
the experiment.
No deaths were observed in
this group. There was an initial weight loss of 1 to 1.5 g,
but the initial weight was regained by Day 6.
An indication of the tumor mass was obtained by weigh
ing the animals. The untreated tumor-bearing
animals
gained weight (average, 12.6 g) from the day of inoculation
until the time of death. In contrast, the weight of the abrintreated tumor-bearing
mice remained fairly constant. Au
topsy revealed, however, that these animals also had a
small amount of ascites at the time of death.
When ricin was used instead of abrin, a considerable
reduction in the formation of ascites was again found, but
the survival time was increased by only 29%.
The effect of abrin and ricin was compared with that of
some commonly used cytostatics. Although the number of
mice in each group was low, the results (Table 1) indicate
that the effect of 5-fluorouracil
and methotrexate was ap
proximately the same as that for abrin, whereas vinblastine
and, in particular,
cyclophosphamide
caused a greater
increase in survival time.
Abrin also inhibited the growth of Lewis lung carcinoma
in B6D2F, mice (Chart 1). However, in spite of the inhibition
of tumor growth, the treated animals lived no longer than
did the untreated controls.
The growth of B16 melanoma transplanted
s.c. into
B6D2F, mice was inhibited after i.v. administration
of abrin
(Chart 2). In this case the survival time increased by approx
imately 36% in the abrin-treated group. At the time of death
of the animals, the tumor size was approximately
equal in
treated and untreated animals.
Effect of Abrin on the Growth in Nude Mice of Tumors
from HeLa Cells and from Abrin-resistant Variants. We
one-tenth the usual concentration
of leucine and 21 mM Ntested the effect of abrin on different toxin-sensitive
and
2-hydroxyethylpiperazine-/V'-2-ethanesulfonic
acid (pH 7.7)
-resistant cell lines in nude mice to see whether the various
was added to each well. Increasing amounts of abrin or
cell lines exhibit similar differences in sensitivity when they
ricin were added, and the samples were incubated at 37°
grow as tumors and when they grow in culture.
for 3 hr. Then 0.5 /¿Ci[14C]leucine (specific activity, 330 Ci/
The resistant variants were selected by growing the cells
mole) was added to each sample, and the incubation was
in the presence of ricin and isolating a clone of the surviving
continued for 1 hr. The experiments were terminated by cells. Since there is evidence that abrin and ricin bind to
removing the medium and adding 1 ml of 0.1 M KOH. The
the same receptors on the cell surface (21, 28) and that
their mechanism of action on the molecular level is identical
3 S. Olsnes, manuscript in preparation.
(1, 17, 22), it was expected that the cell lines selected for
when the cells were attached to the bottom of the wells,
the medium was removed, the wells were rinsed twice with
Hanks' solution, and 1 ml of Eagle's medium containing
DECEMBER
1977
4561
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1977 American Association for Cancer Research.
O. Fodstad et al.
Table 1
agents on the survival time of mice inoculated with Ehrlich
ase/fes C6//S
The mice were given i.p. injections of 1 x 106 Ehrlich ascites cells suspended in 0.2 ml
Effect of various cytostatic
of 0.9% NaCI solution.
The cytotoxic
agents were also given i.p.
animalsDose/in
of in
jection!,"9)0.0110.0110.0110.0090.0070.05075750203000No.
ofmice16141011142293333Survival
time(days)14
inlife
AgentNoneAbrinAbrinAbrinAbrinAbrinRicinMethotrexate5-FluorouracilVinblastineCyclophosphamideTreatmentDay
jection141,
span
(%)6457435093295071107221
0.2°23
±
1.122
±
.420±1
.621±1
51.
0.927
±
51.
121.
6,
2.118
±
61,
1.121
±
0.724
±
41
41,
.
029±
41,
4.545
±
4of
±3.5Increase
" Mean ±S.D.
A-
i.
A
8— ,¿.tS-
71000'S
[A"î
750o>
6EE
I/jfH
•y-
500N"
250oE.Control
AbrinAn'^Q
*
g-
t|2
t
r/°'l
Ô •
4«H*<U
-
i' i —i
i
I\
?/
t/-_a0'""~''
»
HeLa/S
n D-a— o-D-a-0
-A-,B-a-GB—
091**—fc
0)
i" i"""il
li
l!
20
Time
i
20C
t
t
10
\
i
BR"
CR"nt
30
(days)
DRRm
.'
8'SE
Chart 1. Ability of abrin to inhibit the growth of Lewis lung tumor ¡n
mice. Fragments (about 30 mg) of Lewis lung carcinoma were implanted
s.c. into 28 mice. Eighteen mice were given i.v. injections of 7 ng of abrin
per 20 g mouse body weight on Days 1 and 7. The remaining 7 mice were
kept as untreated controls. Tumor size was measured on the days indicated
and is expressed in sq mm. Bars, S.D.
e=5
,1
'
1/6
4Cfl -*j^AI/*',,
-¿yA;
^/
'01
, â„¢'?^ ,i
10
600?|
.l
M/1T
OI/I
'S
200o£..ControlI1'v\I/I,/I
AbrinI
1¿/I11
0
1i/Uft^--^^°
^l,°
I
10
Time
20
25
(days)
Chart 3. Effect of abrin on the growth of various HeLa cell variants in
nude mice. Five x 10* cells were injected s.c. into groups of 8 nude mice.
When the tumor diameter had reached about 4 to 5 mm, 7 ng of abrin per 20
g mouse body weight were injected i.v. into one-half of the mice at the
times indicated with arrows, the remaining animals were used as controls.
Tumor size was measured by calipers every other day. A, untreated mice;
D, mice treated with abrin.
I
20
30
(days)
Chart 2. Ability of abrin to inhibit the growth of B,fi melanoma in mice.
Fragments (about 30 mg) of Blt melanoma were implanted s.c. into 26 mice;
then 7 ng of abrin per 20 g mouse body weight were injected i.v. into 12
mice on Days 1.11, and 15. The remaining 11 mice were used as untreated
controls. Sars, S.D.
resistance to ricin would also have a decreased sensitivity
to abrin.
When the tumors, which all had similar histology, had
reached an easily measurable size, about 4 to 6 mm in
diameter, one-half of the animals were treated with abrin
and one-half were used as untreated controls. Chart 3
shows that the tumors from the parent HeLa cells virtually
stopped growing after the abrin treatment, whereas the
4562
;
15
Time after transplantation
«0•
"O/o
tumors derived from variants resistant to abrin and ricin in
vitro were insensitive and continued to grow at about the
same rate as those in the untreated animals. Chart 4 shows
that the ricin-resistant
variants were also resistant to abrin
in vitro. It is evident that considerably
higher amounts of
abrin were required to inhibit protein synthesis in the ricinresistant HeLa variants than in the parent cells.
Effect of Abrin on the Growth in Nude Mice of Tumors
from BHK Cells and from Abrin-resistant Variants. A series
of toxin-sensitive
and -resistant BHK variants isolated in
the laboratory of Dr. R. C. Hughes (12, 13) were tested to
see whether similar results could be obtained with other
types of cells. The resistant cells, which tolerated 10 to 100
times more ricin than did the parent BHK cells (12) all
proved to be abrin resistant as well (data not shown). The
CANCER
RESEARCH
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1977 American Association for Cancer Research.
VOL. 37
Effect of Abrin on Human Cancers in Nude Mice
to grow at the same rate in the treated and untreated
animals.
The results obtained with the tumors from the different
cell lines derived from HeLa and BHK cells indicate that the
sensitivity to abrin of these tumors grown in nude mice is
determined by the sensitivity of the tumor cells as such.
This supports the view that the sensitivity to abrin of
different tumors growing in nude mice may give relevant
information on the intrinsic sensitivity of the tumor cells to
the toxin.
Effect of Abrin on Human Cancers in Nude Mice. Exper
iments were then performed to study the effect of abrin on
the growth of human tumor tissue transplanted
into nude
mice. The procedure used was similar to that described by
Povlsen and Rygaard (24).
The data in Chart 6 show that injections of abrin had a
pronounced but transient inhibitory effect on the growth of
10
100 T^
10
a human fibrosarcoma.
In contrast, 5-fluorouracil
had no
100
effect on tumor growth.
Toxin added (ng/ml)
The growth of an ovarian sarcoma (Chart 7) was inhibited
Chart 4. Effect of abrin and ricin on protein synthesis in different variants
to a definite, but moderate, extent by abrin. Experiments
of HeLa cells. To HeLa cells and 3 different ricin-resistant variants grown as
monolayers in Linbro tissue culture plates were added, in duplicate wells,
increasing amounts of abrin (A) and ricin (B); after a 3-hr incubation at 37°, on the same tumor carried out by Davy ef al. (3) showed
that this tumor was highly resistant to actinomycin
D and
0.5 ¡id [14C]leucine (specific activity. 330 Ci/mole) was added. The incuba
tion was continued for 1 hr longer, and the acid-precipitable radioactivity
irradiation. It was obtained from a patient who succumbed
was measured. The data are expressed as percentage of the control value
rapidly despite intense treatment.
(no toxin added), which varied between 6.000 and 12,000 cpm in different
An ovarian carcinoma (Chart 8) and a malignant mela
experiments
noma (Chart 9) were both strongly inhibited by abrin. Some
mice carrying the melanoma were treated with DTIC, one
1
1
—" 1
1
1
1 1"B.D 1
of
the most efficient agents in the treatment of malignant
BHKwild
108t421 A
melanoma in humans. DTIC proved to have approximately
/AXD/ayA/^-'
typethe same effect as abrin.
various cell lines were then grown as solid tumors in nude
mice, and one-half the animals were treated with abrin. As
shown in Chart 5, the growth of the parent BHK cells
decreased when the animals were treated with abrin,
whereas all the variants resistant to ricin in vitro continued
~r
,A
' "&
' A/Ä'A
n'tfl
0ET10
l" 'i
-//—I-
G/7Sa£
'
FIBROSARCOMA
R12rJg*i
lj li
'
~ 1
i] 1
S*•
c
CA^Aei-
S-1
iT\
ii
U'D
/-
NUMERI
2 6
eri
i
Jl
]À
i
»/A/°I/'•
(
6^
&
È 3
•5
4*0)
'-P
/
1 2
/"p
2E.S
R?-OIII
oTJS
I1
i'ERH-i
i
100>S
•
E
Rl7-D:
t~-fR22-»
I1
I"T I
1
i
!I —
I
? -
86420n
!
/yi£rA^â.^,,>*-"
»/•'
/D
i . r"T
_J
20
.Aa**C
•
»
— O — B^
1
Time after transplantation
1
1
1
(days)
Chart 5. Effect of abrin on the growth of various BHK cell variants in
nude mice. Five x 10s cells were injected s.c. into groups of 8 nude mice.
When the tumor diameter had reached about 4 to 5 mm, 7 ng of abrin per 20
g body weight were injected i.v. into one-half of the mice at the times
indicated with arrows; the remaining animals were used as controls. Tumor
size was measured by calipers every other day. A. untreated mice; D. mice
treated with abrin.
DECEMBER
1977
30
t
t
)
tttft
Time after
4b
t
50
transplantation
60
70
(days )
Chart 6. Effect of abrin on the growth in nude mice of a fibrosarcoma
from human humérus.After establishing growth of the tumor in nude mice,
as described in "Materials and Methods,' we implanted fragments (about 2
x 2 x 2 mm) of the tumor s.c. into 51 nude mice. On Day 23 after
implantation, when the tumor diameter was about 4 to 5 mm, 26 mice were
treated with 7 ng of abrin per 20 g body weight, and the injection was
repeated on Days 29, 34, and 40 (upper row ol arrows). An additional 5 mice
were treated with 5-fluorouracil [200 ¿igper 20 g body weight per day for 5
days (lower row of arrows)]; the remaining 20 mice were used as controls.
Tumor growth was measured as described in "Materials and Methods."
Bars, S.E. A, untreated mice; O, mice treated with 5-fluorouracil; D, mice
treated with abrin
4563
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1977 American Association for Cancer Research.
O. Fodstad et al.
and measured daily the number of WBC. A certain increase
in the number of leukocytes
occurred
around Days 4
through 7, after which the number returned to normal.
Also, in the control animals a slight increase occurred with
time in the leukocyte number (Chart 11D), probably due to
inflammatory
reaction in the tail after repeated collection
of blood samples. Similar results were obtained with higher
doses, i.e., 12 ng of abrin and 50 ng of ricin. In fact, no
decrease in the number of leukocytes was observed even
after injection of toxin doses leading to death after 3 days.
In contrast, i.v. injection of 8 mg of Cyclophosphamide
per
OVARIAN SARCOMA
/'
r-//-
„Ã
L/A
10
I
Time
20
I
after
30
40
I
I
I
transplantation
50
60
(days)
Chart 7. Effect of abrin on the growth of a human ovarian sarcoma in
nude mice. Fragments (2x2x2
mm) were implanted s.c. into 19 mice; on
Day 15 after implantation, when the tumor diameter was about 4 to 5 mm, 7
ng of abrin per 20 g mouse body weight were injected i.v. into 10 animals.
This was repeated on Days 21, 27, 32. and 41 (arrows). Tumor growth was
measured and is presented as in Chart 6. A. untreated mice; U, mice
treated with abrin.
30
50
40
60
Time
I
CARCINOMA
4
transplantation
SO
60
(days)
Chart 9. Effect of abrin (A) and DTIC (6) on the growth of a human
melanoma in nude mice. Fragments (2x2x2
mm) were implanted s.c.
into 14 nude mice; on Day 3I. when the tumor diameter was about 4 to 5
mm. 7 ng of abrin per 20 g mouse body weight were injected i.v. into 5
mice. The injection was repeated on Days 38 and 47 (A). One mg of DTIC
per 20 g mouse body weight was injected each day for 5 consecutive days
into 4 mice (B); 5 mice were used as controls. Arrows, times of injections.
A. untreated mice; D, mice treated with abrin (XI)or DTIC (B).
-fr-r-
OVARIAN
after
30
40
( I I I I
iH
£ 2
76^Ml</)
SARCOMA.I/Ir/1A/i•
5+
1E1
-Ih
40
30
50
40)m
60
Time
after
transplantation
(days)
the effect
of abrin
on the growth
of Ewing's
sarcoma was tested (Chart 10). Here also a pronounced
but transient inhibition
of tumor growth was observed.
Treatment with actinomycin
D had a somewhat weaker
effect.
Effect of Abrin, Ricin, and Cyclophosphamide
on the
Level of WBC. To study the effect of the toxins on the
WBC we treated B6D2F, mice i.v. with one 7-ng dose of
abrin (Chart 11/4) or two 25-ng doses of ricin (Chart 11S)
4564
/t/1
T^/l°-?/9
i"5"/
°/'x-''
X„,D=ìf-'''
oEA
i'!
T,?
024)E
1nC2
*
,>!/!
i/1Õ
u•EWING's
Chart 8. Effect of abrin on the growth of a human ovarian carcinoma in
nude mice. Fragments of the tumor (2x2x2
mm) were implanted s.c. into
8 nude mice; on Day 32 after implantation, when the tumor diameter was
about 4 to 5 mm, 7 ng abrin per 20 g mouse body weight were injected i.v.
into 5 mice. The injection was repeated on Days 39, 46, and 52 (arrows).
The remaining 3 mice were used as controls. A, untreated mice; D, mice
treated with abrin.
Finally,
<T/1
\^'[
lilt
J-
jt
O-'*
'//1
r
"/'TO'"''
Q'?°
''"
4080till
50
Time
after
60
1
70
transplantation
(days)
Chart 10. Effect of abrin on the growth of an Ewing's sarcoma in nude
mice Fragments (2x2x2
mm) were implanted s.c. into 14 nude mice; on
Day 41, when the tumor diameter was about 4 to 5 mm, 7 ng of abrin per 20
g mouse body weight were injected i.v. into 6 mice. Injection of abrin was
repeated on Days 45. 49, 54, and 60 (arrows). Actinomycin D (4 ¿ig/20g
body weight was injected i.v. into 3 mice; 5 mice were used as controls. A,
untreated mice; O, mice treated with actinomycin; D, mice treated with
abrin.
CANCER
RESEARCH
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1977 American Association for Cancer Research.
VOL. 37
Effect of Abrin on Human Cancers in Nude Mice
«0HO.
A'T
30I
TsSSß
F°
20o
70
,o—
o
1*.
Bh'\*\!.
\¡_IMÕÔ^T,Î-Ô'|"os'1
yi
i
i.
XO
"DS
2!c
-°~6o
/It C
201—i10,
D-
//l
-IO
~O0
Ã-I
ooò°-°Xo/
f
\
T
1.l*û
1,¿.
,
AT'
x°"°
^ilô1
A
..
30
20
Time
10
20
( days )
Chart 11. Effect of abrin, ricin, and cyclophosphamide on the level of
WBC in mice. A, 5 mice each given i.v. injections of 7 ng of abrin; B, 4 mice
each given injections of 25 ng of ricin on Days 1 and 5; C. 3 mice each given
injections of 8 mg of cyclophosphamide; D. 4 mice kept as controls. On the
days indicated, we collected blood samples by cutting short pieces from the
tails of the animals, and we measured the number of WBC in a Coulter
counter.
20 g body weight resulted in a strong but transient decrease
in the number of WBC (Chart 11C) followed by a strong
overshoot reaction around Day 10. Subsequently, the leu
kocyte level decreased to approximately normal. Alto
gether, the data indicate that abrin and ricin, in contrast to
cyclophosphamide, have no depressive effect on the leu
kocyte level in mice.
DISCUSSION
It is now well established that human tumors transplanted
into nude mice maintain their morphological characteris
tics. Even after numerous passages the chromosomal pat
tern is definitely human (5, 25). It is therefore reasonable to
assume that such tumors also retain their biochemical
properties and their sensitivity to chemotherapeutic agents.
The limited data available tend to support this view (16,
29). Recently, Davy ef al. (3) demonstrated a good correla
tion between the sensitivity of different human tumors in
nude mice and the response of these tumors to the same
agents in patients. Several of the human tumors here tested
in the nude mice responded as well or better to abrin than
to various drugs currently used in the chemotherapy of
such tumors. Unfortunately, the effect of abrin on tumor
growth could only be followed for 4 to 7 weeks, due to the
short life span of nude mice kept under our conventional
laboratory conditions. Therefore, in most cases the data do
not allow any conclusion on the duration of the inhibition
of tumor growth.
Giovanella ef al. (5) observed that transplanted human
tumors grow better in nude mice in good health than in
mice in poor condition. Since therapeutic doses of abrin
and other cytostatics temporarily impair the general condi
tion of the animals, it was important to see whether the
growth-inhibiting effect of abrin could be a secondary
effect unrelated to the action of abrin on the tumor cells as
DECEMBER
1977
such. Our demonstration that the sensitivity of the cells to
abrin in culture is correlated with their sensitivity when
they grow as tumors in nude mice indicates that the growthinhibiting effect of abrin is due to a direct effect on the
malignant cells and that the response of the cells is not
demonstrably modified by the host.
Previously, Lin ef al. (10) reported a dramatic effect of
i.p. injection of abrin and ricin into mice bearing Ehrlich
ascites tumor. Our results confirm that abrin has a definite
inhibitory effect on the growth of this tumor in mice, but
the effect was modest compared with that reported by Lin
ef al. (10). The reason for this discrepancy is not clear. In
the present experiments more highly purified preparations
of abrin and ricin were used. However, in terms of the dose
lethal to 50% of animals, the doses were similar to those
used by Lin ef al. Possibly, the difference may be related to
the fact that our tumor line was far more malignant than
that used by Lin ef al. Also, these investigators used a
different mouse strain.
Treatment of Lewis lung carcinoma with abrin reduced
the growth of the primary tumor but did not increase the
life span of the animals. Similarly, Johnson ef al. (8) found
that cyclophosphamide and 5-fluorouracil, which inhibited
the growth of Lewis lung carcinoma, did not prolong the
survival of the animals. The reason may be that Lewis lung
carcinoma metastasizes early to the lungs (11) and that the
métastasesmay be less susceptible to the cancerostatic
agents than is the primary tumor.
Only a modest growth inhibition of B16 melanoma was
seen with abrin. This tumor is rather resistant to a number
of other cytostatic agents, as well. Thus, in the study by
Tarnowski ef al. (31), the most potent agents tested,
namely, cyclophosphamide and busulfan, increased sur
vival only about 30%, i.e., a value close to the one obtained
here with abrin. Corbett ef al. (2) obtained similar results
with Adriamycin and cyclophosphamide.
In most cases the effect of abrin and ricin on tumor
growth was only temporary. One reason for this could be
selection of toxin-resistant cells in the tumor. Another
possibility, which we are now investigating, is that the
toxins induce the formation of neutralizing antibodies. If
the formation of antibodies is indeed the reason for the
decreasing effect of abrin, treatment could be continued
with the immunologically unrelated ricin.
The reason for the selective growth-inhibiting effect of
abrin on malignant tumors is poorly understood. Thus, all
eukaryotic nucleated cells appear to be sensitive to the
toxic effect of abrin and ricin in vitro. One possibility is that
the cells of some tumors may internalize the toxins more
easily than do the normal cells. Thus, Nicolson ef al. (15)
have shown that malignantly transformed 3T3 cells are
more sensitive to ricin than are untransformed cells.
In contrast to most anticancer agents currently used in
humans, abrin and ricin do not interfere primarily with the
synthesis of nucleic acids. It is therefore possible that the
toxins may be useful in the treatment of slowly growing
human tumors. The finding that even high doses of the
toxins give no demonstrable suppressive effect on the
blood level of leukocytes suggests that abrin and ricin may
be of value when bone marrow depression rules out further
treatment with other cytostatic agents.
4565
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1977 American Association for Cancer Research.
O. Fodstad et al.
ACKNOWLEDGMENTS
The technical assistance of Jorunn Jacobsen and Wenche Telle is grate
fully acknowledged.
REFERENCES
1. Benson, S., Olsnes, S., Pihl, A., Skorve, J., and Abraham, A. K. On the
Mechanism of Protein Synthesis Inhibition by Abrin and Ricin. Inhibition
of the GTP Hydrolysis Site on the 60S Ribosomal Subunit. European J.
Biochem., 59. 573-580. 1975.
2 Corbett, T. H.. Griswold, D. P., Mayo, J. G.. Laster, W. R., and Schabel,
F. M.. Jr. Cyclophosphamide-Adriamycin Combination Chemotherapy
of Transplantable Murine Tumors. Cancer Res., 35. 1568-1573. 1975.
3. Davy, M., Brustad, T., and Mossige, J. Irradiation of Human Ovarian
Tumors in Nude Mice. Proceedings of the Second International Work
shop on Nude Mice University of Tokyo Press. Tokyo: in press
4. Fodstad, 0., Olsnes, S.. and Pihl, A. Toxicity, Distribution and Elimina
tion of the Cancerostatic Lectins Abrin and Ricin after Parenteral
Injection into Mice. Brit. J. Cancer, 34: 418-424, 1976.
5. Giovanella. B. C., Stehlin, J. S., and Williams. L. J., Jr. Heterotransplantation of Human Malignant Tumors in "Nude" Thymusless Mice. II.
6.
7.
8.
9.
10.
11.
12
13.
14.
Malignant Tumors Induced by Injection of Cell Cultures Received from
Human Solid Tumors. J. Nati. Cancer Inst.. 52 921-930. 1974.
Gürtler.L., and Steinhoff. D. Hemmung des Tumorwachstums durch
Lectin aus der Bohne von Ricinus commuais. Z. Physiol. Chem., 353:
1521,1972.
Hsu. C.-T., Lin. J.-Y., and Tung, T.-C. Further Report on Therapeutic
Effect of Abrin and Ricin on Human Cancers. J. Formosan Med. Assoc.,
73: 526-542, 1974.
Johnson, R. K.. Inouye, T.. Goldin, A., and Stark, G. R. Antitumor
Activity of W-(Phosphonacetyl)-L-aspartic Acid, a Transition-State Inhibi
tor of Aspartate Transcarbamylase. Cancer Res., 36. 2720-2725, 1976.
Lin, J.-Y., Lin, L.-T., Chen, C.-C.. Tserng. K.-Y.. and Tung, T.-C. The
Inhibitory Effect of Crystalline Ricine on Ehrlich Ascites Cells. J. Formo
san Med. Assoc.. 69: 53-57. 1970.
Lin, J.-Y., Tserng, K.-Y., Chen, C.-C., Lin, L.-T., and Tung, T.-C. Abrin
and Ricin: New Anti-tumor Substances. Nature. 227, 292-293, 1970.
Mayo, J. G., Laster, W. R., Jr., Andrews, C. M., and Schabel, F. M., Jr.
Success and Failure in the Treatment of Solid Tumors. III. "Cure" of
Metastatic Lewis Lung Carcinoma with Methyl-CCNU (NSC-95441). Can
cer Chemotherapy Rept.. 56: 183-195, 1972.
Meager, A., Ungkitchanukit. A., and Hughes, R. C. Variants of Hamster
Fibroblasts Resistant to Ricinus communis Toxin (Ricin). Biochem. J.,
154: 113-124, 1976.
Meager, A., Ungkitchanukit, A., Nairn, R., and Hughes, R. C. Ricin
Resistance of Baby Hamster Kidney Cells. Nature. 257: 137-139, 1975.
Mosinger. M. Sur les RéactionsNeuro-endocriniennes et Génitalesdans
L'intoxication Par la Ricine. Compt. Rend.. 145: 738-740, 1951.
15. Nicolson, G. L., Lacorbiere. M., and Hunter, T. R Mechanism of Cell
Entry and Toxicity of an Affinity-Purified Lectin from Ricinus communis
and Its Differential Effects on Normal and Virus Transformed Fibroblasts.
Cancer Res.. 35. 144-155. 1975.
16 Ohsawa. N . Veyama, Y.. Morita. K.. and Kondo. Y Heterotransplantation of Human Functioning Tumors to Nude Mice. Proceedings of the
Second International Workshop on Nude Mice University of Tokyo
Press. Tokyo: in press
17. Olsnes, S.. Fernandez-Puentes. C.. Carrasco, L., and Vazquez, D.
Ribosome Inactivation by the Toxic Lectins Abrin and Ricm. Kinetics of
the Enzymatic Activity of the Toxin A-Chams. European. J. Biochem.,
60. 281-288. 1975.
18. Olsnes. S.. and Pihl. A. Treatment of Abrin and Ricm with ß-Mercaptoethanol. Opposite Effects on Their Toxicity in Mice and Their Ability to
Inhibit Protein Synthesis in a Cell-free System. Federation European
Biochem. Soc. Letters, 28 48-50, 1972
19. Olsnes. S.. and Pihl. A. Different Biological Properties of the Two
Constituent Peptide Chains of Ricin. A Toxic Protein Inhibiting Protein
Synthesis. Biochemistry. 12: 3121-3126. 1973.
20. Olsnes, S., and Pihl, A. Isolation of Properties of Abrin: A Toxic Protein
Inhibiting Protein Synthesis. Evidence for Different Biological Functions
of Its Two Constituent Peptide Chains. European J Biochem.. 35 179185. 1973.
21. Olsnes, S., Refsnes, K.. and Pihl, A Mechanism of Action of the Toxic
Lectins Abrin and Ricm. Nature, 249: 627-631, 1974.
22. Olsnes, S., Sandvig, L., Refsnes, K., and Pihl, A Rates of Different
Steps Involved in the Inhibition of Protein Synthesis by the Toxic Lectins
Abrin and Ricm. J. Biol. Chem., 251: 3985-3992. 1976.
23. Povlsen. C. O., Jacobsen. G. K.. and Rygaard, J. The Mouse Mutant
Nude as a Model for Testing of Anticancer Agents In: 5th ICLA Sympo
sium. Stuttgart: Gustav Fischer Verlag, 1973.
24. Povlsen, C. 0., and Rygaard, J. Effects of Cyclophosphamide
(Endoxan") on a Burkitt's Lymphoma Serially Grown in Nude Mice. In:
25.
26.
27.
28.
29.
30
31.
32.
Proceedings of the First International Workshop on Nude Mice. pp.
285-292. Stuttgart: Gustav Fischer Verlag, 1974.
Povlsen, C. O., Visfeldt, J., Rygaard. J.. and Jensen, G Growth Patterns
and Chromosome Constitutions of Human Malignant Tumors after Longterm Serial Transplantation in Nude Mice. Acta Pathol. Microbio! Scand
A. 83: 709-716, 1975.
Reddy, V. V. S , Choudry, J. N., Vadlamudi. S , Waravdekor. V. S., and
Goldin. A. Antileukemic and Immunosuppressive Properties of Abrin
and Ricm. Federation Proc.. 32 Abstr. 2936. 1973.
Reddy, V. V., and Sirsi. M. effect of Abrusprecatorius L. on Experimen
tal Tumors. Cancer Res..29: 1447-1451. 1969.
Sandvig, K.. Olsnes. S.. and Pihl. A. Kinetics of Binding of the Toxic
Lectins Abrin and Ricin to Surface Receptors of Human Cells. J. Biol.
Chem.,257: 3877-3984, 1976.
Shimosato. Y.. Kameya, T., Nagai. K.. Hirohashi. S.. Koide. T.. Hayashi.
H.. and Nomura, T. Transplantation of Human Tumors in Nude Mice. J
Nati. Cancer Inst., 56: 1251-1255. 1976.
Sperti. S., Montanaro. L . Mattiolo. A., and Stirpe. F. Inhibition by Ricin
and Protein Synthesis in Vitro. 60S Ribosomal Subunits as Target of the
Toxins. Biochem. J., 736: 813-815. 1973.
Tarnowski, G. S., Schmid, F. A., Capuccino. J. G.. and Stock. C. C.
Chemotherapy Studies in an Animal Tumor Spectrum. II. Sensitivity of
Tumors to Fourteen Antitumor Agents. Cancer Res.. 26: 181-207, 1966.
Tung. T.-C., Hsu, C.-T., and Lin, J.-Y. Therapeutic Effect of Abrin and
Ricin on Human Cancers. J Formosan Med Assoc.. 70: 569-578, 1971.
Fig. 1. Microscopic examination of variants of HeLa cells grown in monolayer and as solid tumors in nude mice. HeLa cells and the abrin-resistant HeLa
R"lll were seeded into culture flasks: after growing to confluence the cells were removed by trypsinization, diluted, and seeded into another culture flask.
Concurrently, 5 x 106 HeLa cells or the abrin-resistant variant cells were injected s.c. into the flanks of nude mice When the tumors had reached a
diameter of approximately 15 mm, they were removed, fixed in formaldehyde, and processed for histological examination. A, HeLa cells grown in
monolayer culture; B. R"lll in monolayer culture; C. HeLa cells grown as solid tumor in nude mice: D. R"lll as solid tumor in nude mice.
4566
CANCER
RESEARCH
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1977 American Association for Cancer Research.
VOL. 37
Effect of Abrin on Human Cancers in Nude Mice
DECEMBER 1977
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1977 American Association for Cancer Research.
4567
Inhibitory Effect of Abrin and Ricin on the Growth of
Transplantable Murine Tumors and of Abrin on Human Cancers
in Nude Mice
Øystein Fodstad, Sjur Olsnes and Alexander Pihl
Cancer Res 1977;37:4559-4567.
Updated version
E-mail alerts
Reprints and
Subscriptions
Permissions
Access the most recent version of this article at:
http://cancerres.aacrjournals.org/content/37/12/4559
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 June 17, 2017. © 1977 American Association for Cancer Research.