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Int. J. Cancer: 67,842-848 (1996)
0 1996 Wiley-Liss, Inc.
’*-
Publication of the InternationalUnion Against Cancer
Publication de I’Union Internationale Contre le Cancer
-il
SYNERGISM BETWEEN N-ACETYLCYSTEINE AND DOXORUBICIN
IN THE PREVENTION OF TUMORIGENICITY AND METASTASIS
IN MURINE MODELS
Silvio DE FLORA’.’,Francesco D’AGOSTINII,
Luciana MASIELLO~,
Daniela GIUNCIUGLIO~
and Adriana ALBINI~
Institute of Hygiene and Preventive Medicine, University of Genoa; and 21stitutoNazionale per la Ricerca sul Cancro, Genoa, Italy.
The thiol N-acetylcysteine (NAC) is a promising cancer
chemopreventiveagent which acts through a variety of mechanisms, including i t s nucleophilic and antioxidant properties. We
have recently shown that NAC inhibits type-IV collagenase
activity as well as invasion, tumor take and metastasis of
malignant cells in mice. NAC is also known to attenuate the
cardiotoxicity of the cytostatic drug doxorubicin (DOX, Adriamycin). The present study was designed t o evaluate whether
the combination of NAC and DOX treatments in mice injected
with cancer cells could affect their tumorigenic and metastatic
properties. Six separate experiments were carried out, using a
total of 291 adult female mice. In experimental metastasis
assays, in which B 16-FIO melanoma cells were injected i.v. into
(CD-I)BR nude mice, DOX significantly reduced the number of
lung metastases when administered i.v. at a dose of 10 mg/kg
body weight, 3 days after the i.v. injection of cancer cells. NAC
inhibited lung metastases when added to the medium of cancer
cells before their i.v. injection. The combined treatment with
D O X and NAC, under various experimental conditions, was
highly effective, showing a synergistic reduction in the number
of mestastases. In tumorigenicity and spontaneous metastasis
assays, in which B 16-BL6 melanoma cells were injected S.C. into
the footpad of C57BLI6 mice, DOX decreased the number of
lung metastases when given i.p. at 2 mg/kg body weight. Oral
NAC exerted significant protective effects, and considerably
prolonged survival of mice. The combined treatment with D O X
and NAC again showed synergistic effects on the frequency and
weight of primary tumors and local recurrences,and completely
prevented the formation of lung metastases in the experiment
in which these end-points were evaluated at fixed times. While
injection of DOX 7 days after implantation of cancer cells failed
to improve the cancer-protective effects of NAC, i t s injection
after I day resulted in a striking inhibition of lung metastases.
These findings demonstrate an evident synergism between
D O X (given parenterally) and NAC (given with drinking water)
in preventingtumorigenicity and metastases. The indications of
these animal studies warrant further evaluation in clinical trials.
Q
1996 Wcley-Liss,Inc.
Numerous studies indicate that N-acetylcysteine (NAC). a
precursor and analogue of reduced glutathione (GSH), can
prevent mutation and cancer through a variety of mechanisms.
The nucleophilic and antioxidant properties of this thiol (De
Flora et al., 1995a, b ) appear to be particularly important.
NAC is considered one of the most promising cancer chemopreventivc agents, and it is currently undergoing clinical trial
for the prevention of cancer and modulation of intermediate
biomarkers in the carcinogenic process (van Zandwijk et al.,
1993; Kelloff et al., 1994).
We have recently shown, in both in vitro and in animal
models, that NAC can also exert protective effects in the most
advanced stages of carcinogenesis, invasion and metastasis.
Zymographic analyses showed that NAC selectively inhibited
type-IV collagenases (72-kDa gelatinase A or MMP2 and the
92-kDa gelatinase B or MMP9) produced by many types of
cancer cells (Albini et al., 1995). This is in agreement with a
preliminary report from another laboratory showing NAC
inhibition of human myeloid cell MMP2 using both ELISA and
zymography (Kalebic et al., 1994). It is well known that
production of these metalloproteinases by malignant metastatic cells plays a fundamental role in the degradation of
basement membrane (Kohn and Liotta, 1995), a key barrier to
metastasis formation. Accordingly, NAC efficiently inhibited
the chemotactic and invasive activity of malignant cells of
either human or murine origin in the Boyden chamber (Albini
et al., 1995), an in vitro system predictive of metastatic potential.
Moreover, the number of experimental lung metastases was
sharply reduced when cancer cells were pre-treated with NAC
and resuspended in NAC-enriched medium before i.v. injection into nude mice (Albini et al., 1995). In tumorigenicity experiments, in which malignant murine cells were injected S.C. or i.m.
into mice, the oral administration of NAC significantly decreased
the weight of local primary tumors and caused a dose-dependent
delay in their formation. The number of spontaneous lung
metastases produced by melanocytic cancer cells was also
decreased to a significant extent (Albini et al., 1995).
Doxorubicin (DOX, Adriamycin) is an anthracyclinic antibiotic with anti-tumoral activity which is extensively used in the
chemotherapy of a variety of solid cancers and of cancers of
the hematopoietic system (Carter, 1975; Arcamone, 1985).
DOX has a dual mechanism of action. The ability of its molecule to
intercalate between adjacent DNA base pairs results in inhibition
of DNA synthesis and of DNA-dependent RNA synthesis,
whereas the monoelectronic reduction of its anthracyclinic
ring generates free radicals responsible for DNA fragmentation and damage to cell membranes and proteins (Olson et al.,
1980).A limitation to the clinical use of D O X is that this agent
may cause a potentially lethal and dose-dependent congestive
cardiomyopathy, that is ascribed to the production of reactive
species as well as to the inhibitory effect of this drug on the
limited enzymatic capacity of the heart to detoxify reactive
oxygen metabolites (Doroshow et al., 1981).
Several studies (Kimball et al., 1979; Olson et al., 1980,1983;
Doroshow et al., 1981; Graeff and Scheuler, 1986) showed that
D O X cardiotoxicity in mice is attenuated by NAC administration. Furthermore, NAC decreased D O X genotoxicity by
inhibiting its mutagenicity in Salmonella typhimurium, both in
the presence and in the absence of an exogenous metabolic
system (De Flora et al., 1991a), and by reducing the elevated
frequency of micronuclei induced in mice by D O X in bonemarrow cells (Sohn et al., 1993). The protective effects of NAC
are not accompanied by a loss of DOX therapeutic efficacy. In
fact, survival of DOX-treated mice, either with P388 leukemia
cells (Doroshow et al., 1981) or Ehrlich ascites tumor (Freeman et al., 1980; Olson et al., 1980), was not significantly
affected by additional treatment with NAC at doses that
inhibited D O X cardiotoxicity.
We show here that NAC and D O X can act synergistically in
the prevention of primary tumors, local recurrences and lung
metastases formed in mice by malignant melanoma cells. In 3
experiments, cancer cells were injected i.v. in order to by-pass
formation of primary tumors and evaluate the effect of the 2
drugs, either individually or in combination, on the occurrence
To whom correspondence and reprint requests should be sent, at
the Institute of Hygiene and Preventive Medicine, University of
Genoa, Via A. Pastore 1.1-16132 Genoa, Italy. Fax: 39 10 353 8504.
Received April 17, 1996 and in revised form May 24,1996.
SYNERGISM BETWEEN N-ACETYLCYSTEINE AND DOXORUBICIN
843
TABLE 1-EXPERIMENTAL METASTASES IN FEMALE (CD-1)BRNUDE MICE RECEIVING AN I.V.
INJECTION OF ~ 1 6 . ~ 1CELLS
0
(5 x 104 CELLSIMOUSE)
Experiment
number
Treatment groups
1
Controls (untreated)
DOX (5 mglkg i.p., +24 hr)
NAC (2 glkg p.0.. -31+25 days)
DOX + NAC
Controls (0.15 M NaCl i.p.)
DOX (10 mgikg i.v., +3 days)
NAC (1 g/kg i.p., -8 hr/+8 days)
DOX NAC
Controls (0.15 M NaCl i.p.)
DOX (1 mg/kg i.v., +24 hr)
NAC (10 mM in the cell medium)
DOX + NAC
2
+
3
M,ce
(number)
o
1-10
11-50
251
Number of lung
metastases
(mean -t SE)
13
11
12
12
15
15
15
15
0
2
0
2
5
lo2
5
5
5
7
5
2
0
3
0
1
0
0
0
3
0
2
0
1
0
3
0
4
3
0
0
54.2 23.1
13.1 ? 11.6
42.1 2 15.8
10.9 k 13.6’
12.2 2 6.6
0.7 k 0.3’
25.1 2 10.2
0.07 k 0.075
36.8 ? 14.6
29.2 k 13.7
2.5 2 0.76
0.6 2 0.3’
11
11
11
11
Mice with metastases (number)
5
14j
4
3
4
7
4
3
5
7
5
4
1
2
5
7
4
*
Statistical analysis:
Exp. 1: ‘p = 0.09 (Student’s t-test) andp = 0.10 (Mann-Whitney test) as compared to controls.
Exp. 2: lp = 0.04 as compared to controls (Fisher’s exact test).-?p = 0.10 (Student’s t-test) andp =
0.02 (Mann-Whitney test) as compared to controls.-$ = 0.002 as compared to both controls and
NAC alone, and p = 0.09 as compared to DOX alone (Fisher’s exact test).+ = 0.08 (Student’s
t-test) andp = 0.003 (Mann-Whitney test) as compared to controls. p = 0.06 (Student’s t-test) and
p = 0.19 (Mann-Whitney test) as compared to DOX a1one.p = 0.02 (Student’s t-test) andp = 0.003
(Mann-Whitney test) as compared to NAC alone.
Exp. 3:6p = 0.03 (Student’s t-test) andp = 0.49 (Mann-Whitney test) as compared to controls.’p = 0.02 (Student’s t-test) a n d p = 0.11 (Mann-Whitney test) as compared to contro1s.p = 0.05
(Student’s t-test) andp = 0.03 (Mann-Whitney test) as compared to DOX a1one.p = 0.03 (Student’s
t-test) andp = 0.07 (Mann-Whitney test) as compared to NAC alone.
of experimental lung metastases. In 3 other experiments, the
cancer cells were injected S.C.in order to evaluate the effect of
treatments on the formation of local primary tumors and, in
one experiment, also on formation of local recurrences, along
with the modulation of spontaneous lung metastases. The
choice of doses, time schedules and administration routes of
NAC and DOX was based on our previous experience (Albini
et af., 1995; and data not shown) as well as on data from the
literature, and was optimized throughout the study in order to
evaluate different experimental conditions.
MATERIAL A N D METHODS
Drugs
D O X was used in the form of an injectable commercial
product, containing 10 mg of compound per vial (Adriblastina,
Farmitalia-Carlo Erba, Milan, Italy). DOX was dissolved in
distilled water just before injection. In the experiments involving oral administration, NAC was used in the form of a
commercial product containing 200 mg of compound per
confection (Fluimucil, Zambon, Vicenza, Italy), which was
dissolved in drinking water. In the experiments involving the
parenteral (i.p. or i.v.) administration of NAC, a pure laboratory reagent was used (Sigma, St. Louis, MO) dissolved either
in PBS, pH 7.4, and taken to neutrality by adding 0.1 N NaOH
(tumorigenicity experiments), or in 0.15 M NaCl regulated to a
final pH of 7.0 (experimental metastasis assays).
Animals
A total of 291 adult female mice (Charles River, Calco,
Lecco, Italy) were used, including 152 (CD-l)BR nude mice,
aged 7 weeks. average weight approximately 25 g, and 139
C57BL/6 mice, aged 6-8 weeks, average weight approximately
20 g. (CD-l)BR and C57BL/6 mice were used in the experiments evaluating the modulation of experimental metastasis
and spontaneous metastasis, respectively. Nude mice were
housed in sterile cages equipped with filtering covers, 3 mice to
a cage, at a temperature of 26-2S°C, with a relative humidity of
55%, a ventilation accounting for 15 air-renewal cycles per
hour, and a 12-hr day/night cycle. They received a special
sterile diet (Mucedola, Settimo Milanese, Milan) and sterilized drinking water a d libitum. C57BL/6 mice were housed 5 to
a cage, at a temperature of 25-27”C, with a relative humidity of
55%, a ventilation accounting for 15 air-renewal cycles per
hour, and a 12-hr day-night cycle. They received a standard
rodent diet (MIL, Morini, S. Polo d’Enza, Reggio Emilia,
Italy), and drinking water a d libitum throughout the duration
of the experiments. In the experiments involving the killing of
mice at a scheduled time, animals were anesthetized with ethyl
ether and subsequently killed by cervical dislocation. Housing
and all treatments of animals were in accordance with the
national and European Community guidelines (D.L. 271 1/92
No. 116; 861609iEEC Directive).
Experimental metastasis assays
B16-Fl0 murine melanoma cells were resuspended in serumfree Dulbecco’s MEM and injected i.v. in the lateral tail vein of
nude mice, in a volume of 100 pl ( 5 x lo4 cells/mouse). As
reported in Table I, each of the 3 experiments of this type
involved 4 groups of animals: one control group (either
untreated mice or mice receiving i.p. o r i.v. injections of 0.15 M
NaCI); one group treated with DOX only; one group treated
with NAC only; and one group treated with both drugs. In
experiment 1 D O X was administered i.p. in a single dose (5
mgikg body weight) 24 hr after injection of cancer cells,
whereas NAC was administered with drinking water, at a
calculated daily dose of 2 g/kg body weight. starting 3 days
before injection of cancer cells and continuing throughout the
duration of the experiment. In experiment 2, D O X was
administered i.v. in a single dose (10 mg/kg body weight) 72 hr
after injection of cancer cells, whereas NAC was administered
daily in i.p. injections (1 g/kg body weight), starting 8 hr before
injection of cancer cells and continuing for 8 days. In experiment 3, DOX was administered i.v. in a single dose (1 mg/kg
body weight) 24 hr after injection of cancer cells, whereas NAC
was added to the medium of cancer cells, at a 10 rnM
concentration.
The animals included in each experiment were killed at the
time of the earliest natural death in which lung metastases
were detected, ie., 25 days (experiment l), 29 days (2), and 27
844
DE FLORA ETAL.
TABLE 11 - PRIMARY TUMORS, LOCAL RECURRENCES AND SPONTANEOUS METASTASES IN FEMALE C57BLl6 MICE RECEIVING AN
S.C. INJECTION OF B16-BL6 CELLS (2-5 x I @ CELLSIMOUSE)
nF:ker
4
5
6
Treatment groups
Controls
DOX (10 mgikg
i.v., +24 hr)
NAC(2g/kgp.o.,
-48 hri8 weeks)
DOX + NAC
Controls
DOX (10 mg/kg
i.v.. +24 hr)
NAC’(2g/kg p.o.,
-72 hriend)
DOX + NAC
Controls
DOX (10 mgikg
i.v.. +7 davs)
NAC’(2 gikg p.o.,
-72 hr/end)
DOX + NAC’
Primary tumors
Local recurrences
Frequency
Weight (g)
(%)
(mean 2 SE)
Lung metastases
Frequency
Number
(IU)
(mean SE)
Survival
(days)
Mice
(number)
Frequency
(a)
Weight (8)
(mean 2 SE)
13
13
100.0
92.3
0.29 2 0.06
0.21 t 0.05
61.5
69.2
2.5 f 0.7
2.2 2 0.7
61.5
75.0
21.2 2 2.2
32.6 f 12.2
56 killing
56 {killing{
15
93.3
0.28 t 0.04
53.3
2.8 2 0.8
45.5
12.9 t 7.8
56 (killing)
12
13
15
33.3’
100.0
100.0
0.04 0.02’
3.0 2 0.9
2.2 2 0.6
8.33
NA*
NA*
0.2 2 0.24
NA*
NA*
05
Ob
72.7
76.9
20.3 f 8.8
23.0 f 10.0
56 (killing)
45.2 2 2.1
51.3 t 1.97
14
100.0
1.9 2 0.6
NA*
NA*
18.28
9.5 2 8.3’
60.1 2 3.71°
15
13
15
100.0
100.0
100.0
2.0 f 0.5
3.0 2 0.9
2.3 ? 0.6
NA*
NA*
NA*
NA*
NA*
NA*
53.8
72.7
63.6
1.6 2 0.7’’
20.3 2 8.8
12.6 + 9.0
56.9 f 3.412
45.2 2 2.1
49.0 t 2.8
14
100.0
1.9 2 0.6
NA*
NA*
18.2K
9.5 f 8.39
60.1 t 3.7’O
14
100.0
2.3 t 0.7
NA*
NA*
50.0
*
17.9 f 9.6
(mean
SE)
58.1 2 2.9’’
“NA = not applicable.
Statistical analysis:
Exp. 4: ’ p = 0.0005 as compared to controls,p = 0.003 as compared to DOX alone, andp = 0.003 as compared to NAC alone (Fisher’s
exact test).-$ = 0.0009 (Student’s t-test) andp = 0.0002 (Mann-Whitney test) as compared to controls;p = 0.007 (Student’s t-test) and
p = 0.002 (Mann-Whitney test) as compared to DOX alone; p = 0.0001 (Student’s t-test) and p = 0.0001 (Mann-Whitney test) as
compared to NAC aIone.Jp = 0.01 as compared to controls,p = 0.003 as compared to DOX alone, andp = 0.02 as compared to NAC
= 0.005 (Student’st-test) andp = 0.02 (Mann-Whitney test) as compared to contro1s.p = 0.009 (Student’s
alone (Fisher’s exact te~t).-~p
t-test) andp = 0.009 (Mann-Whitney test) as compared to DOX alone;p = 0.008 (Student’s t-test) andp = 0.03 (Mann-Whitney test) as
compared to NAC alone.-’p = 0.002 as compared to controls,p = 0.0005 as compared to DOX alone, andp = 0.05 as compared to NAC
alone (Fisher’s exact test).-6p = 0.03 (Student’s t-test) andp = 0.009 (Mann-Whitney test) as compared to contro1s.p = 0.01 (Student’s
t-test) andp = 0.003 (Mann-Whitney test) as compared to DOX a1one;p = 0.12 (Student’s t-test) andp = 0.14 (Mann-Whitney test) as
compared to NAC alone.
Experiments 5 and 6: ’p = 0.04 (Student’s t-test) andp = 0.003 (Mann-Whitney test) as compared to controls.-*p = 0.02 as compared
to controls (Fisher’s exact t e ~ t ) . - ~=p 0.41 (Student’s t-test) and p = 0.03 (Mann-Whitney test) as compared to controls.-lOp = 0.002
(Student’s t-test) andp = 0.002 (Mann-Whitney test) as compared to controls.-1$ = 0.03 (Student’s t-test) andp = 0.02 (Mann-Whitney
test) as compared to controls. p = 0.04 (Student’s t-test) and p = 0.01 (Mann-Whitney test) as compared to DOX alone.-12p = 0.009
(Student’s t-test) and p = 0.01 (Mann-Whitney test) as compared to c o n t r ~ l s . - ~ ~=p 0.001 (Student’s t-test) and p = 0.003
(Mann-Whitney test) as compared to contro1s.p = 0.03 (Student’s t-test) andp = 0.04 (Mann-Whitney test) as compared to DOX alone.
days (3) after injection of cancer cells. All mice were necropsied and the lungs were removed, rinsed in PBS, and fixed with
buffered formalin. The total number of visible surface tumor
colonies was scored with the aid of a dissecting microscope.
Tumorigenicity and spontaneous metastasis assays
In 3 additional experiments (3-6) B16-BL6 murine melanoma cells were resuspended in serum-free Eagle‘s M E M and
injected S.C. in the footpad of the right hind leg, in a volume of
50 4, containing 2 x lo5 cells/mouse. As shown in Table 11,
the animal groups were similar to those designed for experimental metastasis assays. NAC was administered with drinking
water at a calculated dose of 2 gikg body weight, starting 48-72
hr before injection of cancer cells and continuing until the end
of the experiment. DOX was administered i.v. (10 mgikg body
weight) either 24 hr (experiments 4 and 5) or 7 days (experiment 6) after the injection of cancer cells.
In experiments 5 and 6, the mice were kept until spontaneous death, in order to evaluate the effect of treatments on
survival. Accordingly, both primary tumors (frequency and
weight) and lung metastases were scored at the time of death.
In contrast, in experiment 4 the footpad affected by the
primary tumor was removed after 4 weeks and, after 4 more
weeks, all animals were killed. The locally formed primary
tumors and local recurrences in the leg stump were excised,
examined to exclude the presence of ulcerations, necrosis or
infections and weighed. The lungs were removed, rinsed in
PBS, fixed in buffered formalin, and examined for metastases
by surface inspection with a dissecting microscope.
Statistics
In each experiment we evaluated the statistical significance
of the differences concerning the investigated parameters in
the mice treated individually with either DOX or NAC as
compared to controls. Moreover, the effects of combined
treatments with the 2 drugs were compared with the data
recorded both in controls and in each of the individual
treatment groups.
Differences in the frequency of mice carrying primary
tumors, local recurrences or lung metastases, as related to the
treatment group, were evaluated by Fisher’s exact test. The
differences in the mean weight of primary tumors or local
recurrences, in the mean number of lung metastases and in
mean survival times, were evaluated by both Student’s f-test
and the non-parametric Mann-Whitney U-test. Further, in
experiments 5 and 6 the survival frequency of mice within each
experimental group was compared on a daily basis by xz
analysis.
RESULTS
Experimental metastasis
Three experiments were carried out to evaluate the combined effects of NAC and DOX on the induction of experimen-
SYNERGISM BETWEEN N-ACETYLCYSTEINE AND DOXORUBICIN
845
an even more striking reduction in the weight of local tumors,
not only as compared to controls but even as compared to each
of the 2 individual drug treatments. Similarly, both frequency
and weight of local recurrences were significantly lower in mice
receiving the combined treatments than in control mice and in
mice treated with either of the 2 drugs. None of the 12 mice
receiving the combined treatment suffered from lung metastases, which resulted in a significant reduction in the frequency
of mice bearing metastases as compared to each of the other
groups, and a significant reduction in number of metastases as
compared to both controls and DOX-treated mice. A 1.6-fold
decrease in the number of lung metastases was observed in
NAC-treated mice as compared to controls, which was not
statistically significant.
Oral NAC (2 g/kg body weight) was quite effective in
enhancing survival of mice in experiments 5 and 6, as assessed
both by evaluating the mean survival time compared to
controls (Table 11) and by comparing daily the survival curves
inthese2groups(Fig. 1,curvesa andb). DOX(10mgikgbody
weight i.v.). also prolonged survival of mice when administered
24 hr after the injection of cancer cells, though to a lower
extent. This resulted in a significant amelioration of the
survival curve, especially during the first 50 days (Fig. 1, curve
c), and in a significant enhancement of the mean survival time
(Table 11, experiment 5 ) . Conversely, treatment with D O X 7
days after injection of cancer cells did not affect either the
survival curves (Fig. 1, curve e ) or the mean survival time
(Table 11, experiment 6). Combination of NAC and DOX
treatments did not further enhance survival as compared to
NAC alone. Nevertheless, it is noteworthy that the combined
treatments improved survival over treatment with DOX alone
given 24 hr (Fig. I, curve d versus curve c ) , or 7 days (Fig. 1,
curve f versus curve e ) after injection of cancer cells. It is also
noteworthy that a combination of NAC with D O X (after 7
days) enhanced the survival time as compared to controls,
whereas treatment with D O X alone was unsuccessful (Table
11, experiment 6).
When comparing the treatment groups included in experiments 5 and 6, it should be kept in mind that primary tumors
and lung metastases were evaluated in each animal at death.
Accordingly, when survival times were significantly enhanced,
even similar carcinogenicity data (tumor size and number of
metastases) actually reflect a slower development of the
cancer. For the same reason, the observed protective effects
are even more remarkable than those inferred from statistical
analyses. In this light, the non-significant decreases in the
weight of primary tumors, compared to controls, are noteworthy in the groups of mice treated with either NAC, DOX after
24 hr, or the combination of NAC with each of the 2 DOX
Spontaneous metastasis
Three additional experiments, 2 of which partially over- treatments, since time of death was significantly delayed in all
lapped (5 and 6), were designed in order to evaluate the effects these groups. Even more important is the significant inhibition
of NAC and DOX, both individually and in combination, on both in the frequency (4.8-fold) and in the number (2.1-fold) of
the formation of local primary tumors and on the subsequent lung metastases in mice receiving oral NAC. A further inhibispread of lung metastases (Table 11). Female C57BL/6 mice tion in the number of lung metastases was recorded in mice
received an S.C. injection of 2-5 x lo5 cells/mouse in the treated with both NAC and D O X 24 hr after injection of
footpad. In 2 experiments ( 5 and 6), animals were kept until cancer cells. In this group the number of metastases was
spontaneous death, when the weight of the primary tumor and significantly lower when compared not only to controls (12.7the number of metastases were recorded. In experiment 4, the fold) but also to DOX-treated mice (14.4-fold), and was also
paw was removed 4 weeks after injection of cancer cells, and considerably, though not significantly, decreased when comthe primary tumor was excised and weighed. After a further 4 pared to NAC-treated mice (5.9-fold).
weeks all animals were killed and lung metastases were scored.
At killing, any local recurrences at the stump of the paw were
DISCUSSION
recorded.
In experiment 4 the weight of primary tumors 4 weeks after
Modulation by NAC and/or D O X of tumorigenicity and
injection of cancer cells was rather low, and was not affected by formation of experimental o r spontaneous lung metastases in
the individual treatments with either D O X or NAC. In mice treated with malignant melanoma cells was evaluated
contrast, the combined treatment produced a significant and under a variety of experimental conditions. O n the whole, the
marked inhibition in the frequency of tumor-bearing mice and results obtained provide evidence of a synergism between
tal metastases m female (CD-l)BR mice. The results of these
experiments are summarized in Table I.
In experiment 1, D O X was given in a single i.p. injection (5
mg/kg body weight), 24 hr after injection of cancer cells. NAC
was administered daily with drinking water (2 g/kg body
weight), starting 3 days before injection of cancer cells and
continuing until the end of the experiment (25 days). Under
these conditions, compared to control mice injected with
cancer cells but receiving no further treatment, D O X and
NAC decreased the average number of lung metastases
4.1-fold and 1.3-fold, respectively. Such a decrease was not
statistically significant. The combined treatment decreased
lung metastases 5.0-fold, which was still not statistically significant but approached the significance threshold.
In experiment 2, D O X was given i.v. at a higher dose (10
mg/kg body weight), 3 days after injection of cancer cells. This
resulted in a significant decrease both in the frequency of mice
bearing metastases (from 66.7% to 33.3%) and in the mean
number of metastases per animal, which was decreased 17.4fold. NAC, which was administered daily i.p. (1 g/kg body
weight), starting 8 hr before the injection of cancer cells and
continuing for 8 days, did not affect the frequency of animals
with metastases. The average number of metastases in these
animals was even higher, but not significantly so. The combination of the 2 drugs had striking protective effects, the frequency
of mice bearing metastases dropping to 6.7%, a decrease which
was highly significant with respect to both controls and
NAC-treated mice and approached significance when compared to results obtained with D O X alone. The mean number
of metastases was as much as 174 times lower than in controls,
367 times lower than in NAC-treated mice, and 10 times lower
than in DOX-treated mice.
Due to the strong effect of D O X alone in experiment 2, in
experiment 3 the drug was given i.v. at a 10-fold lower dose (1
mg/kg body weight), 24 hr after injection of cancer cells.
Under these conditions, D O X was almost ineffective in modulating the induction of lung metastases. NAC (10 mM) was
dissolved in the medium of cancer cells, which resulted in a
significant, 14.7-fold decrease of metastases, as compared to
controls receiving untreated cancer cells. The cells suspended
in NAC had equivalent viability and the same plate efficiency
as untreated cells (data not shown). Injection 24 hr later of
D O X which, as already mentioned, was ineffective per se,
produced a marked synergistic effect. In fact, the decrease in
the number of metastases was 61.3-fold compared to controls,
48.7-fold compared to D O X alone, and 4.2-fold compared to
NAC alone. All these differences were statistically significant.
846
DE FLORA E T A L .
I
I
100
I
I
I
I
\
4
o Controls
itrols (a)
80
o NAC (b)
60
40
20
I
I
100
80
o NAC + Doxo 1 (d)
60
40
20
0
100
80
o NAC + Doxo 7 (4
60
40
20
0
30
40
50
60
70
80
90
Time (days)
FIGURE1 - Survival time of C57BL/6 mice after S.C. injection of B16-BL6 melanoma cells in the footpad (5 x lo5 cells/mouse), as
related to the treatment group. Curves: (a) controls (untreated mice); (b) mice receiving daily NAC (2 g/kg body weight) with drinking
water, starting 3 days before injection of cells; (c) mice receiving a single i.v. injection of DOX (10 mg/kg body weight) 1 day after
injection of cancer cells; (d) a combination of treatments b and c; (e) mice receiving a single i.v. injection of DOX (10 mg/kg body weight)
7 days after injection of cancer cells; and (f) a combination of treatments b and e. Filled symbols indicate those days in which the percent
survival of treated mice was significantly higher than survival of control mice, as assessed by x2 analysis. The mean (2SE) survival times
and their statistical comparisons are reported in Table I1 (experiments 5 and 6).
SYNERGISM BETWEEN N-ACETYLCYSTEINE AND DOXORUBICIN
these 2 drugs in preventing advanced stages of the carcinogenic process.
In the 3 assays evaluating experimental metastasis in nude
mice receiving cancer cells i.v., the treatments varied according
to DOX doses (1 to 10 mg/kg body weight) and administration
routes (i.p. or i.v.), and according to NAC doses (1 or 2 g/kg
body weight) and administration routes (p.o., i.p. or i.v. in the
medium of cancer cells).
The individual treatment with DOX significantly reduced
the number of lung metastases only when the drug was
administered i.v. at a dose of 10 mg/kg body weight, 3 days
after the i.v. injection of cancer cells. The individual treatment
with NAC significantly inhibited lung metastases only when
the thiol was added to the medium of cancer cells before their
i.v. injection. This finding confirms, also from a quantitative
point of view, the conclusions of a previous experiment with a
similar design (Albini et al., 1995).
The combined treatment with DOX and NAC was much
more effective than either of the 2 individual treatments alone.
In particular, in experiment 1 the decrease in lung metastases
was 5.0-fold greater than in controls with both drugs (DOX i.p.
and NAC p.o ), 4.1-fold greater than with DOX alone, and
1.3-fold greater than with NAC alone. In the other 2 experiments, in which DOX was administered i.v., the combined
effect of the 2 drugs was not only more than additive but even
more than multiplicative. The number of metastases in mice
treated with DOX, NAC and the combined drugs was 17.4,0.5
and 174.3 times lower than the control data in experiment 2,
and 1.3, 14.7 and 61.3 times lower than the control data in
experiment 3, thus showing clear synergism.
In the 3 assays evaluating the tumorigenicity and formation
of spontaneous metastases in mice receiving S.C. injections of
cancer cells, NAC was always given daily with drinking water at
the dose of 2 g/kg body weight, whereas DOX was given at a
dose of 10 mg/kg body weight, 1 or 7 days after injection of
cancer cells.
The individual treatment with DOX did not significantly
affect the frequency or weight of primary tumors and local
recurrences, or the frequency of lung metastases. In agreement
with the results obtained in a previous study (Albini et al.,
1995). the individual treatment with NAC exerted a significant
protective effect. The most striking finding in experiments 5
and 6, in which 2 groups (controls and NAC) overlapped, was
that administration of NAC with drinking water considerably
prolonged the survival times in the animals injected S.C. with
cancer cells. This is a very important effect which highlights the
protective properties of this thiol against cancer even in later
stages of carcinogenesis. The 1.6-fold lower weight of primary
tumors in NAC-treated mice, although not statistically significant compared to controls, is indeed remarkable, since the
tumors were evaluated at the time of death of the animals,
which was delayed on an average 15 days by NAC treatment.
Keeping this in mind, even more impressive was the finding
that both the frequency and number of lung metastases were
significantly lower in NAC-treated mice.
The combined treatment with DOX and NAC displayed
synergistic effects in the tumorigenicity experiment (4) in
which fixed times were scheduled for assessing primary tumors
at the site of S.C.injection (4 weeks), local recurrences and lung
metastases (8 weeks). In fact, in this experiment neither DOX
nor NAC alone significantly affected the investigated parameters, although NAC decreased 1.6-fold the number of lung
metastases. In contrast, the combination of DOX and NAC
significantly decreased both frequency and weight of primary
tumors and local recurrences, and completely prevented the
formation of lung metastases. Combination of oral NAC with
DOX i.v., either 24 hr (experiment 5) or 7 days (experiment 6)
847
after injection of cancer cells, enhanced the survival of animals
as compared with treatments with DOX alone. Administration
of the cytostatic drug after 7 days failed to improve the
cancer-protective effects of NAC. In contrast, its administration after 24 hr in NAC-treated mice resulted in more than
multiplicative effects on the reduction of lung metastases at
death, which, compared to controls, varied 0.9-fold in DOXtreated mice, 2.1-fold in NAC-treated mice, and as much as
12.7-fold in mice receiving both drugs.
On the whole, the results of the 6 experiments herein
reported confirm our previous conclusion (Albini et al., 1995)
that NAC can inhibit tumor take and metastases of malignant
cells in murine models. In addition, there is sound evidence
that NAC potentiates the effects of DOX in the same models,
not only when administered with the medium of cancer cells
but also when supplied with drinking water, which appears to
be an outstanding characteristic, especially when dealing with
a drug like NAC whose safety and tolerability have been
established in more than 30 years of clinical use, mainly in the
treatment of respiratory disorders. The possibility of combining these 2 drugs according to a single administration schedule
warrants further studies, which are now in progress.
Concerning the rationale for the observed synergism, some
hypotheses can be raised, taking into account the mechanisms
of the 2 drugs. As already mentioned, the cytotoxic action of
DOX is due to intercalation between adjacent DNA base pairs
and to generation of free radicals in cancer cells (Olson et al.,
1980). The key mechanism of NAC action at this stage of the
carcinogenic process is presumably the inhibition of type-IV
collagenases (Albini et al., 1995; Kalebic et al., 1994), which is
expected to result in a reduced take of transplanted cancer
cells, in attenuated invasiveness of the malignant cells forming
the local primary mass, and in reduced formation of metastases. Inhibition by NAC of type-IV collagenases, which are
produced by B16 melanoma cells (Albini et al., 1995), can be
ascribed to the fact that sulfhydryl groups chelate a zinc ion
which is essential for the activity of these enzymes (StetlerStevenson et aL, 1989). Additionally, NAC is likely to regulate
collagenase production at the transcription level. In fact, this
thiol has been shown to inhibit the induction of the transcription factor activator-protein-1 (AP-l), a heterodimeric complex encoded by the proto-oncogenes c-jun and c-fos which is
involved in collagenase gene transcription (Devary et al., 1991;
Pinkus et a l , 1993; Bergelson et al., 1994). AP-1 can bind to the
promoter region of intermediate genes required for other cell
functions, including cell proliferation, particularly in the transition from the Go to GI phases of the cell cycle (Angel and
Karin, 1991). Accordingly, it has been suggested that thiol
levels are important in protecting cells against the proliferative
effects of carcinogens (Janssen et aL, 1995).
A tentative explanation for the synergism between NAC and
DOX is that inhibition of type-IV collagenases by the thiol may
render the cancer cells more accessible and vulnerable to the
action of the cytostatic drug. In addition, NAC is an effective
inhibitor of reactive oxygen species (De Flora et al., 1991b),
which may play an important role in basement membrane
degradation when released by cancer cells (Iwagachi et al.,
1993), and which are also involved both in DOX cardiotoxicity
(Doroshow et aL, 1981) and in its cytotoxic activity (Olson et
aL, 1980). It cannot be excluded that the cytotoxic action of
DOX in cancer cells may be better expressed when its
oxidative component is attenuated. It has been demonstrated
in an in vitro test system that the generation of free radicals by
DOX is responsible for an increased invasive capacity of tumor
cells, thus suggesting that a treatment with DOX which does
not eliminate all tumor cells may increase the incidence of
invasion and metastasis (Imamura et al., 1990). Interestingly,
this effect was partially inhibited by NAC in the same system
848
DE FLORA ETAL.
(Imamura et al., 1990). In any case, it is important that not only
is the protection by NAC against DOX cardiotoxicity not
accompanied by a loss of chemotherapeutic efficacy, as has
been assessed by evaluating the survival of mice transplanted
with murine tumors (Freeman et al., 1980; Doroshow et al.,
1981; Olson et al., 1980), but that, using suitable models, it is
even possible to show a synergism between these 2 drugs in
preventing invasion and metastases of cancer cells.
Thus, in addition to being considered one of the most
promising cancer chemopreventive agents (van Zandwijk et al.,
1993; Kellolf et al., 1994; De Flora et al., 1995a, b ) , inhibiting
via various mechanisms the carcinogenesis process before the
stage of malignancy, NAC displays encouraging properties,
bothper se and in combination with a classical chemotherapeutic agent, as an agent attenuating the invasive and metastatic
potential of malignant cells.
ACKNOWLEDGEMENTS
This study was supported by the CNR Targeted Projects
“Prevention and Control of Disease Factors-FATMA” and
“Clinical Applications of Oncological Research-ACRO” and
the Italian Ministry of Health (Targeted Project “Innovative
Therapies”). D. Giunciuglio is recipient of a fellowship from
the Associazione Italiana per la Ricerca sul Cancro (AIRC).
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