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Cancer Research Laboratory, Mercy Cancer Institute, The Mercy Hospital of Pittsburgh, Pittsburgh, PA 15219
ABSTRACT
This study describes the novel anticarcinogenic activity of diallyl disulfide, a naturally occurring organosulfide from garlic. Oral administration of diallyl disulfide resulted in a dose-dependent and significant inhibition of the growth of H-ras oncogene transformed NIH 3T3 cells implanted in nude mice. The effect of diallyl disulfide was apparent in terms of delay in the appearance of measurable tumors, tumor volume and tumor weight. On the other hand, the growth of H-ras oncogene transformed tumors was not inhibited by dipropyl disulfide, a naturally occurring saturated analog of diallyl disulfide. The diallyl disulfide–mediated inhibition of H-ras oncogene transformed tumor growth correlated with the inhibition of p21H-ras membrane association. The levels of membrane-associated p21H-ras were markedly lower in the tumors of diallyl disulfide–treated mice than in those of controls. An opposite trend, however, was evident for the cytosolic p21H-ras. The results of this study indicate that diallyl disulfide inhibits the growth of H-ras oncogene transformed tumors in vivo by inhibiting the membrane association of p21H-ras and that the allyl group may be an important determinant in the inhibitory effect of this organosulfide on tumor growth.
KEY WORDS: • garlic • organosulfides • diallyl disulfide • H-ras • growth inhibition
The ras family of protooncogenes encode 21-kDa
proteins (p21ras), which play an important role
in the transduction of extracellular signals to the cell nucleus
(Barbacid 1987
, McCormick 1993
).
Oncogenic ras, caused by a single point mutation in a normal
ras gene, has been detected in 20–30% of human cancers
(Barbacid 1987
). Plasma membrane association of mutated
p21ras, encoded by oncogenic ras, is
essential for its cell transformation activity (Der and Cox 1991
, Kato et al. 1992
). A lipid
post-translational modification (farnesylation) has been shown to
be critical for the plasma membrane association of
p21ras, and this reaction is catalyzed by a
specific cytosolic enzyme, farnesyltransferase (Der and Cox 1991
, Kato et al. 1992
, Manne et al. 1990
, Moores et al. 1991
, Schaber et al. 1990
). The farnesyl pyrophosphate pool originates from the
mevalonic acid pathway of cholesterol biosynthesis (Goldstein and Brown 1990
). Mevalonate, a precursor of various cellular
isoprenoids, including farnesyl pyrophosphate, is synthesized from
3-hydroxy-3-methylglutaryl coenzyme A (HMG-Co A), a reaction catalyzed
by HMG-Co A reductase (Goldstein and Brown 1990
).
Therefore, inhibition of p21ras farnesylation can
be achieved either by inhibiting farnesyltransferase activity or by
lowering the farnesyl pyrophosphate pool through inhibition of
HMG-Co A reductase activity.
Naturally occurring organosulfides from garlic, such as diallyl
disulfide (DADS), have been shown to be potent inhibitors of chemically
induced cancers in several different animal tumor bioassay systems,
including those in which tumorigenesis is associated with the
activation of ras oncogenes (Reddy et al. 1993
, Sparnins et al. 1988
, Sumiyoshi and Wargovich 1990
). In this communication, we report that DADS
administration inhibits the growth of H-ras oncogene
transformed tumors in nude mice by inhibiting membrane association of
tumoral p21H-ras.
MATERIALS AND METHODS
Effect of organosulfides on H-ras oncogene transformed tumor growth in vivo.
The effect of organosulfides on the growth of H-ras
oncogene transformed tumors in vivo was evaluated using a nude mouse
xenograft assay. Female athymic mice
(Balb/c-nu/nu) were purchased from the
Frederick Cancer Research and Development Center, Frederick, MD. The
use of mice for these studies was approved by the Institutional Animal
Care and Use Committee. The H-ras oncogene transformed
NIH 3T3 cells were generously provided by Dr. J. M. Pipas,
University of Pittsburgh Medical Center, Pittsburgh, PA. Monolayer
cultures were maintained in Dulbecco’s modified Eagle’s medium
supplemented with 10% fetal bovine serum and antibiotics. Cells
(
106) were implanted subcutaneously on both left and
right flanks of nude mice, and the mice were divided into two groups.
The experimental mice were treated orally with the desired
concentration of DADS or dipropyl disulfide (suspended in 0.1 mL
soybean oil) three times per week beginning the day of tumor cell
implantation. The control mice received soybean oil alone. Tumor size
was recorded three times per week. Tumor volume was calculated using
the following equation: tumor volume (mm3) = (length
x width2)/2. Significanct differences in tumor volume
between control and treated group were determined by two-factor
repeated-measures ANOVA followed by Fisher’s Least Significant
Difference test for multiple comparisons. Mice were killed by cervical
dislocation at specified time points, and liver and tumor tissues were
removed. The tissues were washed thoroughly with ice-cold PBS and
stored at -80°C until used. Significanct differences in tumor wet
weight between control and treated group were determined by Student’s
t test.
Determination of p21H-ras levels, and farnesyltransferase and HMG-Co A reductase activities.
The cytosolic and membrane fractions from tumor tissues of control and
DADS-treated mice were prepared by the method of Khan et al. (1995)
as described previously by Singh et al. (1996)
. The levels of cytosolic and membrane-associated
p21H-ras were determined by the method of James et al. (1994)
with some modifications described by Khan et al. (1995)
. The farnesyltransferase activity in the tumor
cytosol was determined by the method of Khan et al (1995)
. The HMG-CoA reductase activities in the microsomal
fractions of the liver and tumor tissues were determined by the method
of Favata et al. (1987)
. Significance differences in
farnesyltransferase and HMG-Co A reductase activities between
control and DADS-treated groups were assessed by Student’s
t test
RESULTS AND DISCUSSION
Oral administration of DADS (33 µmol, three times per week beginning the day of tumor cell implantation) resulted in a marked delay in the appearance of measurable tumors (data not shown). For example, although tumor measurement was possible in 50% of the control mice at d 7, none of the DADS-treated mice had measurable tumors on that day. Similarly, compared with 92% of the control mice with measurable tumors at d 11, tumor measurement was possible in only 30% of the DADS-treated mice.
Figure 1
shows the effect of DADS treatment on the growth of H-ras
oncogene transformed tumors in nude mice; this growth was significantly
inhibited by DADS administration. For example, 21 d after tumor
cell implantation, the average tumor volume in control mice was 3345
± 415 mm3, whereas the average tumor volume
in DADS-treated mice was 1631 ± 313 mm3 (P
< 0.05). A significant difference in average tumor volume between
control and DADS-treated mice was also evident on d 16 and 18. The
average tumor volumes in control mice on d 16 and 18 were higher by
2.8- and 2.2-fold, respectively, compared with DADS-treated
mice.
|
5.2 g over a
period of 21 d, the average body weight gain in DADS-treated
mice was 3.6 g. After correction for the wet tumor weight (the
average wet tumor weights in control and DADS-treated mice were 3.1
± 0.3 and 1.6 ± 0.3 g, respectively), the final body
weights of the control and DADS treated mice were also comparable,
indicating that DADS treatment did not cause any weight loss.
The dose-response for the tumor growth inhibitory activity of DADS
was also investigated, and the results are shown in Figure 2
. Oral administration of DADS resulted in a dose-dependent
inhibition of H-ras oncogene transformed tumor growth in
nude mice. For example, the average wet tumor weight in the control
mice was higher by 3.5- and 5.6-fold compared with mice treated with
16.5 and 33 µmol DADS, respectively (P < 0.05).
|
To ascertain whether DADS-mediated suppression of H-ras
oncogene transformed tumor growth in vivo resulted from inhibition of
p21H-ras membrane association, the levels of
cytosolic and membrane-bound p21H-ras were
determined in the tumors of control and DADS treated mice; the results
are shown in Figure 3
. Western blot analysis revealed that the levels of
membrane-associated p21H-ras were markedly
lower in the tumors of DADS-treated mice than in those of controls.
An opposite trend, however, was evident for the cytosolic
p21H-ras. The levels of cytosolic p21H-ras were
markedly lower in the tumors of control mice than in those of the
DADS-treated group.
|
, the HMG-Co A reductase activity was significantly lower (77–79%)
in both liver and tumor tissues of DADS-treated mice compared with
the controls. On the other hand, the cytosolic farnesyltransferase
activity was significantly higher in the tumors of DADS-treated
mice than in those of controls (Table 1)
. These results suggest that
the DADS-mediated inhibition of H-ras oncogene
transformed tumor growth may not be associated with the inhibition of
farnesyltransferase activity.
|
Medicinal uses of the vegetables of the genus Allium,
such as garlic, have been noted throughout recorded history. Among
several biological effects observed is the prevention of chemically
induced cancers in laboratory animals by organosulfides from these
vegetables (Reddy et al. 1993
, Sparnins et al. 1988
, Sumiyoshi and Wargovich 1990
,
Wargovich et al. 1988
, Wattenberg et al. 1989
). The results of this study suggest that the consumption
of these vegetables may be beneficial not only for preventing the
occurrence of malignancy, but also in controlling the growth of tumors
that harbor ras mutations.
FOOTNOTES
1 Presented at the conference "Recent Advances on the Nutritional Benefits Accompanying the Use of Garlic as a Supplement" held November 15–17, 1998 in Newport Beach, CA. The conference was supported by educational grants from Pennsylvania State University, Wakunaga of America, Ltd. and the National Cancer Institute. The proceedings of this conference are published as a supplement to The Journal of Nutrition. Guest editors: John Milner, The Pennsylvania State University, University Park, PA and Richard Rivlin, Weill Medical College of Cornell University and Memorial Sloan-Kettering Cancer Center, New York, NY. 
2 Supported, in part, by USPHS grant CA 55589, awarded by the National Cancer Institute.
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)
control, and (•) DADS-treated mice (33 µmol,
three times per week beginning the day of tumor cell implantation).
Points represent means ± SEM (n = 10–12). *P < 0.05, control vs. DADS-treated
mice. Data reproduced with permission from Singh et al. (1996)
