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Experimental Therapeutics |
Herbert Irving Comprehensive Cancer Center [H. S., Y.K., J-W. S., I. B. W.] and Department of Pathology [V. M.], College of Physicians and Surgeons, Columbia University, New York, New York 10032; Department of Medicine, St. Luke’s-Roosevelt Hospital Center, New York, New York 10025 [H. S., S. F. M., P. R. H.]; Nutrition Research Laboratory and the Clinical Nutrition Research Unit [J. T. P., R. S. R.] and Flow Cytometry Core Facility [T. D.], Memorial Sloan Kettering Cancer Center, New York, New York 10021
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ABSTRACT |
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INTRODUCTION |
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To further elucidate the antiproliferative activity of naturally occurring water-soluble constituents of garlic, we examined the effects of both SAC and SAMC on cell proliferation, cell cycle kinetics, and apoptosis in two well-characterized human colon cancer cell lines, SW-480 and HT-29. In addition, because several signaling molecules (the c-Fos/Jun complex, bcl-2, and jun kinase) contain cysteine domains (14 , 15) that are sensitive to changes in sulfhydryl/disulfide oxidation, we also assayed the effects of these compounds on cellular levels of GSH. We hypothesized that changes in the intracellular redox environment induced by allylsulfides may alter the activity of specific signal transduction factors. Because of the current interest in the chemoprevention of colon cancer using sulindac or its metabolites (16 , 17) , we also compared the effects of these two allylsulfides with those of SS in SW-480 and HT-29 cells.
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MATERIALS AND METHODS |
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Cell Lines and Culture Conditions.
SW-480 and HT-29 human colon cancer cell lines were obtained from the
American Type Culture Collection and maintained in DMEM with 10% fetal
bovine serum (Life Technologies, Inc.) in 180-ml tissue culture flasks
(Nunclon, Rochester, NY) at 37°C in an atmosphere
5%-CO2 and air. Cells were fed fresh medium with
serum every 2–3 days and split when 50% confluent. All studies were
performed with exponentially growing cells at 50% confluence. SW-480
and HT-29 cells were seeded in triplicate in 6-well (3.5-cm diameter)
cell culture plates (Becton Dickinson) at a concentration of
3 x 104 cells/well. After 24 h, cells were treated with increasing concentrations of SAC or SAMC
(50–250 µM) or SS (100 and 200 µM). Cells
were harvested by trypsinization at 0, 24, and 48 h
postadministration of each compound and cell numbers were determined
using a Coulter Counter (Coulter Electronic, Inc.).
Assessment of Cell Cycle Distribution and Apoptosis.
SW-480 and HT-29 cells that were cocultured with either SAMC or SAC
and/or SS were analyzed, in parallel with cells grown in the absence of
these compounds, using a standard flow cytometric procedure (18
, 19)
to determine effects on cell cycle distribution and
apoptosis. Both adherent and floating cells were collected and fixed
with 70% ethanol. The conditions for PI staining and data acquisition
have been described previously (20)
. Apoptotic cells were
considered to constitute the sub-G1 cell
population. All experiments were performed in triplicate and gave
similar results. Cells were also fixed and stained with a 1:250
dilution (20 µg/ml) of a MPM-2 mouse antibody (Upstate Biotechnology,
Lake Placid, NY) and FITC-labeled goat antimouse IgG, diluted 1:100 (2
µg/ml; Rockland, Gilbertsville, PA). The PI-staining after
MPM-2-staining was performed with a concentration of PI (5 µg/ml)
that was lower than that used for routine flow cytometric studies.
FISH.
SW-480 and HT-29 cells, before and after a 24-h treatment with 200
µM SAMC, were harvested when 50% confluent, and
deposited on slides. FISH analysis was performed using standard methods
(21)
. Spectrum orange- and spectrum green-labeled
centromeric probes for chromosomes 11, 12, 16, and 17 were obtained
from Vysis (Downer Grove, IL). Double-color FISH was performed to
identify and enumerate chromosome numbers, and the signals were
captured and analyzed using an Applied Imaging CytoVision Capture
workstation (Santa Clara, CA). At least 1000 cells were analyzed per
experiment.
Protein Extraction and Western Blotting.
Cells in their log phase of growth were collected and proteins
extracted for immunoblotting as described previously (20)
.
Samples of 50–100 µg were subjected to SDS-PAGE, transferred to
nylon membranes, incubated overnight at 4°C in blocking buffer (50
mM Tris, 200 mM NaCl, 0.2% Tween 20, and 3%
BSA) and reincubated for 1 h with the indicated antibodies.
Polyclonal antisera to the Bcl-2 gene family of proteins
(Bcl-2, Bax, Bcl-x, and Bak) were kindly provided by Dr. John C. Reed
(Burnham Institute, La Jolla, CA) and used at a concentration of
1:1500, as described previously (22)
. Immunoblotting of
actin (1:5000 dilution; Sigma Chemical Company, St. Louis, MO) was
performed to verify equivalent loading of protein on the gels.
GSH Determinations.
Cellular concentrations of GSH were measured using a Perkin-Elmer
high-performance liquid chromatograph equipped with a
four-channel coulometric array detector (ESA, Inc., Chelmsford, MA;
Ref. 23
). Cells were plated in 150-mm culture dishes at
concentrations to yield 50% confluence within 48 h. Media were
aspirated and replaced with fresh media containing either PBS, 200
µM SAC, 200 µM SAMC, or 200
µM SS or combinations of SAC/SS (200 µM/100
µM) and SAMC/SS (200 µM/100
µM). At 3 and 24 h postincubation, cells were
harvested by gentle scraping, washed twice with ice-cold PBS, and
centrifuged to obtain a final cell pellet. After removal of the
supernatant fraction, the cells (5–10 x 105) were lysed in 0.25 ml of 200 mM
methane sulfonic acid containing 5 mM
diethylenetriaminepentaacetic acid and centrifuged at 10,000 x g for 10 min. Precipitates were dissolved in 0.1
N NaOH and saved for protein determinations,
which were measured by a spectrophotometric quantitation method using
the BCA protein assay reagent (Pierce Chemical Co., Rockford,
IL). After a 1:3 dilution of the supernatant fractions with the mobile
phase, aliquots were injected onto an Ultrasphere 5 microns,
4.6 x 250 mm, C18 column, and then eluted with a mobile
phase of 50 mM
NaH2PO4, 0.05
mM octane sulfonic acid, and 2% acetonitrile (pH
2.7) at a flow rate of 1 ml/min. The four-channel CoulArray detectors
were set at 525, 625, 725, and 825 mV, respectively. Peak areas were
analyzed using ESA, Inc., software. The final concentrations of GSH
were reported as nmol/mg protein.
Assays for JNK1 Activity.
Assays for JNK1 kinase activity were performed at 30, 60, and 120 min
posttreatment of the cells with 200 µM of SAC, SAMC, or
SS. Cell pellets were suspended in 500 µl M2 lysis buffer [20
mM Tris (pH 7.6) containing 0.5% NP-40, 250 mM
NaCl, 3 mM EDTA, 3 mM EGTA, 1 mM
DTT, 0.5 mM phenylmethylsulfonyl chloride, 1 µg/ml
leupeptin, 20 µl of a 0.2 M solution of
-glycerophosphate, and 5 µl of 0.1 M sodium
vanadate]. Cellular JNK1 was precipitated by incubation of 200
µg of the cell protein extract with an anti-JNK antiserum (Santa
Cruz, Santa Cruz, CA) and protein A-Sepharose (Sigma, St. Louis, MO)
beads in M2 lysis buffer for 2 h with rotation at 5°C. The
precipitates were washed twice with M2 buffer and twice with kinase
buffer [20 mM Tris (pH 7.5) containing 20 mM
-glycerophosphate, 10 mM
p-nitrophenylphosphate, 10 mM
MgCl2, 1 mM DTT, 50 µM
sodium vanadate, and 20 µM ATP] leaving a 25 µl final
volume of beads and buffer. The kinase assay mix, containing 1 µg of
GST-c-Jun (New England Biolab., Beverly, MA) as the substrate and 1
µl of 
-32P-ATP (Amersham, Piscataway, NJ) in
25 µl of kinase buffer per reaction, was added to each tube on ice
(50 µl total volume). The kinase reaction was then performed at
30°C for 20 min and terminated by adding SDS sample buffer. The
reaction mixtures were boiled for 5 min and analyzed by SDS-PAGE
(10%). The extent of protein phosphorylation was determined using
autoradiography.
Assays for Caspase Activity.
After treatment with SAC, SAMC, or SS, the cells were washed twice with
ice-cold PBS and lysed in caspase buffer [50 mM
HEPES-KOH (pH 7.4) containing 0.1%
3-[(3-cholamidopropyl)dimethylammonio]-1 -propanesulfonic acid, 100
mM NaCl, 1 mM EDTA, 10 mM DTT, and
10% glycerol]. The lysates were sonicated for 5 s and then
placed on ice for 10 min. The homogenates were centrifuged at
14,000 x g for 2 min and the supernatant
fraction saved for analysis. Caspase activity was determined using a
fluorometric assay (24)
. Briefly, 50 µg of total
protein, determined by the Bio-Rad Protein Assay (Bio-Rad, Richmond,
CA), were incubated with 10 µg of a fluorogenic peptide substrate,
Ac-DEVD-AFC (PharMingen, San Diego, CA), with caspase buffer, in a
total volume of 1 ml. After a 1-h incubation at 37°C, the release of
7-amino-4-methylcoumarin was determined spectrofluorometrically
using an excitation wavelength of 400 nm and an emission wavelength of
505 nm, as suggested by PharMingen (San Diego, CA).
Cotreatment with SAC Plus SS or SAMC Plus SS.
In studies to determine whether SAC or SAMC enhanced the induction of
apoptosis by SS, SW-480 and HT-29 colon cancer cells were treated with
three different concentrations of SS (100, 150, and 200
µM), alone or in combination with 100 or 200
µM SAC or SAMC, for 48 h. Apoptosis was then
assessed by DNA flow cytometric analysis, as described above.
Statistical Analyses.
Data were analyzed by using the Statgraphics version 4.0 program
(Statistical Graphics Corporation, Rockville, MD). Data are expressed
as mean ± SD. Dunnett’s t test was
used to compare the significance of differences between PBS-treated
controls and SAC, SAMC, or SS-treated cells.
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RESULTS |
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160 µM for SW-480
cells and 175 µM for HT-29 cells (Fig. 1)
. To examine the mechanism responsible for this growth inhibition, cell
cycle distribution was evaluated using flow cytometry. The addition of
200 µM SAMC caused an accumulation of cells in
G2-M with both the SW-480 and HT-29 cells,
although this effect was less pronounced in HT-29 cells. Thus, at
24 h, 71% of the SAMC-treated SW-480 cells were in
G2-M versus 16% of the control cells;
and 30% of the SAMC treated HT-29 cells were in
G2-M versus 12% of the control cells.
There was a reciprocal decrease in the number of cells in
G1 in both cell types (Fig. 2A)
. These changes were much less striking at 48 h (Fig. 2A)
, perhaps because of the onset of apoptosis, as described
below.
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and no significant change in cell cycle distribution (data not shown).
We included SS for comparison and found that it also inhibited growth,
with an IC50 value similar to that of SAMC (Fig. 1)
, and it also inhibited cell cycle progression; but with this agent,
the arrest was in G1 (data not shown), as
reported previously (25)
.
Evidence that SAMC Specifically Arrests Cells in the G2
or M Phases.
The above studies indicated that, in contrast to SS, SAMC arrests cells
in G2-M (Fig. 2A)
. Conventional DNA
flow cytometry does not distinguish cells that are in the
G2 phase from cells that are in the M phase,
because cells in both phases have the same DNA content. Therefore, we
used an MPM2 antibody that reacts specifically with phosphoproteins
present in M-phase cells (26)
in combination with DNA flow
cytometry to further analyze the effects of SAMC. Using this technique,
we found that after treating the SW-480 cells with 200
µM SAMC, there was a significant increase of
cells in the G2 phase but not in the M phase when
compared with control untreated cells (Fig. 2B)
. However,
with 300 µM SAMC there was an increase of cells
in both G2 and M (Fig. 2B)
. With the
HT-29 cells, 200 µM SAMC caused a reduction of cells in the
G2 phase and an increase of cells in the M phase.
These changes were further exaggerated when HT-29 cells were treated
with 300 µM SAMC (Fig. 2B)
. Thus,
SAMC causes SW-480 cells to accumulate in both G2
and M, but causes HT-29 cells to accumulate in M. As expected, SS (200
µM) did not cause an increase of cells in
G2 or in M (Fig. 2B)
, but instead most
of the treated cells accumulated in G1 (data not
shown).
It is well known that cells in anaphase have twice the number of
centromeres as those in metaphase or in stages before metaphase.
Therefore, using the criterion of number of centromeres, it was of
interest to determine whether some of the cells that accumulated in the
M phase after treatment with SAMC were actually in anaphase.
Exponentially growing cultures of either SW-480 or HT-29 cells were
treated with SAMC (200 µM) for 24 h or with only the
solvent, and then double-color FISH analysis was performed with two
sets of centromeric probes, CEP12/16 and CEP 11/17. The number of
targeted centromeres in untreated SW-480 and HT-29 cells varied between
2 and 4, which is consistent with the aneuploid nature of these cells.
In the SAMC-treated SW-480 cells there was an increase in the number of
centromeres. Thus, the frequency of cells in anaphase was 7.6% in the
SAMC-treated SW-480 cells compared with 1.9% in the untreated cells
(Fig. 3)
. However, HT-29 cells did not display an increase in the number of
cells in anaphase after treatment with SAMC (data not shown). These
data suggest that the SAMC-induced G2-M blockade
in SW-480 cells, as demonstrated by flow cytometry (Fig. 2A)
, is associated with a cell cycle delay in anaphase.
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. This effect was greater in the SW-480 cells than in the HT-29 cells,
which is consistent with the fact that HT-29 cells were also somewhat
more resistant with respect to growth inhibition (Fig. 1)
. With both
cell types, the increase in apoptosis was apparent at 24 h, and
the extent of apoptosis increased further at 48 h (Fig. 4)
. On the
other hand, SAC did not induce apoptosis (Fig. 4)
, which is consistent
with its failure to cause growth inhibition in these cell lines (Fig. 1)
. SS (200 µM) produced about twice the level of
apoptosis obtained with the same concentration as SAMC (Fig. 4)
. It
was, therefore, of interest to examine the combined effects of SAMC and
SS on apoptosis when each was tested at 200 µM. With the
SW-480 cells, the combined effects of SAMC plus SS on apoptosis were
close to additive, and with HT-29 cells the combined effects were
supra-additive (Fig. 4)
. As expected, the effects of SAC plus SS were
similar to those of SS alone, because SAC alone did not induce
apoptosis (Fig. 4)
.
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. As expected, SAC did not induce caspase-3 activity in both cell lines
at all three time points. In SW-480 cells, SAMC and SS induced a 2.5-
and 3.1-fold increase, respectively, in caspase-3 activity at 24 h
with no induction at earlier time points. In the HT-29 cell line, the
increase in caspase-3 activity at 24 h after SAMC treatment was
significantly less than in SW-480 cells, inasmuch as there was only a
1.3-fold increase at 24 h. SS caused a 3.3-fold increase in
caspase-3 activity in HT-29 cells at 24 h. Thus, there was a
direct association between induction of caspase-3-like activity and
apoptosis. Additional studies using caspase inhibitors may help to
clarify whether the activation of caspase-3-like proteases are required
for the induction of cell death by SAMC.
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Effect of Garlic Derivatives on Cellular Levels of GSH.
Because several signal proteins contain redox-sensitive sites that can
be modified by oxidative stress, we measured the concentration of GSH
in cells treated for either 3 or 24 h with 200 µM
SAC, SAMC, or SS. Fig. 6
indicates that with both SAC and SAMC there was an increase in GSH
within 3 h and an additional increase with SAMC at 24 h, in
both SW-480 and HT-29 cells. The effect of SAMC was about twice that of
SAC. SS had no effect at 3 h, but it caused a 2- to 3-fold
increase at 24 h. In HT-29 cells, the combined effects of SS plus
SAC or SS plus SAMC on GSH levels were approximately additive.
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. Treatment of the cells with 200 µM SAMC led to an
2.6-fold increase in JNK 1 activity in SW-480 cells at the
120-min time point (Fig. 7)
. With the HT-29 cells, the induction of JNK
1 activity was only about 1.8-fold (data not shown). Treatment
with 200 µM SAC had no effect in either cell type, and
treatment with 200 µM SS led to an 1.9-fold activation
within 30 min in both cell types. The fact that SAMC gave a stronger
induction of JNK1 activity in SW-480 cells than in HT-29 cells is again
consistent with its more potent antiproliferative effects in SW-480
cells (Figs. 1
and 2)
.
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DISCUSSION |
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Our results with SAC are consistent with previous reports (9) that also failed to show growth-inhibitory effects of SAC in HCT15 colon cancer cells or in lung and skin carcinoma cell lines. However, in previous studies, comparable doses of SAC did have antiproliferative effects on neuroblastoma (11) , melanoma (10) , and prostate carcinoma cells (28) . Therefore, SAC may be effective as an antiproliferative agent in specific cell lines and/or under specific conditions. The inhibitory effects on cell proliferation obtained with SAMC in the present study are consistent with results obtained previously with allyldisulfide derivatives. Thus, previous studies indicated that erythroleukemia (HEL; Ref. 29 ), promyelocytic leukemia (HL60; Ref. 30 ), and colon carcinoma (HCT15, SW-480, and HT-29) cells (9 , 31) exposed to the diallyldisulfide ajoene ([E,Z]-4,5,9-trithiadodeca-1,6,11-triene-9-oxide), or to SAMC, exhibited marked decreases in the number of cells in the G1 phase and a corresponding accumulation of the cells in G2-M. By contrast, human umbilical vascular endothelial cells and smooth-muscle cells appeared to arrest in G1 when treated with these compounds (12) .
The inhibitory effects of SAMC on HT-29 cells were less pronounced than
those on SW-480. HT-29 cells have a normal k-ras gene and
express Cox-1 and Cox-2, whereas SW-480 cells have an oncogenic
k-ras mutation and do not express Cox-2 (31)
.
Studies by Singh et al. (33)
demonstrated that
diallyldisulfide appears to suppress the growth of H-ras
oncogene-transformed tumors in nude mice by decreasing the association
of the p21H-ras protein with the plasma membrane. Because,
however, HT-29 cells are responsive to the inhibitory effects of SAMC
(Fig. 1)
, this inhibition is not entirely dependent on ras
transformation. Likewise, Cox-2 may not play a critical role, because
HCT116 colon cancer cells do not express Cox-2, (34)
, and
yet we found that they are sensitive to induction of apoptosis and
changes in cell cycle progression when treated with SAMC. Therefore,
the reason for the greater resistance of HT-29 cells to SAMC is not
apparent at the present time.
It is of interest that when treated with SAMC, both the SW-480 and
HT-29 cells accumulated in the G2-M phases of the
cell cycle when examined by DNA flow cytometry (Fig. 2A)
.
This is in contrast to the effects of sulindac, SS, and
sulindac-sulfone, which usually cause cells to arrest in
G1 (25)
. Using FISH analysis to
examine the number of centromeres (Fig. 3)
, and flow cytometry after
staining the cells with an antibody that reacts only with
phosphoproteins present in the M phase (Fig. 2B)
, we
obtained evidence that after treatment with 300
µM SAMC, an appreciable fraction of the SW-480
and HT 29 cells were actually arrested in the M phase. Studies are in
progress to determine whether this reflects an effect of SAMC on
microtubules per se, or on other proteins that control the M
phase.
Recent studies have indicated that c-jun H-terminal kinases, also
referred to as stress-activated protein kinases, are frequently
involved in cellular responses to various environmental stresses,
including agents that induce apoptosis (35
, 36)
. Yu
et al. (37
, 38)
found that
phenethylisothiocyanate, which is present in cruciferous vegetables,
induces apoptosis with concomitant activation of JNK1 and suggested
that this plays a role in the chemopreventive effect of this compound.
In the present study, we found that treatment of either SW-480 or HT-29
cells with SAMC, but not with SAC, caused, within 30 to 60 min, the
activation of JNK1. SS had a similar and even more rapid effect (Fig. 7
; Ref. 27
). Studies are in progress to examine the
significance of this finding with respect to growth inhibition, cell
cycle arrest, and apoptosis.
It has been suggested that the anticarcinogenic effect of allylsulfide
derivatives is mediated by the induction of GSH (28
, 39
, 40)
because this endogenous tripeptide thiol compound can
detoxify various carcinogens, serves as an intracellular antioxidant,
and also regulates DNA and protein synthesis (41)
. Indeed,
we found that the treatment of SW-480 or HT-29 cells with SAMC led to
increased cellular levels of GSH. However, this was also seen with SAC
(Fig. 6)
; and yet the latter compound did not have an antiproliferative
effect. Therefore, it seems unlikely that the cell cycle arrest and
apoptotic effects of SAMC are solely attributable to the increase in
GSH. At the same time, SAMC, SAC, and other compounds in garlic may, by
enhancing cellular levels of GSH, play a protective role in the early
phases of carcinogenesis by scavenging free radicals. Thus, specific
allylsulfide compounds in garlic, like SAC, might inhibit initiation of
the carcinogenic process whereas others, like SAMC, might, through
their antiproliferative effects, inhibit tumor promotion and/or
progression. Our finding that SS also induces GSH in colon cancer cells
within 24 h after treatment is consistent with previous data
indicating that nonsteroidal anti-inflammatory drugs can induce GSH and
glutathione S-transferase in the digestive tract
(42)
.
Finally, we examined whether SAC or SAMC can augment the apoptotic
effect of SS. Sulindac and sulindac derivatives are known to cause
growth inhibition and to induce apoptosis in colon cancer cells
(43
, 44) , and sulindac has been used for the suppression
of adenoma formation in patients with familial adenomatous polyposis
(45)
. We found that, when added together, SAMC and SS had
an approximately additive effect on the extent of apoptosis in both
SW-480 and HT-29 cells (Fig. 4)
. Recent studies indicate that the
combination of lovastatin with SS is also additive with respect to
induction of apoptosis in colon cancer cells (46)
.
Taken together, these cell culture studies suggest that SAMC may be useful in colon cancer chemoprevention when used alone or in combination with other compounds, e.g., sulindac or its derivatives. Obviously, additional studies are required to evaluate the efficacy of SAMC in suitable experimental animal systems.
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FOOTNOTES |
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1 This research was supported by NIH Grant
CA-63467 (to I. B. W.) and NIH Grant CA-29502 (to R. S. R.); awards
from the National Foundation for Cancer Research and the T. J. Martell
Foundation (to I. B. W.); awards from Wakunaga of America Co., Ltd.,
and the Frank J. Scallon Medical Research Foundation, (to J. T. P.);
and the Ronald and Susan Lynch Foundation, the Sunny and Abe Rosenberg
Foundation, the Rosenfeld Heart Foundation, and the Allen Foundation
(to R. S. R.). 
2 To whom requests for reprints should be
addressed, at Herbert Irving Comprehensive Cancer Center, Columbia
University, 701 West 168th Street, New York, NY 10032. Phone:
(212) 305-6921; Fax: (212) 305-6889; E-mail: weinstein{at}cuccfa.ccc.columbia.edu
3 The abbreviations used are: GSH, reduced
glutathione; SAC, S-allylcysteine; SAMC, S-allylmercaptocysteine, SS,
sulindac sulfide; PI, propidium iodide; JNK1,
c-Jun-NH2-terminal kinase; FISH, fluorescence
in situ hybridization.
Received 6/23/00. Accepted 11/13/00.
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radiation. Duration of JNK activation may determine cell death and proliferation. J. Biol. Chem., 271: 31929-31936, 1996.This article has been cited by other articles:
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M. Shimizu, A. Deguchi, J. T.E. Lim, H. Moriwaki, L. Kopelovich, and I. B. Weinstein (-)-Epigallocatechin Gallate and Polyphenon E Inhibit Growth and Activation of the Epidermal Growth Factor Receptor and Human Epidermal Growth Factor Receptor-2 Signaling Pathways in Human Colon Cancer Cells Clin. Cancer Res., April 1, 2005; 11(7): 2735 - 2746. [Abstract] [Full Text] [PDF]
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M. Shimizu, M. Suzui, A. Deguchi, J. T. E. Lim, D. Xiao, J. H. Hayes, K. P. Papadopoulos, and I. B. Weinstein Synergistic Effects of Acyclic Retinoid and OSI-461 on Growth Inhibition and Gene Expression in Human Hepatoma Cells Clin. Cancer Res., October 1, 2004; 10(19): 6710 - 6721. [Abstract] [Full Text] [PDF]
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 D. Xiao, J. T. Pinto, J.-W. Soh, A. Deguchi, G. G. Gundersen, A. F. Palazzo, J.-T. Yoon, H. Shirin, and I. B. Weinstein Induction of Apoptosis by the Garlic-Derived Compound S-Allylmercaptocysteine (SAMC) Is Associated with Microtubule Depolymerization and c-Jun NH2-Terminal Kinase 1 Activation Cancer Res., October 15, 2003; 63(20): 6825 - 6837. [Abstract] [Full Text] [PDF]
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L. M. Knowles and J. A. Milner Diallyl Disulfide Induces ERK Phosphorylation and Alters Gene Expression Profiles in Human Colon Tumor Cells J. Nutr., September 1, 2003; 133(9): 2901 - 2906. [Abstract] [Full Text] [PDF]
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F. G. Bottone Jr., J. M. Martinez, J. B. Collins, C. A. Afshari, and T. E. Eling Gene Modulation by the Cyclooxygenase Inhibitor, Sulindac Sulfide, in Human Colorectal Carcinoma Cells: POSSIBLE LINK TO APOPTOSIS J. Biol. Chem., July 3, 2003; 278(28): 25790 - 25801. [Abstract] [Full Text] [PDF]
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Poster Abstracts J. Nutr., November 1, 2002; 132(11): 3540S - 3556. [Full Text] [PDF]
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F. G. Bottone Jr., S. J. Baek, J. B. Nixon, and T. E. Eling Diallyl Disulfide (DADS) Induces the Antitumorigenic NSAID-Activated Gene (NAG-1) by a p53-Dependent Mechanism in Human Colorectal HCT 116 Cells J. Nutr., April 1, 2002; 132(4): 773 - 778. [Abstract] [Full Text] [PDF]
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 K. Antman, M. C. Benson, J. Chabot, D. Cobrinik, V. R. Grann, J. S. Jacobson, A. K. Joe, A. E. Katz, K. Kelly, A. I. Neugut, et al. Complementary and Alternative Medicine: The Role of the Cancer Center J. Clin. Oncol., September 15, 2001; 19(90001): 55s - 60. [Full Text] [PDF]
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