1392
Diosgenin, a Steroid Saponin of Trigonella foenum graecum
(Fenugreek), Inhibits Azoxymethane-Induced Aberrant
Crypt Foci Formation in F344 Rats and Induces
Apoptosis in HT-29 Human Colon Cancer Cells
Jayadev Raju, Jagan M.R. Patlolla, Malisetty V. Swamy, and Chinthalapally V. Rao
Division of Nutritional Carcinogenesis, Institute for Cancer Prevention, American Health Foundation Cancer Center, Valhalla, New York
Abstract
Trigonella foenum graecum (fenugreek) is traditionally
used to treat disorders such as diabetes, high choles-
terol, wounds, inflammation, and gastrointestinal ail-
ments. Recent studies suggest that fenugreek and its
active constituents may possess anticarcinogenic poten-
tial. We evaluated the preventive efficacy of dietary
fenugreek seed and its major steroidal saponin constit-
uent, diosgenin, on azoxymethane-induced rat colon
carcinogenesis during initiation and promotion stages.
Preneoplastic colonic lesions or aberrant crypt foci
(ACF) were chosen as end points. In addition, we
assessed the mechanism of tumor growth inhibition
of diosgenin in HT-29 human colon cancer cells. To
evaluate the effect of the test agent during the initiation
and postinitiation stages, 7-week-old male F344 rats
were fed experimental diets containing 0% or 1% fen-
ugreek seed powder (FSP) or 0.05% or 0.1% diosgenin
for 1 week and were injected with azoxymethane (15
mg/kg body weight). Effects during the promotional
stage were studied by feeding 1% FSP or 0.1% diosgenin
4 weeks after the azoxymethane injections. Rats were
sacrificed 8 weeks after azoxymethane injection, and
their colons were evaluated for ACF. We found that,
by comparison with control, continuous feeding of 1%
Introduction
Colon cancer is considered a preventable disease (1).
However, there seems to be no decline in the incidence
of colon cancer, and many of the risk factors associated
with colon cancer prevail (2). Diet-based strategies hold
promise for both prevention and treatment of colon
cancer (1, 3). In this regard, plant-derived diets contain-
ing phytochemicals could be used in preventive strat-
egies to reduce the risk and inhibit or retard the
development of colon cancer. Trigonella foenum graecum,
commonly called fenugreek, is a leguminous plant native
Received 1/23/04; revised 3/3/04; accepted 3/10/04.
Grant support: USPHS grant CA-80003 from the National Cancer Institute.
The costs of publication of this article were defrayed in part by the payment of
page charges. This article must therefore be hereby marked advertisement in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Requests for reprints: Chinthalapally V. Rao, Division of Nutritional Carcinogenesis,
Institute for Cancer Prevention, American Health Foundation Cancer Center,
1 Dana Road, Valhalla, NY 10595. Phone: 914-789-7196; Fax: 914-592-6317.
E-mail: crao@ifcp.us
Copyright D 2004 American Association for Cancer Research.
Cancer Epidemiol Biomarkers Prev 2004;13(8). August 2004
Cancer Epidemiology, Biomarkers & Prevention
FSP and 0.05% and 0.1% diosgenin suppressed total
colonic ACF up to 32%, 24%, and 42%, respectively
(P V 0.001 to 0.0001). Dietary FSP at 1% and diosgenin
at 0.1% fed only during the promotional stage also
inhibited total ACF up to 33% (P V 0.001) and 39%
(P V 0.0001), respectively. Importantly, continuous
feeding of 1% FSP or 0.05% or 0.1% diosgenin reduced
the number of multicrypt foci by 38%, 20%, and 36%
by comparison with the control assay (P V 0.001). In
addition, 1% FSP or 0.1% diosgenin fed during the
promotional stage caused a significant reduction
(P V 0.001) of multicrypt foci compared with control.
Dietary diosgenin at 0.1% and 0.05% inhibited total
colonic ACF and multicrypt foci formation in a dose-
dependent manner. Results from the in vitro experi-
ments indicated that diosgenin inhibits cell growth
and induces apoptosis in the HT-29 human colon can-
cer cell line in a dose-dependent manner. Furthermore,
diosgenin induced apoptosis in HT-29 cells at least in
part by inhibition of bcl-2 and by induction of caspase-3
protein expression. On the basis of these findings, the
fenugreek constituent diosgenin seems to have poten-
tial as a novel colon cancer preventive agent. (Cancer
Epidemiol Biomarkers Prev 2004;13(8):1392 – 8)
to many Asian, Middle Eastern, and European countries
(4). The seeds and leaves of fenugreek are edible and are
used as condiments and as Ayurvedic medicine in the
Indian subcontinent to treat diabetes, high cholesterol,
wounds, inflammation, and gastrointestinal ailments (4).
Fenugreek seeds have been successfully tested in labo-
ratory animals and in humans with type 1 and type 2
diabetes as a hypoglycemic agent (5-7). The potential of
fenugreek seeds to modulate several enzymes, including
those associated with glucose and lipid metabolism, has
been described earlier (8). Among bioactive compounds
isolated from fenugreek seeds are protodioscin, trigoneo-
side, diosgenin, yamogenin, and others (9, 10).
Extracts of fenugreek seeds and some of their saponin
constituents have been found to have anticarcinogenic
potency in different settings (11, 12). Fenugreek seed
extract has been evaluated in the Ehrlich ascites car-
cinoma model in BALB/c mice, where it effected 70%
inhibition of tumor cell growth compared with controls
(11). The findings of Hibasami et al. (12) suggest that
growth inhibition of human leukemia HL-60 cells by

protodioscin, isolated from fenugreek seeds, results from
the induction of apoptosis. Diosgenin [(25R)-5-spirosten-
3h-ol], a steroid sapogenin constituent of fenugreek
seeds, is a precursor of steroid hormones, such as
progesterone, and anti-inflammatory steroids, such as
cortisone (13). Figure 1 illustrates the chemical structure
of diosgenin. Moalic et al. (14) reported that diosgenin
inhibits cell proliferation in the human osteosarcoma
1547 cell line by induction of apoptosis and G1 phase cell
cycle arrest. Furthermore, in the osteosarcoma 1547 cell
line, it was showed that diosgenin caused cell cycle arrest
and apoptosis principally by increasing the expression
of the tumor suppressor oncoprotein p53 (15). On the
basis of the information described above, diosgenin and
other fenugreek seed constituents possess anticarcino-
genic properties, suggesting their potential role as
suitable phytochemicals for colon cancer prevention.
However, to our knowledge, the colon cancer inhibitory
properties of diosgenin and other fenugreek seed
constituents have not been studied in detail, and there
is no single study to ascertain that the anticancer
capabilities of diosgenin showed earlier in vitro will
prevail in an in vivo setting.
Colon carcinogenesis is a multistep process involving
sequential change of normal colonic epithelial cells into
preneoplastic, neoplastic, and metastatic states (16).
Aberrant crypt foci (ACF) are preneoplastic lesions of
the colon in mice, rats, and humans that are regarded
as valuable biomarkers in screening potential chemo-
preventive agents (reviewed in refs. 17, 18). The molec-
ular basis for inhibition of preneoplastic and neoplastic
lesions by potential chemopreventive agents is currently
being explored. Changes pertaining to aberrant cell
growth and proliferation that lead to tumorigenicity
have been identified as early as in the formation of
ACF per se (19-22). Mechanisms involved in the inhi-
bition of cell proliferation and induction of apoptosis are
recognized as being pivotal. Bcl-2 and caspase-3 among
others have been implicated as molecular mediators of
apoptosis (reviewed in refs. 23, 24). Colon tumors are
characterized by an overexpression of bcl-2, whereby
apoptosis is down-regulated (25), and chemopreventive
agents decrease the expression of bcl-2 in human colon
cancer cells, thus augmenting apoptosis (26, 27). By
contrast, the proapoptotic caspase-3 is down-regulated
in colon tumors, and its expression is increased by cancer
preventive agents (reviewed in ref. 24). Several studies
have used the measurement of apoptosis in colon cancer
cells induced by chemopreventive agents to assess the
efficacy of such agents (26-29). Whether diosgenin
inhibits colon tumor cell growth by the induction of
apoptosis is not known. The present study was therefore
designed to evaluate the efficacy of dietary fenugreek
Figure 1. Structure of diosgenin.
Cancer Epidemiol Biomarkers Prev 2004;13(8). August 2004
Cancer Epidemiology, Biomarkers & Prevention 1393
seed and its major steroid saponin constituent diosgenin
in inhibiting or retarding ACF formation during ini-
tiation/postinitiation and promotional stages of azoxy-
methane-induced rat colon carcinogenesis. In addition,
we determined the effect of diosgenin on inhibiting
cell growth and modulating the expression of bcl-2 and
caspase-3 in HT-29 human colon cancer cells.
Materials and Methods
Animals, Care, and Diets. Seven-week-old male F344
rats were procured from Charles River Laboratories
(Kingston, NY) and housed in suspended cages f10 cm
above bedding trays with a 12-hour light/dark cycle in
the animal housing facility of the Institute for Cancer
Prevention (Valhalla, NY). Temperature and relative
humidity were controlled at 21jC and 55%, respectively.
All animals were acclimatized to the above conditions
for 1 week with free access to standard laboratory rodent
chow and drinking water until initiation of the exper-
iment. Animals were cared for according to the guide-
lines of the American Council on Animal Care. Diets
were based on modified AIN-76A containing 5% corn
oil by weight (30). Fenugreek seeds were a gift from
Dr. Peter R. Chang (Agriculture and Agro-Food Canada
Research Center, Saskatoon, SK), and diosgenin was
purchased from Sigma Chemical Co. (St. Louis, MO). The
control diet contained no fenugreek seed powder (FSP)
or diosgenin. The experimental diets contained 1% FSP
or 0.05% or 0.1% diosgenin (w/w). Diets were prepared
twice each week and were stored at 4jC until used. Rats
were allowed ad libitum access to the respective diets
and tap water, and food cups were replenished with
fresh diets thrice weekly. The stability of diosgenin in
the diet was established by testing the experimental diet
kept at room temperature for a period of 1 week. Each
day, diet samples were collected, extracted, and analyzed
by high-performance liquid chromatography according
to the method of Artuno et al. (31). Based on the results,
even after 7 days at room temperature, >95% diosgenin
were recoverable from the feed, suggesting the reason-
able stability of diosgenin at room temperature.
Experimental Design. The experimental protocol is
shown in Fig. 2. Rats were randomized into groups
receiving either the control diet (n = 30) or diets con-
taining 1% FSP or 0.05% or 0.1% diosgenin (n = 10 per
group). Beginning 1 week later, all rats were s.c. injected
with azoxymethane once a week for 2 weeks at a dose of
15 mg/kg body weight. Four weeks after the second
injection, rats intended for promotion stage testing were
switched from control diet to experimental diets, in
this case, either 1% FSP or 0.1% diosgenin (n = 10 per
group). All animals were sacrificed by CO2 asphyxiation
8 weeks after azoxymethane injection. The colons were
removed, flushed with ice cold PBS, and slit open along
the length from the anus to the cecum on an ice-cold
glass plate. The colons were examined for any macro-
scopic changes and were fixed flat between filter papers
in 70% ethanol and coded for blind scoring.
Quantification of ACF. Topographical analysis of
the colonic mucosa according to Bird (32) was done after
a minimum of 24 hours in 70% ethanol. Colons were
1394 Colon Cancer Preventive Effects of Diosgenin/Fenugreek
Figure 2. In vivo experimental protocol.
stained with 0.2% methylene blue solution for 5 to 10
minutes, placed mucosal side up on a microscopic slide,
and viewed under a light microscope. The total number
of ACF in the entire colon was determined in every 2 cm
section with the distal colon as the starting point and
through to the proximal end of the colons. ACF were
categorized into those with crypt multiplicity of 1, 2, 3,
and z4.
Cell Culture and Treatments. HT-29 human colon
cancer cells obtained from American Type Culture
Collection (Manassas, CA) were maintained in McCoy’s
5A medium (Life Technologies, Inc., Grand Island, NY)
with 10% fetal bovine serum in a humidified atmosphere
of 95% air and 5% CO2 at 37jC. All studies were done
with cells at f70% to 80% confluence. Stock solution of
10 2 mol/L diosgenin was prepared in ethanol. Cells
were treated with either diosgenin or ethanol; the
latter at a final concentration of 0.1% was added to the
cells not treated with diosgenin.
Toxicity and Cell Proliferation Assays. The 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT) assay and trypan blue exclusion method were
done to assess the effect of diosgenin on toxicity and cell
proliferation, respectively. In both methods, quadruplet
samples were run for each concentration of diosgenin
and for each time point, and the experiment was re-
peated thrice. For the MTT assay, HT-29 cells were
seeded in 96-well culture plates and treated with 0 to
100 Amol/L diosgenin for 24 hours. Then, 20 AL of MTT
(5 mg/mL stock) solution were added to the wells and
incubated at 37jC for 5 hours. Thereafter, the medium
was gently removed from the wells, and 200 AL of DMSO
were added to each well to dissolve the purple formazan
crystals. The absorbance at 570 nm was recorded using
the Dynatech MR5000 spectrophotometer (Dynatech
Laboratories, Inc., Chantilly, VA). For the trypan blue
exclusion method, HT-29 cells were seeded in six-well
culture plates, treated with 0 to 100 Amol/L diosgenin,
and incubated for 18, 24, 36, and 48 hours. At each time
point, cells were washed with PBS and trypsinized. One
hundred microliters of the cell suspension were mixed
with 100 AL of trypan blue dye (0.4% trypan blue in
saline), a small aliquot was applied to a hemocytometer,
and live cells were counted with the coverslip on.
Apoptosis Assay by Acridine Orange/Ethidium
Bromide Staining. HT-29 cells treated with 0, 20, 40, or
Cancer Epidemiol Biomarkers Prev 2004;13(8). August 2004
60 Amol/L diosgenin for 24 hours were washed with PBS
and trypsinized. Twenty-five microliters of the cell sus-
pension (f0.5  106 per mL) were incubated with 1 AL
of acridine orange/ethidium bromide (one part each of
100 Ag/mL acridine orange and 100 Ag/mL ethidium
bromide in PBS) just prior to microscopy. A 10 AL aliquot
of the gently mixed suspension was placed on micro-
scope slides, covered with glass slips, and examined
under an Olympus AX70 microscope (Tokyo, Japan)
connected to a digital imaging system with SPOT RT
Software version 3.0. Acridine orange is a vital dye that
will stain both live and dead cells, whereas ethidium
bromide will stain only those cells that have lost their
membrane integrity. Live cells stain uniformly green and
can be distinguished from apoptotic cells as they exhibit
yellow to orange coloration depending on the degree of
loss of membrane integrity due to costaining with
ethidium bromide.
Western Blot Analyses. Whole cell lysates of treated
and untreated HT-29 cells were prepared with lysis
buffer containing protease inhibitors. Total proteins were
quantified using the Bio-Rad Protein Assay reagent (Bio-
Rad Laboratories, Hercules, CA). Volumes of whole
cell lysates containing 100 Ag protein were heated for
4 minutes at 80jC with 2 Laemmli sample buffer
(Sigma Chemical) and separated by 10% SDS-PAGE
using the Mini-Protean Bio-Rad II System. The separated
proteins were transferred to Hybond enhanced chemilu-
minescence nitrocellulose membranes (Amersham Life
Technologies, Arlington Heights, IL). These membranes
were blocked for 1 hour at room temperature with 5%
skim milk powder and probed with primary antibodies
at 4jC overnight on a shaker. The primary antibodies
were rabbit anti-bcl-2 and anti-caspase-3 (sc-492 and
sc-7148, respectively; Santa Cruz Biotechnology, Santa
Cruz, CA) at 1:500 dilutions. Blots were washed and
incubated with secondary anti-rabbit antibody conjugat-
ed with horseradish peroxidase (Santa Cruz Biotechnol-
ogy) at 1:2,500 dilution for 1 hour at room temperature.
After washing, the blots were incubated with Super-
Signal West Pico Chemiluminescent Substrate (Pierce
Chemical Co., Rockford, IL) for 5 minutes and exposed
to photographic film to detect protein bands.
Results
General Observations. To ascertain that dietary
fenugreek seed or diosgenin had no negative effect on
body weight gain or eating habit, all rats were moni-
tored on a routine basis. The initial body weight (mean F
SE) before dietary interventions with fenugreek seed or
diosgenin and azoxymethane injection was 117.20 F
2.18. At the time of termination, there was no significant
difference in body weights of control and treated rats
(Table 1). The food intake of animals in the experi-
mental groups did not vary. Fenugreek seed at 1% and
diosgenin at 0.05% or 0.1% were well tolerated and
caused no adverse effects in F344 rats.
Effect of FSP and Diosgenin on Colonic ACF during
Initiation/Postinitiation Stages. We used the well-
established, short-term protocol of the azoxymethane-
induced rat colon carcinogenesis model to determine

Table 1. Body weights of animals treated with or
without fenugreek seed or diosgenin at either
initiation/postinitiation or promotion stage (n = 10
per group)
Group Mean F SE Body Weight (g)
Control 282.0 F 4.19
Intervention at initiation/
postinitiation stages
0.05% Diosgenin 282.5 F 4.63
0.1% Diosgenin 277.7 F 5.48
1% Fenugreek seed 290.1 F 6.04
Intervention at promotion stages
0.1% Diosgenin 278.0 F 8.80
1% Fenugreek seed 289.9 F 5.92
the efficacy of fenugreek seed and diosgenin to inhibit
the formation or retard the development of ACF. Dietary
1% FSP and 0.05% and 0.1% diosgenin given continu-
ously for 8 weeks suppressed total colonic ACF to
32%, 24%, and 42% (P V 0.001 to 0.0001), respectively,
compared with control group (Fig. 3). Importantly, the
continuous feeding of 1% FSP or 0.05% or 0.1% dios-
genin significantly lowered the number of multicrypt
foci or large ACF (with crypt multiplicity z4) by 38%,
20%, and 36% compared with control (P V 0.001; Fig. 4).
Dietary diosgenin inhibited total colonic ACF and mul-
ticrypt ACF formation in a dose-dependent manner
(Figs. 3 and 4).
Effect of FSP and Diosgenin on Colonic ACF during
Promotion Stages. To determine the effects of fenugreek
seed and diosgenin at the promotional stages, groups
of rats were given dietary 1% FSP and 0.1% diosgenin
4 weeks after azoxymethane injections for 4 weeks. In
this protocol, 1% FSP and 0.1% diosgenin inhibited
total ACF up to 33% (P V 0.001) and 39% (P V 0.0001),
respectively (Fig. 3). Similar to the effects observed in
the initiation/postinitiation stages, 1% FSP and 0.1%
diosgenin resulted in 25% and 32% lower (P V 0.001)
incidence of multicrypt ACF than were seen in positive
controls (Fig. 4).
Effect of Diosgenin on HT-29 Human Colon Cancer
Cell Proliferation and Apoptosis. To explore the anti-
cancer potential of diosgenin in human colon cancer
cells, we conducted several in vitro experiments. We
examined the cytotoxic effects of 0 to 100 Amol/L
Figure 3. Effect of dietary FSP and diosgenin on azoxy-
methane-induced colonic ACF formation: total ACF data.
Columns, mean; bars, SE. *, P < 0.001, **, P < 0.0001,
significantly different from control.
Cancer Epidemiol Biomarkers Prev 2004;13(8). August 2004
Cancer Epidemiology, Biomarkers & Prevention 1395
Figure 4. Effect of dietary FSP and diosgenin on azoxy-
methane-induced colonic ACF formation: multicrypt foci data.
Columns, mean; bars, SE. *, P < 0.001, **, P < 0.0001,
significantly different from control.
diosgenin (24-hour treatment) on HT-29 cells using the
MTT cytotoxicity assay. A dose-dependent MTT reduc-
tion (or color change from yellow to purple) was
observed in diosgenin-treated cells (Fig. 5). On 24-hour
exposure to diosgenin, MTT activity reduced by z50%
was achieved at the higher concentrations (i.e., z80
Amol/L). However, compared with the control, 20 to 60
Amol/L diosgenin reduced the MTT activity only by
f5% to 30% (Fig. 5). Next, we examined dose-dependent
and time-dependent effects of diosgenin on the prolifera-
tion of HT-29 cells using the trypan blue dye exclusion
method. Diosgenin caused a significant time-dependent
and dose-dependent decrease in the proliferation of
HT-29 cells (Fig. 6). Twenty-four-hour exposure to
diosgenin (20 to 100 Amol/L) inhibited cell proliferation
compared with untreated cell growth (taken as 0%;
Fig. 6). This inhibition was 21%, 68%, 82%, and 100% for
20, 40, 60, and 80 Amol/L diosgenin, respectively (Fig. 6).
To determine whether the inhibition of cell prolif-
eration by diosgenin was due to the induction of
apoptosis, we assessed the latter with the acridine
orange/ethidium bromide method. Figure 7 summarizes
the apoptotic effects of diosgenin in HT-29 cells. A dose-
dependent increase in induction of apoptosis was
observed when HT-29 cells were treated with diosgenin
Figure 5. Cytotoxic effects of various doses of diosgenin in
HT-29 cells as assessed by MTT activity assay. Points, mean;
bars, SE.
1396 Colon Cancer Preventive Effects of Diosgenin/Fenugreek
Figure 6. Time-dependent and dose-dependent effects of
diosgenin on HT-29 cell proliferation as measured by trypan
blue exclusion method. Points, mean %; bars, SE.
at 0, 20, 40, and 60 Amol/L for 24 hours. Compared with
the control, 42% and 62% of the cell population in 40 and
60 Amol/L diosgenin-treated cells displayed apoptosis,
respectively.
Diosgenin Modulates the Protein Expression of bcl-2
and Caspase-3 in HT-29 Cells. Western blot analyses
of the apoptosis regulatory proteins bcl-2 and caspase-3
were conducted using HT-29 cells treated with or
without 24-hour diosgenin. We found that bcl-2 protein
was significantly decreased in a dose-dependent man-
ner by 0, 20, 40, and 60 Amol/L diosgenin (Fig. 8); by
contrast, these doses of diosgenin significantly increas-
ed caspase-3 expression in HT-29 cells again in a dose-
dependent manner (Fig. 8).
Figure 7. Acridine orange/ethidium bromide staining of HT-29
cells to detect apoptosis induced by different doses of diosgenin:
(A) 0 Amol/L, (B) 20 Amol/L, (C) 40 Amol/L, and (D) 60 Amol/L.
Live cells are uniformly green, whereas apoptotic cells (arrows)
are characterized by yellow-orange staining due to chromatin
condensation and loss of membrane integrity. Magnification
400.
Cancer Epidemiol Biomarkers Prev 2004;13(8). August 2004
Figure 8. Dose-dependent modulation of bcl-2 and casapse-3
proteins by Western blot analyses in HT-29 cells.
Discussion
The main objective of this study was to evaluate the
potential efficacy of fenugreek seed, a commonly used
herb, and its steroid saponin constituent, diosgenin, in
preventing colon carcinogenesis in vivo and to under-
stand the anticancer mechanisms of diosgenin in vitro.
To our knowledge, this is the first study demonstrating
that fenugreek seed and diosgenin have the potential
to prevent colon cancer. Using the azoxymethane-
induced rat colon carcinogenesis model, we show that
dietary fenugreek seed and diosgenin reduce or retard
the appearance of colonic ACF when given during
the initiation/postinitiation stages and even when given
only during the promotional stage. In addition, diosge-
nin inhibits cell proliferation and induces apoptosis in
HT-29 human colon cancer cell lines. The induction of
apoptosis by diosgenin is in part affected by its ability to
suppress the expression of the antiapoptotic bcl-2 while
increasing the expression of the proapoptotic caspase-3.
Fenugreek seeds and their active constituents have
been reported to be excellent antidiabetic agents based
on several in vivo studies, including human intervention
studies (reviewed in ref. 5), and their possible mecha-
nisms of action as antidiabetics have been described
(8, 33). In a 90-day subchronic study, rats fed fenugreek
seeds, at doses between 1% and 10% in pure diet, had
no toxic effects (34). In the present study, 1% fenugreek
seed was used in the bioassay with rats. The level of
diosgenin in fenugreek seeds ranges from f0.42% to
0.75% depending on the cultivars and seed quality (35).
Because the entire seed was used, active seed contents
other than diosgenin might influence ACF modulation;
therefore, we selected doses at higher levels of diosgenin
(0.1% and 0.05%) than are actually found in 1% fenu-
greek diet (f0.004% to 0.007%). Moreover, in a previous
study, no acute toxic effects were reported when rats
were given dietary diosgenin at 1%, 0.2%, or 0.05% doses
(36). As our results indicate, no acute or chronic distress
was observed in diosgenin-treated or fenugreek seed –
treated animals.
Colon carcinogenesis is a multistep event; it involves
the transformation of normal colonic epithelial cells into
a preneoplastic state and progresses toward advanced
neoplasia (16). We observed a significant inhibition of
the initiation and development of total and large colonic

ACF when 1% fenugreek or 0.1% or 0.05% diosgenin
were given during either initiation/postinitiation or pro-
motion stage. The ability to cause regression or retard the
appearance of ACF in either stage marks fenugreek seed
and diosgenin as likely colon cancer preventive agents.
The latter is being confirmed through tumorigenesis
studies in our laboratory using the azoxymethane-
induced colon cancer model in rats with intervention
strategies directed toward initiation/postinitiation and
promotion/progression stages. In the present study,
ACF were classified according to their size; ACF with
one to three crypts and those with four or more crypts
were designated as either ‘‘small’’ or ‘‘large’’ (multi-
crypt), respectively. Fenugreek seed and diosgenin
significantly reduced the number of large ACF in both
intervention strategies used. This suggests that these
agents would be effective not only in preventing the
appearance of ACF but plausibly also in retarding the
growth and progression of large ACF, including those
of the intermediate and advanced type. This aspect is
very important considering that a large portion of the
population at risk for colon cancer is characterized by the
presence of polyps and large ACF in their colons (37, 38).
It is also important with regard to the dose levels of
diosgenin used that there were no differences in ACF
numbers between rats treated with 0.1% and those re-
ceiving 0.05% of the agent; hence, the lower dose seems
sufficient to block ACF formation during the early stages
of colon carcinogenesis. However, the dose responses to
0.05% or 0.1% diosgenin in inhibiting tumor parameters
require further evaluation.
Our in vitro data indicate dose-dependent inhibition of
HT-29 human colon cancer cell proliferation by diosge-
nin. Diosgenin exhibited cytotoxicity only at the higher
concentrations (80 to 100 Amol/L); however, concentra-
tions as low as 40 Amol/L were capable of inhibiting cell
proliferation by z50%. Furthermore, diosgenin induced
apoptosis in HT-29 cells; this is at least in part mediated
by the suppression of bcl-2 and the induction of caspase-
3 proteins. As mentioned earlier, colonic tumors poten-
tiate their growth and survival by suppressing apoptosis
(23, 24). How diosgenin regulates bcl-2 or caspase-3
expression during colon carcinogenesis in vivo is unclear
and warrants further investigation. Other mechanism(s)
of diosgenin that could possibly be involved in the
inhibition of HT-29 cells could be those relating to
modulation of cyclooxygenase-2 and the activation of
nuclear factor-nB, p53, or p21 expression as shown earlier
in the inhibition of osteosarcoma cells (14).
In summary, our results show for the first time that (a)
fenugreek seed and diosgenin inhibit early events of
azoxymethane-induced colon cancer when given during
either initiation/postinitiation or promotion stage and (b)
diosgenin exhibits anticancer effects by blocking the
proliferation of HT-29 human colon cancer cells and
induces apoptosis in part by modulating bcl-2 and
caspase-3 expression in vitro. How much of the dietary
diosgenin is absorbed into the system and how it is
retained, metabolized, or excreted by rats during azoxy-
methane-induced colon carcinogenesis are currently
under investigation in our laboratory. Phytochemicals
or food-based compounds hold promise for novel strate-
gies in cancer chemoprevention and control (reviewed in
ref. 39). Whereas the clinical potential of fenugreek seeds
Cancer Epidemiol Biomarkers Prev 2004;13(8). August 2004
Cancer Epidemiology, Biomarkers & Prevention 1397
and their active constituents in the control of hypercho-
lesterolemia or diabetes has been documented (reviewed
in ref. 5), the findings of this study and those of earlier
ones demonstrating the anticancer properties of fenu-
greek constituents and diosgenin (11, 12, 14, 15) have
potential clinical relevance for cancer prevention and
control. Thus, the role of fenugreek seed and its main
active constituent diosgenin as new supplements in diet-
based preventive/therapeutic strategies to potentially
alleviate human colon cancer remains an important field
of study for future investigations.
Acknowledgments
We thank Ilse Hoffman for editorial expertise, Dr. Arun Sharma
and Barbara Simi for help, and the staff of the Research Animal
Facility of the Institute for Cancer Prevention for providing
technical assistance in the animal study.
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