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