Diosgenin, a Steroid Saponin of Trigonella foenum graecum (Fenugreek), Inhibits Azoxymethane-Induced Aberrant Crypt Foci Formation in F344 Rats ...

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Diosgenin, a Steroid Saponin of Trigonella foenum graecum (Fenugreek), Inhibits Azoxymethane-Induced Aberrant Crypt Foci Formation in F344 Rats ...
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

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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

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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

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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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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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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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                                                Cancer Epidemiol Biomarkers Prev 2004;13(8). August 2004

                 Downloaded from cebp.aacrjournals.org on January 25, 2021. © 2004 American Association for Cancer
                                                            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.

Cancer Epidemiol Biomarkers Prev 2004;13:1392-1398.

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