Diosgenin, a Steroid Saponin of Trigonella Foenum Graecum

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: [email protected] (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

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 rats were procured from Charles River Laboratories basis of the information described above, diosgenin and f other fenugreek seed constituents possess anticarcino- (Kingston, NY) and housed in suspended cages 10 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 j properties of diosgenin and other fenugreek seed humidity were controlled at 21 C 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 j being explored. Changes pertaining to aberrant cell twice each week and were stored at 4 C 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

60 Amol/L diosgenin for 24 hours were washed with PBS and trypsinized. Twenty-five microliters of the cell suspension (f0.5 Â 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 microscope 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 z and 4. 4 minutes at 80jC with 2Â 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 j were blocked for 1 hour at room temperature with 5% of 95% air and 5% CO2 at 37 C. All studies were done with cells at f70% to 80% confluence. Stock solution of skim milk powder and probed with primary antibodies j 10À2 mol/L diosgenin was prepared in ethanol. Cells at 4 C 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

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 azoxymethane-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 proliferation 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 V V Fig. 6). This inhibition was 21%, 68%, 82%, and 100% for total ACF up to 33% (P 0.001) and 39% (P 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% V eration by diosgenin was due to the induction of diosgenin resulted in 25% and 32% lower (P 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 azoxymethane-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.

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

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, diosgenin 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 mechanisms 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% fenugreek 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)0Amol/L, (B)20Amol/L, (C)40Amol/L, and (D)60Amol/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 400Â. the initiation and development of total and large colonic

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

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’’ (multicrypt), 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 appearance of ACF but plausibly also in retarding the technical assistance in the animal study. 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 population at risk for colon cancer is characterized by the 1. Giovannucci E. Modifiable risk factors for colon cancer. Gastro- presence of polyps and large ACF in their colons (37, 38). enterol Clin North Am 2002;31:925-43. It is also important with regard to the dose levels of 2. Jemal A, Murray T, Samuels A, Ghafoor A, Ward E, Thun MJ. Cancer statistics, 2003. CA Cancer J Clin 2003;53:5-26. diosgenin used that there were no differences in ACF 3. Milner JA, McDonald SS, Anderson DE, Greenwald P. Molecular numbers between rats treated with 0.1% and those re- targets for nutrients involved with cancer prevention. Nutr Cancer ceiving 0.05% of the agent; hence, the lower dose seems 2001;41:1-16. sufficient to block ACF formation during the early stages 4. Chevallier A. Encyclopedia of herbal medicine. New York (NY): Dorling Kindersley Publishing, Inc.; 2000. p. 271. of colon carcinogenesis. However, the dose responses to 5. Basch E, Ulbricht C, Kuo G, Szapary P, Smith M. Therapeutic 0.05% or 0.1% diosgenin in inhibiting tumor parameters applications of fenugreek. Altern Med Rev 2003;8:20-7. require further evaluation. 6. Sharma RD, Raghuram TC, Rao NS. Effect of fenugreek seeds on blood glucose and serum lipids in type I diabetes. Eur J Clin Nutr Our in vitro data indicate dose-dependent inhibition of 1990;44:301-6. HT-29 human colon cancer cell proliferation by diosge- 7. Madar Z, Abel R, Samish S, Arad J. Glucose-lowering effect of nin. Diosgenin exhibited cytotoxicity only at the higher fenugreek in non-insulin dependent diabetics. Eur J Clin Nutr 1988; concentrations (80 to 100 Amol/L); however, concentra- 42:51-4. A 8. Raju J, Gupta D, Rao AR, Yadava PK, Baquer NZ. Trigonella foenum tions as low as 40 mol/L were capable of inhibiting cell graecum (fenugreek) seed powder improves glucose homeostasis in proliferation by z50%. Furthermore, diosgenin induced alloxan diabetic rat tissues by reversing the altered glycolytic, gluco- apoptosis in HT-29 cells; this is at least in part mediated neogenic and lipogenic enzymes. Mol Cell Biochem 2001;224:45-51. by the suppression of bcl-2 and the induction of caspase- 9. Murakami T, Kishi A, Matsuda H, Yoshikawa M. Medicinal foodstuffs. XVII. Fenugreek seed (3): structures of new furostanol- 3 proteins. As mentioned earlier, colonic tumors poten- type steroid saponins, trigoneosides Xa, Xb, XIb, XIIa, XIIb, and XIIIa, tiate their growth and survival by suppressing apoptosis from the seeds of Egyptian Trigonella foenum graecum L. Chem Pharm (23, 24). How diosgenin regulates bcl-2 or caspase-3 Bull (Tokyo) 2000;48:994-1000. expression during colon carcinogenesis in vivo is unclear 10. Yoshikawa M, Murakami T, Komatsu H, Murakami N, Yamahara J, Matsuda H. Medicinal foodstuffs. IV. Fenugreek seed (1): structures and warrants further investigation. Other mechanism(s) of trigoneosides Ia, Ib, IIa, IIb, IIIa, and IIIb, new furostanol saponins of diosgenin that could possibly be involved in the from the seeds of Indian Trigonella foenum graecum L. Chem Pharm inhibition of HT-29 cells could be those relating to Bull (Tokyo) 1997;45:81-7. modulation of cyclooxygenase-2 and the activation of 11. Sur P, Das M, Gomes A, et al. Trigonella foenum graecum (fenugreek) n seed extract as an antineoplastic agent. Phytother Res 2001;15:257-9. nuclear factor- B, p53, or p21 expression as shown earlier 12. Hibasami H, Moteki H, Ishikawa K, et al. Protodioscin isolated from in the inhibition of osteosarcoma cells (14). fenugreek (Trigonella foenum graecum L.) induces cell death and In summary, our results show for the first time that (a) morphological change indicative of apoptosis in leukemic cell line H-60, but not in gastric cancer cell line KATO III. Int J Mol Med fenugreek seed and diosgenin inhibit early events of 2003;11:23-6. azoxymethane-induced colon cancer when given during 13. Norton SA. Useful plants of dermatology. III. Corticosteroids, either initiation/postinitiation or promotion stage and (b) strophanthus, and dioscorea. J Am Acad Dermatol 1998;38:256-9. diosgenin exhibits anticancer effects by blocking the 14. Moalic S, Liagre B, Corbiere C, et al. A plant steroid, diosgenin, induces apoptosis, cell cycle arrest and COX activity in osteosarcoma proliferation of HT-29 human colon cancer cells and cells. FEBS Lett 2001;506:225-30. induces apoptosis in part by modulating bcl-2 and 15. Corbiere C, Liagre B, Bianchi A, et al. Different contribution of caspase-3 expression in vitro. How much of the dietary apoptosis to the antiproliferative effects of diosgenin and other plant diosgenin is absorbed into the system and how it is steroids, hecogenin and tigogenin, on human 1547 osteosarcoma cells. Int J Oncol 2003;22:899-905. retained, metabolized, or excreted by rats during azoxy- 16. Fearon ER, Vogelstein B. A genetic model for colorectal tumori- methane-induced colon carcinogenesis are currently genesis. Cell 1990;61:759-67. under investigation in our laboratory. Phytochemicals 17. Bird RP. Role of aberrant crypt foci in understanding the patho- or food-based compounds hold promise for novel strate- genesis of colon cancer. 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Downloaded from cebp.aacrjournals.org on September 30, 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.

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