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Cell Biology International (2005) 29, 687–694 (Printed in Great Britain)
Chemopreventive activities of Trigonella foenum graecum (Fenugreek) against breast cancer
Amr Amina*, Aysha Alkaabia, Shamaa Al‑Falasia and Sayel A. Daoudb
aBiology Department, UAE University, P.O. Box 17551, Al-Ain, United Arab Emirates
bHistopathology Department, Twam Hospital, P.O. Box 15258, Al-Ain, United Arab Emirates


Abstract

Cancer is the second leading cause of death worldwide. Conventional therapies cause serious side effects and, at best, merely extend the patient's lifespan by a few years. Cancer control may therefore benefit from the potential that resides in alternative therapies. There is thus an increasing demand to utilize alternative concepts or approaches to the prevention of cancer. In this report, we show a potential protective effect of Fenugreek seeds against 7,12-dimethylbenz(α)anthracene (DMBA)-induced breast cancer in rats. At 200mg/kg b.wt., Fenugreek seeds' extract significantly inhibited the DMBA-induced mammary hyperplasia and decreased its incidence. Epidemiological studies also implicate apoptosis as a mechanism that might mediate the Fenugreek's anti-breast cancer protective effects. To our knowledge, this is the first study that suggests significant chemopreventive effects of Fenugreek seeds against breast cancer.


Keywords: Breast cancer, Fenugreek, Chemoprevention, Rat.

*Corresponding author. Tel.: +971 3 7134389; Fax: +971 3 7671291.


1 Introduction

Breast cancer is the third most common cancer in the world with about 48 000 women dying of breast cancer every year. Over 50% of breast cancer incidence occur in the developed countries (Castro et al., 2001; Dagar et al., 2001). In the past few years, incidence of breast cancer has been dramatically increasing in developed countries (Antismog, 2003). Breast cancer is also one of the most common types of cancers in the Middle East region. During the last two decades, the United Arab Emirates (UAE), like many other gulf countries, has witnessed rapid development in many aspects of life. Increased development has led to a parallel increase in major public health problems including breast cancer (El-Helal, 1997; Denic and Bener, 2001).

Along with the conventional means of cancer control, the concept of chemoprevention has recently gained scientific recognition worldwide (Chemoprevention Working Group, 1999). The main objective of cancer chemoprevention research is to advance knowledge in identifying and characterizing entities that might reduce the risk of the human population developing cancer. Therefore, it is of interest to explore the possibility of using phytochemicals or other dietary chemicals as chemopreventive agents. Further, the study of the biological effects of these phytochemicals at cellular level provides the molecular basis for their anti-disease function and helps to establish the platform for generating more potent chemopreventive and even chemotherapeutic agents (Gosslau and Yu-Chen, 2004). In theory, cancer chemoprevention can be defined as an intervention in the carcinogenic process by a chemical that either blocks neoplastic process induction or prevents transformed cells from progressing to malignant phenotype. It may also encompass a reversal of the process of progression (Chow et al., 2003; Kelloff et al., 1994). In practice, a potential intervention agent must enhance the physiological processes protecting humans against pre-neoplastic cell progression or neoplastic cell growth.

Legumes are rich in nutrients such as digestible protein with good array of amino acids and minerals. Leguminous seeds have been reported to be excellent sources of energy in animal and human diets. This explains why considerable research has been directed to harnessing the potential of these seeds in animal or human diets (Kelloff et al., 1992).

Fenugreek has been widely cultivated in Asia, Africa and Mediterranean countries for the edible and medicinal values of its seeds. In Chinese traditional medicine, the seeds of this plant have been prescribed as a tonic for stomach disorders, and the whole aerial part of the plant is used as a folk medicine for the treatment of renal diseases in the Northern-East region of China. Many phytochemical studies on constituents of the seeds have been reported (Mohamad et al., 2004).

Trigonella foenum graecum seeds have been shown to lower blood glucose level and partially restore the activities of key enzymes of carbohydrate and lipid metabolism close to normal values in various animal model systems. The components responsible and the mechanism by which Fenugreek exerts these effects in not clearly understood. However, several studies have shown the presence of steroid saponins in Fenugreek seeds. Saponin compounds, diasgenin, alkaloids and trigonelline, inhibit intestinal glucose uptake in vitro. 4-Hydroxyisoleucine, a modified amino acid extracted and purified from Fenugreek seeds, also displayed an insulinotropic property in vitro, stimulated insulin secretion in vivo and improved glucose tolerance in normal rats and dogs and in rat model of type 2 diabetes mellitus. Besides 4-hydroxyisoleucine, arginine and tryptophan are the other amino acids having antidiabetic and hypoglycemic effect. In addition to this, many trace elements, which are the components of Fenugreek, have been found to possess antidiabetic effects (Agbede and Aletor, 2005). However, little research has been carried out on the effect of this herbal plant on cancer. This study investigates the anti-cancer activities of the Fenugreek on DMBA-induced breast cancer in a mammalian model.

Animal models have helped demonstrating different classes of chemicals to act as initiators and induce mammary cancer. Polycyclic aromatic hydrocarbons (PAHs) are toxic compounds commonly found in the environment. PAHs are genotoxic and capable of forming carcinogen-DNA adducts in human or animal tissues. Once these chemicals are consumed, our body will metabolize and transform these compounds into DNA-attacking mutagens. Epidemiological studies have revealed that PAHs can form adducts in humans. Higher amounts of benzyl-[α]-pyrenelike DNA adducts have been found in human breast tumors than in normal breast tissues. As one of the PAHs, DMBA has been shown to affect cellular signaling pathways to induce apoptosis. It is also known to induce cytotoxicity in various cell types, including mouse epidermis, hepatoma, and fibroblasts (Ko et al., 2004). The DMBA-induced rat mammary carcinomas have been shown to arise from the ductal elements of the mammary gland (Chow et al., 2003).

In this report, we investigate the protective effects of T. foenum graecum against the development of breast cancer in rats using the DMBA-induced mammary tumor model.

2 Materials and methods

2.1 Plant extract

Fenugreek seeds were purchased from commercial sources. Before analysis, the dry beans were ground three times with an electric coffee grinder (Krups, Germany) and were extracted in warm water, filtered and lyophilized.

2.2 Chemicals

DMBA was purchased from Sigma, USA; sesame oil and the rest of chemical reagents were purchased from local stores.

2.3 Animals

Seven- to eight-weeks-old female Wistar rats (170–200g) were housed in a pathogen-free environment at the animal house of the Faculty of Medicine in UAE University. Twenty milligram/head of DMBA was administered by gastric intubation to designated rat groups (Fig. 1).


Fig. 1

Experimental design. Rats were fed with 200mg/kg b.wt. tested herb (Group A) or tested herb+20mg/head DMBA (Group B) or sesame oil (Group C) or 20mg/head DMBA only (Group D).


2.4 Experimental protocol

Eight-week-old female Wistar rats were maintained in the Animal Unit of UAE University (UAEU). Rats were housed under constant environmental conditions (photoperiod, temperature, air humidity, food). At 9 weeks of age, each rat of designated groups was treated with 20mg of DMBA (Takahashi et al., 2000; Fig. 1) (Sigma Chemical Co, St. Louis, MO) alone or 7 days after daily administration of Trigonella seed extract dissolved in olive oil.

The rats were palpated for tumor detection twice weekly throughout a 120-day observation period. Once DMBA-induced rat mammary carcinomas had developed, the tumors' growth was checked daily. All grossly observed (i.e., macroscopically visible) mammary tumors were measured after dissection (Fig. 2). The tumors were harvested and the rats were sacrificed for necropsy. The first palpable tumor was observed 110 days post-carcinogen administration. Histological examination of the DMBA-induced rat mammary carcinomas was performed on sections stained with hematoxylin and eosin.


Fig. 2

Isolated tumor (A) from a Wistar rat after tumor (arrow) induction by DMBA (B).


2.5 Immunohistochemistry

Immunohistochemical analyses were done on sections of mammary glands. α-Actin, progesterone receptor (PR) and E-cadherine (E-cad) were analyzed using avidin–biotin complex (ABC) method. After deparaffinization, 4μm-thick sections were sequentially treated with 0.3% H2O2, for 10min, and then blocked with 10% goat or horse serum in PBS for 20min. On thawing, the sections were rinsed in PBS and treated with primary monoclonal antibodies of mouse anti-PR (Dako; diluted 1:100), mouse anti-actin (smooth muscles) (Dako; diluted 1:00) and mouse anti-E-cadherine (Dako; diluted 1:100). Bound IgG was detected with biotinylated goat anti-rabbit IgG (Vector Lab.; diluted 1:100) followed by ABC-peroxidase (Vector Lab.) and diaminobenzidine (Sigma). Sections were then counterstained with hematoxylin. As a negative control, unimmunized rabbit serum was substituted for the primary antibody.

2.6 Light and electron microscopy

Tissues from DMBA-induced or non-induced Wistar rats were fixed in 10% formalin and processed for sectioning and hematoxylin staining. For electron microscopy, 1mm3 of tumor tissue or monolayers of culture cells were fixed by modified Karnovsky's fixative (2.5% glutaraldehyde, 2% paraformaldehyde in 0.1M sodium cacodylate buffer, pH 7.4). Samples were fixed at 4°C for 4–12h. Material was then rinsed briefly in buffer, post-fixed in 1% buffered osmium tetroxide, and dehydrated in graded ethanol. Specimens were then rinsed in propylene oxide and infiltrated and embedded in Araldite resin. Representative sections were prepared using an LKB Ultratome III. Thick sections (1μm) were stained with toluidine clue or Ladd's multiple stains (Paragon Stain, Ladd Research Industries, Burlington, VT). Thin sections (800Å) were stained with aqueous uranyl acetate, counterstained with Reynolds' lead citrate, and examined in Philips CM10 transmission electron microscope (FMHS, UAE University).

3 Results

3.1 Fenugreek extract slows down the progression of mammary carcinogenesis

All tumors observed at autopsy were encapsulated and of solid consistency. There was no gross evidence of acute toxicity after the administration of DMBA or herb. The final incidence of palpable carcinomas in DMBA-induced rats ranged from 40% to 80%. No mammary tumor development was seen in the control group or herb alone treated group (Fig. 3A) during the experimental period. On the other hand, mammary tumors were induced in two DMBA-treated groups; the cumulative incidences with and without herb (Fig. 3B–D) were 40% and 80%, respectively. The first tumor was found at 5 weeks in both groups, and then the incidences increased time-dependently throughout the experiment. The mean numbers of tumors per tumor-bearing rats were 3.5 in Group D, compared with 2.0 in Group B, and the mean tumor weights in Groups D and B were 5.0 and 3.0g, respectively (data not shown). Histologically, mammary tumors in Group D ranged from florid epithelial hyperplasia to fibroadenomas (Fig. 3B and C), while in Group B, only mild-to-moderate hyperplasia were observed (Fig. 3D). No metastases in distant sites were observed in any tested group.


Fig. 3

Hematoxylin and eosin stained sections of representative lesions and normal rat mammary gland. (A) Normal ductal structures of the mammary gland. (B) Mammary ducts with moderate hyperplasia found in DMBA-treated rats. (C) Florid epithelial hyperplasia of DMBA-induced breast tumor with secondary lumina and increased numbers of active cells both at the basal lamina and away from it. (D) Mammary ducts with mild hyperplasia (see also inset) found in DMBA-treated rats after being fed with the tested extract.


3.2 Expression of actin, PR and E-cad

Actin was clearly expressed both in the myoepithelial and luminal cells. Expression of PR was, however, less evident and restricted to the ductal side branching cells. E-cad was hardly expressed by luminal epithelial cells that represent the normal sites of its expression (Fig. 4A–C).


Fig. 4

Pattern of immunostaining obtained with monoclonal antibody specific for α-actin (clone 1 A4), progesterone receptors and E-cadherine in ductal carcinoma in situ of mammary gland in DMBA-induced rats. (A) Expression of cytoplasmic α-actin in myoepithelial and luminal cells. (B) Low expression of progesterone receptors in ductal glands. (C) Expression of E-cadherine by luminal epithelial cells.


3.3 Effect of Fenugreek on the subcellular structure of mammary tumor

In order to further understand the mechanism by which tested herb impedes tumor progression, we analyzed the ultrastructural morphology of normal, DMBA-induced and protected mammary cell structures by transmission electron microscopy. Normal breast epithelial tissues were organized with a high cuboidal layer of luminal cells separated at intervals from the basement membrane by a discontinuous layer of myoepithelial cells (Fig. 5A). The nuclei of the luminal epithelial cells tended to be rounded and more basal. Tumor cells were round to polygonal in shape, with a centrally located nucleus. Chromatin tended to be marginated and nuclei were highly folded with a predominant number having large eccentrically placed nucleoli (Fig. 5B; inset). Mitochondria, intercellular junctions and cytoplasmic vesicles were also identified.The cytoplasm of mammary cells of protected rats had an exceptionally large number of vacuoles (Fig. 5C).


Fig. 5

Electron micrograph of (A) normal breast ductal epithelium consists of a layer of luminal cells subtended by a layer of myoepithelial cells (arrowhead) and basement membrane (arrow); (B) DMBA-induced mammary tumor with large nuclei (N) that possess many folding (also see inset) producing a large surface area and hyper-chromatographic nucleoli; and (C) mammary tumor of protected rat exhibiting cells with large nuclei (N) with peripheral hetero-chromatin and highly vacuolated cytoplasm (arrowheads).


Unlike controls, tumor cells showed an abundant supply of mitochondria was present to support the high metabolic demands of the cells (Fig. 6A and B). The ultrastructure of protected cells showed a notable number of fragmented nuclei as well as an extensive network of cytoplasmic vacuoles. Several of these vacuoles contained cytoplasmic matter and organelles within them, suggesting that they are autophagic in nature (Fig. 6C). Notably, cytoplasmic vacuolation is a characteristic of alternative death processes, including autophagic death and para-apoptosis (Debnath et al., 2002).


Fig. 6

Electron micrograph of (A) normal breast ductal epithelium with limited number of mitochondria (arrowhead); (B) DMBA-induced mammary tumor with vast numbers of mitochondria (arrowhead); (C) mammary tumor of protected rat with number of mitochondria (arrowhead); and (D) mammary tumor from protected rats with dying cells exhibiting fragmented nuclei (white arrow) and cytoplasmic vacuolization, including autophagic vacuoles (black arrows).


Electron microscopic sections manifested an abundance supply of large nucleoli in the folded nuclei, vesicles encompassing secretions and mitochondria. These findings led us to a conclusion for these cells being highly metabolic and malignant in nature. The cells and the nuclei characteristically display pleomorphism—variation in size and shape. Characteristically the nuclei contain an abundance of DNA and are extremely dark staining, suggestive of hyperchromatic features. The nuclei are disproportionately large for the cell, and the nuclear-to-cytoplasmic ratio may approach 1:1 instead of the normal 1:4 or 1:6. The nuclear shape is usually extremely variable, and the chromatin is often coarsely clumped and distributed along the nuclear membrane. Large nucleoli are usually present in these nuclei (Chow et al., 2003; Cotran et al., 2000).

4 Discussion

Fenugreek has primarily been described as an anti-hyperglycemic herb in humans and in laboratory animals (Bordia et al., 1997; Sharma et al., 1990). Its cholesterol-reducing effect is also well established (Sharma, 1984). Fenugreek has also shown an overall stimulatory effect on the specific as well as non-specific immune functions in mice (Bin-Hafeez et al., 2003). The main chemical constituents of T. foenum graecum are fibers, flavonoids, polysaccharides and saponins (Jayaweera, 1981; Yoshikawa et al., 1997). Some of the constituents might be having mitogenic effects, which in turn lead to stimulatory effects on the immunocompetent cells. Some of these constituents also possess antioxidant properties and they may induce the immunostimulant effects (de La Fuente and Victor, 2000; Ruby et al., 1995; Devasagayam and Sainis, 2002). Both pro-oxidant and antioxidant effects of flavonoids have previously been identified (Shen et al., 2004; Rice-Evans, 2001; Ross and Kasum, 2002; Shen et al., 2002).

Apoptosis is a type of cell death, and agents with the ability to induce apoptosis in tumors have the potential to be used for anti-tumor therapy. Flavonoids produce several biological effects, and the apoptosis-inducing activities of flavonoids have been identified in several previous studies (Chen et al., 2003; Shen et al., 2003).

Flavonoids and catechins were first shown to be apoptotic in human carcinoma cells (Ahmad et al., 2000). Similar observation has since been extended to lung tumor cell lines (Yang et al., 1998), colon cancer cells, breast cancer cells, prostate cancer cells (Paschka et al., 1998) stomach cancer cells (Okabe et al., 1999), brain tumor cells (Yokoyama et al., 2001), head and neck squamous carcinoma (Masuda et al., 2001) and cervical cancer cells (Ahn et al., 2003). Genistein, quercetin, rutin, and other food flavonoids have been shown to inhibit carcinogenesis in animal models (Gee et al., 2002). They all induce apoptosis in tumor cells (Katdare et al., 2002; Upadhyay et al., 2001; Choi et al., 2001; Iwashita et al., 2000). It appears that these flavonoids can also differentially induce apoptosis in cancer cells, but not in their normal counterparts.

The ultrastructure of mammary acini from protected rats showed dying cells with large numbers of cytoplasmic vacuoles; some of these vacuoles appear autophagic. Recently, alternative cell death processes have been recognized in epithelial cells, including autophagy and para-apoptosis (Bursch et al., 2000; Leist and Jaattela, 2001; Sperandio et al., 2000). These pathways can be activated in parallel with apoptosis, and significant crosstalk between apoptotic and alternative death pathways may exist (Lee and Baehrecke, 2001). Thus, herbal-induced autophagic or “type II” cell death may also contribute to the cell death and hence inhibiting the DMBA-induced tumor progression. T. foenum graecum has also been shown to have stimulatory effects on macrophages. Phagocytosis and killing of invading microorganisms by macrophages constitute body's primary line of defense against infections (VanFurt, 1982).

The present study establishes that T. foenum graecum has appreciable anti-cancer activity. It is not possible to identify the most effective anti-cancer constituent of T. foenum graecum at this point. However, based on the published studies, flavonoids seem to be most likely candidates eliciting anti-tumorigenic effect. Administration of Fenugreek to man is simple, since its seeds and leaves are used as common dietary constituents in many parts of the world. Further investigations are underway to unravel the molecular mechanism that mediates the legume's anti-cancer protective effects. In addition, further studies are underway to isolate and characterize the Fenugreek's active ingredients that contributes to its preventive effects.

Acknowledgements

This work was financially supported by SURE program for summer 2003 at the UAE University. We thank Dr. Rengasamy Padmanabhan (Anatomy Department, FMHS) for his technical and administrative support. We are also grateful to Mr. Shafii and Mr. Nasir (Animal House, FMHS) for their technical assistance throughout this work. We are also indebted to Ms. Rajaa (Biology Department) for her help with the reagents and equipments and to Mr. Armstrong (EM facility, FMHS) for his technical assistance.

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Received 13 September 2004/13 February 2005; accepted 18 April 2005

doi:10.1016/j.cellbi.2005.04.004


ISSN Print: 1065-6995
ISSN Electronic: 1095-8355
Published by Portland Press Limited on behalf of the International Federation for Cell Biology (IFCB)