Evaluation of chemopreventive and cytotoxic effect of lemon seed extracts on human breast cancer (MCF-7) cells

https://doi.org/10.1016/j.fct.2011.10.057Get rights and content

Abstract

Extracts from lemon seed were investigated for the radical scavenging activity and apoptotic effects in human breast adenocarcinoma (MCF-7) cells and non-malignant breast (MCF-12F) cells for the first time. Defatted seed powder was successively extracted with ethyl acetate (EtOAc), acetone, methanol (MeOH), and MeOH:water (80:20). The chemical constituents were identified and quantified by LC-MS and HPLC analysis, respectively. The highest radical scavenging activity of 62.2% and 91.3% was exhibited by MeOH:water (80:20) at 833 μg/mL in 1,1-diphenyl-2-picryl hydrazyl (DPPH) and 2,2′-azino-di-(3-ethylbenzothiazoline)-6-sulfonic acid (ABTSradical dot+), respectively. In addition, the MeOH:water (80:20) extract showed the highest (29.1%, P < 0.01) inhibition of MCF-7 cells in MTT assay. Treatment of the MeOH:water (80:20) extract induced DNA fragmentation and poly(ADP-ribose) polymerase (PARP) cleavage. Increased levels of Bax and cytosolic cytochrome C and decreased levels of Bcl2 were also observed in MeOH:water (80:20) treated MCF-7 cells. In conclusion, the MeOH:water (80:20) extract from lemon seed has potent antioxidant activity and induces apoptosis in MCF-7 cells, leading to the inhibition of proliferation. These results suggest that aglycones and glucosides of the limonoids and flavonoid present in MeOH:water (80:20) extract may potentially serve as a chemopreventive agent for breast cancer.

Highlights

► Aqueous methanol extract from lemon showed the highest radical scavenging activity. ► Lemon extracts are an excellent source to induce apoptosis in MCF-7 cancer cells. ► Lemon fractions are non-toxic to human normal breast cells. ► Lemons are a potential source to prevent human MCF-7 breast cancer cells.

Introduction

Epidemiological studies have demonstrated the inverse correlation between increasing consumption of fruits and vegetables and incidences of breast cancer risk (Gandini et al., 2000, Steinmetz and Potter, 1996). Recent cohort studies reported that consumption of fruits and vegetables may not have a significant influence in reducing the risk for breast cancer (Van Gils et al., 2005). However, several bioactive compounds derived from fruits and vegetables, including flavonoids (Conklin et al., 2007, Wang et al., 2010), polyphenols (Thangapazham et al., 2007), and vitamins (Ooi et al., 2010, Richard et al., 2010), were evaluated for inhibition of breast cancer cell growth and metastasis in in vitro and in vivo model systems. Despite conflicting reports, fruits and vegetables are commonly recognized for their health benefits. While this is well understood, the mechanisms by which certain bioactive compounds in fruits and vegetables reduce the risk of cancer, as well as their absorption by the human body, are yet to be determined.

Breast cancer is one of the most common cancers among women and arises from many genetic, familial, hormonal, and environmental factors (Harris et al., 2001). Unfortunately, reducing the risk of breast cancer is difficult due to most cases involve complex hormonal responses, increased obesity rates, and high blood estrogen levels for postmenopausal women (Yager and Davidson, 2006). After the discovery in 1973 of the biochemical interaction of the estrogen receptor (ERα) with hormones (Jensen and DeSombre, 1973), ERα antagonists, including tamoxifen and letrozole were utilized for the treatment of breast cancer. While hormone therapy is currently the most prevalent breast cancer treatment, new models need to be explored (Benson et al., 2006).

Citrus fruits, as a major contributor to human diet, have received attention by researchers due to their multitude of bioactive compounds. Recent in vitro studies suggest these bioactive compounds contain health-promoting properties and have potential relevance for antioxidant, anti-proliferative, and anti-viral agents, as well as for the prevention of cardiovascular diseases (Roy and Saraf, 2006). In our previous reports, bioactive compounds from citrus, such as limonoids, flavonoids (naringin), and carotenoids (lycopene, lutein), were determined to suppress the growth rate of human breast cancer (Tian et al., 2001), colon cancer (Jayaprakasha et al., 2007, Jayaprakasha et al., 2008, Jayaprakasha et al., 2010), neuroblastoma cells (Poulose et al., 2006) and rat prostate carcinoma cells (Gunasekera et al., 2007) using in vitro models, as well as azoxymethane-induced aberrant crypt foci in an in vivo study (Vanamala et al., 2006).

Apoptosis is an important regulatory mechanism in the development of tissues, involving biological events such as chromosome condensation, DNA laddering, membrane blebbing, and cytochrome C release, which leads to the removal of unnecessary cells (Yan and Shi, 2005). It is well known that cancer occurs due to either mitochondria-generated reactive oxygen species (ROS), DNA damage, apoptosis, or necrosis (Simon et al., 2000). Furthermore, studies have supported that ROS production, lipid peroxidation, and mitochondria function are related to many diseases, including cancer, diabetes, and neurodegenerative disorder (Benz and Yau, 2008). Recently, research from our lab has provided clear evidence that certain citrus bioactive compounds induce significant increase in the activity of detoxifying enzymes such as glutathione S-transferase and quinone reductase (Perez et al., 2009).

Citrus is grown commercially in more than 140 countries around the world. Among citrus, oranges, grapefruits, and lemons are considered to be the top three fruits for consumption throughout the world (Spreen, 2003). Lemons are widely consumed as culinary fruit and their economic importance has steadily increased due to the fresh fruit juice industry, medicinal folklore, and the ingredients market. During the processing of lemons, more than 50% of the fruit weight is discarded as waste (Manthey and Grohmann, 2001). Currently, these byproducts such as molasses and peels are used as animal feed, health beneficial compounds (Li et al., 2006), and fuel utilization (Alva, 1994). On the other hand, among the different parts of the lemon fruit, seeds are one of the major byproducts which do not have significant use. The current report is an attempt towards utilization of seeds for determining the health beneficial properties from agro-food industrial byproduct. This will add economic benefits to citrus processing industry, citrus growers, and human society. Based on this information, we focused on evaluating the bioactivity of the lemon seeds. Despite several uses of lemons, very little information is available about the health-promoting properties and the mechanism of action of lemon bioactive components compared to other citrus fruits such as oranges and grapefruits.

To the best of our knowledge, there are no reports on the effect of lemon bioactive compounds on growth of human breast cancer and non-malignant cells. Therefore, we investigated the antioxidant potential and growth inhibition of human breast cancer and non-malignant cells using different polar extracts from lemon seed. Furthermore, the bioactive compounds were identified and quantified by LC-MS and HPLC analysis.

Section snippets

Chemicals

All solvents used in this study were analytical grade and purchased from Fisher Scientific (Fair Lawn, NJ)). The following chemicals were purchased from Sigma (St. Louis, MO): 1,1-diphenyl-2-picryl hydrazyl (DPPH); 2,2′-azino-di-(3-ethylbenzothiazoline)-6-sulfonic acid (ABTSradical dot+); Folin–Ciocalteu; Dulbecco’s Modified Eagle Medium (DMEM); trypsin–EDTA; penicillin; streptomycin; 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT); and dithiothreitol (DTT). Fetal bovine serum (FBS) was

Extraction and identification of bioactive components in lemon seed

Successive solvent extraction of the lemon seed in a Soxhlet apparatus resulted in obtaining a yield of 78.62 g (3.27% w/w), 16.37 g (0.68% w/w), 221.50 g (9.23% w/w), and 402.60 g (16.78% w/w) for the EtOAc, acetone, MeOH, and MeOH:water (80:20) extracts, respectively. To investigate the effects of lemon seed components on human breast cancer (MCF-7) and non-malignant (MCF-12F) cells, all the extracts were freeze-dried, and chemical constituents of lemon seed extracts were identified by LC-MS

Conclusion

Bioactive components in lemon seed extracts could be a good source of antioxidants and induce apoptosis in MCF-7 breast cancer cells through the mitochondrial apoptosis pathway attributed from the increased Bax, decreased Bcl2, released cytochrome C in cytosol, and cleaved PARP. Moreover, this research demonstrated for the first time that lemon bioactive compounds are non-toxic to non-malignant breast cells. Future studies should aim to purify and identify compounds present in MeOH:water

Conflict of Interest

The authors declare that there are no conflict of interest.

Acknowledgements

This project was supported by the USDA-NIFA No. 2010-34402-20875, “Designing Foods for Health,” through the Vegetable and Fruit Improvement Center.

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