Cancer Letters

Cancer Letters

Volume 241, Issue 1, 8 September 2006, Pages 124-134
Cancer Letters

Cranberry phytochemical extracts induce cell cycle arrest and apoptosis in human MCF-7 breast cancer cells

https://doi.org/10.1016/j.canlet.2005.10.027Get rights and content

Abstract

Breast cancer is the most commonly diagnosed cancer in women in the US and is one of the leading causes of death due to cancer. Epidemiological studies have consistently suggested the inverse association between cancer risk and intake of fruits and vegetables. These health benefits have been linked to the additive and synergistic combination of phytochemicals in fruits and vegetables. Cranberries have been shown to possess anti-carcinogenic activities such as inhibition of growth of several cancer cell lines, and inhibition of ornithine decarboxylase (ODC) activity in vitro. However, the molecular mechanisms of the anti-cancer properties of cranberry phytochemical extracts have not been completely understood. Our data showed that cranberry phytochemical extracts significantly inhibited human breast cancer MCF-7 cell proliferation at doses of 5 to 30 mg/mL (P<0.05). Apoptotic induction in MCF-7 cells was observed in a dose-dependent manner after exposure to cranberry phytochemical extracts for 4 h. Cranberry phytochemical extracts at a dose of 50 mg/mL resulted in a 25% higher ratio of apoptotic cells to total cells as compared to the control groups (P<0.05). Cranberry phytochemical extracts at doses from 10 to 50 mg/mL significantly arrested MCF-7 cells at G0/G1 phase (P<0.05). A constant increasing pattern of the G1/S index was observed in the cranberry extract treatment group while the G1/S ratio of the control group decreased concomitantly between 10 and 24 h treatment. After 24-h exposure to cranberry extracts, the G1/S index of MCF-7 cells was approximately 6 times higher than that of the control group (P<0.05). These results suggest that cranberry phytochemical extracts possess the ability to suppress the proliferation of human breast cancer MCF-7 cells and this suppression is at least partly attributed to both the initiation of apoptosis and the G1 phase arrest.

Introduction

Breast cancer is the most commonly diagnosed invasive cancer in women in the US and is one of the leading causes of death due to cancer [1]. Breast cancer is extremely difficult to treat due to several distinct classes of tumors that exhibit different treatment response [2]. The non-steroidal anti-estrogen medicine, Tamoxifen, has been reported to be effective in only one-third of the breast cancer patients [3]. Thus, searching for new alternative agents for the prevention and treatment of breast cancer is in great need.

An alternative strategy to lower the risk of cancer is through the dietary modification. It has been suggested that one-third of all cancer deaths could be avoided through this approach [4], [5]. Epidemiological studies have consistently shown that increased consumption of fruits and vegetables is associated with reduced risk of developing cancer [6], [7]. It has been suggested that phytochemicals of fruits and vegetables are primary contributors to these health benefits of fruits and vegetables in the prevention of cancers [8], [9], [10]. Direct associations between increased consumption of fruits and vegetables and decreased risk of breast cancer have been observed in several studies [2], [11], [12]. In a review focusing on the survival of breast cancer patients and their intake of fruits and vegetables, five of the eight cohort studies showed an inverse relationship between vegetable and fruit intake and survival of breast cancer patient, with a 20–90% reduction in death risk [13].

Two major antioxidants, vitamin C and β-carotene, have long been thought to be key components responsible for the chemopreventative effects of fruits and vegetables [14], [15], [16]. Much research has been devoted to identifying the key components and mechanisms that contribute to the health benefits of cranberries [17], [18]. However, increasing evidence suggested that these key factors are much more complex in scope, interaction and magnitude than what current research approaches commonly recognize. Increasingly, recent research suggests that the health benefits of fruits and vegetables are due to the additive and synergistic effects of multiple phytochemicals, rather than to a single source, such as vitamin C or β-carotene [8]. Single compounds or supplement pills simply could not mimic the health benefits provided by the complex of over 8000 different phytochemicals in foods. We have proposed that the benefit of a diet rich in fruit and vegetables is attributed to the complex mixture of phytochemicals present in whole foods, which could not be substituted by single compounds or supplement pills [19].

The bioactivity and mechanism of cranberries against urinary tract infections have been extensively studied and well documented [20]. Cranberry juice has been reported to inhibit copper-induced low-density lipoprotein (LDL) oxidation [21], [22]. In previous studies, we demonstrated that cranberries contained abundant phenolic compounds and possessed the highest level of antioxidant activity among the commonly consumed fruits and vegetables tested [23], [24]. Much research has associated dietary antioxidants with prevention of cancer [25], [26], and a high plasma antioxidant level has been positively associated with a lowered risk of breast cancer [27]. Cranberries are abundant in antioxidants; however, there is little available research on the effects and mechanisms of cranberries on the prevention of breast cancer.

Induction of apoptosis and inhibition of tumor cell proliferation have been used as markers for evaluation of phytochemical anti-cancer activities [28], [29], [30], and many chemotherapy agents exert their effects by interruption of cell cycle progression or by induction of apoptosis in cancer cells. An imbalance between cell proliferation and apoptosis has been implicated in breast cancer development [31]. The cell cycle is simply the program for cell growth and cell proliferation. Thus, cell cycle modulation could serve as an effective method in the regulation of cell proliferation. Several typical events of early cell-cycle regulation are also associated with apoptosis [32]. The growth inhibition of human breast cancer MCF-7 cells by resveratrol could be partially explained by the bioactive effects of this intensively studied phytochemical on cell-cycle control and apoptosis induction [33]. Recently, we demonstrated that cranberry extracts exhibited potent anti-proliferative activity towards human liver cancer HepG2 cells [23] and human colon cancer Caco-2 cells (data not published). The objective of the present study was to determine: (1) if cranberry extracts could inhibit human breast cancer MCF-7 cell proliferation; (2) if the exhibited anti-proliferative activity of cranberry extracts is associated with cell-cycle regulation and apoptosis induction.

Section snippets

Chemicals

Aluminum chloride, sodium hydroxide, methyl tert-butyl ether, methanol and acetone were purchased from Fisher Scientific (Pittsburgh, PA). Gallic acid and metaphosphoric acid was obtained from ICN Biomedical Inc. (Costa Mesa, CA). Sodium nitrite, (+)-catechin, Folin–Ciocalteu reagent, hydrochloric acid, propidium iodide, methyl green, and Triton X-100 were purchased from Sigma Chemical Company (St Louis, MO). All reagents used in the study were of analytical grade.

Preparation of cranberry extracts

Cranberries of the Early Black

Results

Total amounts of major phytochemicals, phenolics, flavonoids and anthocyanins, in cranberry extracts were quantified. The total phenolic content of cranberry extracts was 570.4±21.1 mg gallic acid equivalents/100 g fresh cranberries. The total flavonoid content of cranberry extracts was 161.6±20.3 mg of catechin equivalents/100 g fresh cranberries. Total monomeric anthocyanins of cranberry extracts was 92.0±4.2 mg of cyaniding-3-glucoside equivalent/100 g fresh cranberries.

The anti-proliferative

Discussion

Epidemiological studies have consistently shown that the consumption of fruits and vegetables reduce the risk of developing cancers [7], [39]. In a report reviewing 12 major case–control studies of breast cancer, a significant inverse association was observed between fruit and vegetable intake and breast cancer risk [14]. Vitamin C and β-carotene are extensively used as markers of fruit and vegetable intake. However, vitamin C supplementation showed no effects on prevention of cancer and

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