Original Contribution
Reactive oxygen species-mediated breast cell carcinogenesis enhanced by multiple carcinogens and intervened by dietary ergosterol and mimosine

https://doi.org/10.1016/j.freeradbiomed.2014.12.015Get rights and content

Highlights

  • Coexposure to multiple carcinogens enhanced breast cell carcinogenesis.

  • Carcinogen-induced, mutual interplay with the ERK pathway reinforced ROS elevation.

  • Reinforced ROS and ERK activation led to increased DNA oxidation and damage.

  • Ergosterol and mimosine blocked carcinogen-induced ROS and ERK activation.

  • Ergosterol and mimosine blocked carcinogen-induced DNA oxidation and damage.

Abstract

Most breast cancers occur sporadically due to long-term exposure to low-dose carcinogens in the diet and the environment. Specifically, smoke, polluted air, and high-temperature cooked meats comprise multiple carcinogens, such as 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), benzo[α]pyrene (B[α]P), and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). We sought to determine if these carcinogens act together to induce breast cell carcinogenesis, and if so, whether noncytotoxic dietary agents could intervene. We demonstrated that coexposure to physiologically achievable doses of NNK, B[α]P, and PhIP (NBP) holistically enhanced initiation and progression of breast cell carcinogenesis. Reactive oxygen species (ROS) and activation of the ERK pathway were transiently induced by NBP in each exposure, and cross talk between reinforced ROS elevation and ERK activation played an essential role in increased DNA oxidation and damage. After cumulative exposures to NBP, this cross talk contributed to enhanced initiation of cellular carcinogenesis and led to enhanced acquisition of cancer-associated properties. Using NBP-induced transient changes, such as ROS elevation and ERK pathway activation, and cancer-associated properties as targeted endpoints, we revealed, for the first time, that two less-studied dietary compounds, ergosterol and mimosine, at physiologically achievable noncytotoxic levels, were highly effective in intervention of NBP-induced cellular carcinogenesis. Combined ergosterol and mimosine were more effective than individual agents in blocking NBP-induced transient endpoints, including ROS-mediated DNA oxidation, which accounted for their preventive ability to suppress progression of NBP-induced cellular carcinogenesis. Thus, dietary components, such as mushrooms containing ergosterol and legumes containing mimosine, should be considered for affordable prevention of sporadic breast cancer associated with long-term exposure to environmental and dietary carcinogens.

Introduction

Breast cancer is the most common type of cancer and second leading cause of cancer-related death among North American and European women [1]. Most breast cancers occur sporadically due to chronic exposure to multiple environmental carcinogens; this multistep process results in the transformation of breast cells from noncancerous to precancerous and then to cancerous [2], [3]. We have developed a breast cell carcinogenesis model to mimic sporadic breast cancer development associated with long-term exposure to low doses of carcinogens [4], [5], [6], [7], [8], [9]. In this model, we repeatedly expose immortalized, noncancerous, human breast epithelial cells to physiologically achievable doses of carcinogens to progressively induce cellular acquisition of various cancer-associated properties for studies to reveal cellular, biochemical, and molecular changes. Then, we use these changes as targeted endpoints to identify preventive agents capable of intervening in cellular carcinogenesis [4], [5], [6], [7], [8], [9].

The mammary carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5–b]pyridine (PhIP) is the most abundant heterocyclic amine produced in high-temperature cooked meat, and following cooked meat consumption, PhIP can reach nanomolar levels in circulating plasma in humans [10]. In rats, gastric administration of PhIP induces mammary tumors [11]. In addition, epidemiological studies have indicated a close association between well-done meat consumption and human breast cancer risk [12], [13]. The tobacco-specific nitrosamine ketone 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is widely accepted as a lung carcinogen and can be detected at picomolar concentrations in the body fluids of tobacco users [14]. Despite evidence that gastric administration into rats resulted in mammary tumor development, NNK is not recognized as a mammary carcinogen [15]. Although the link between smoking and breast cancer is controversial, recent studies have indicated that exposure to tobacco smoke can increase breast cancer risk, especially in postmenopausal women [16], [17]. Benzo[α]pyrene (B[α]P) is a polycyclic aromatic hydrocarbon in smoke produced from the incomplete combustion of organic materials, such as fossil fuels and tobacco [18]. B[α]P can be found at picomolar concentrations in human fat and liver and is classified as a weak mammary carcinogen in humans [18], [19], [20], [21]. American lifestyles involve frequent consumption of high-temperature cooked meats containing PhIP and wide exposure to smoke and polluted air containing NNK and B[α]P. However, whether these carcinogens may act together to cause breast cell carcinogenesis needs to be verified.

Epidemiological and experimental evidence suggests that certain dietary compounds possess anticancer activities [3]. Ergosterol is a sterol that forms the major constituent of the fungal cell membrane [22]. Common dietary sources of ergosterol include mushrooms, morels, and truffles, as well as products made with yeasts such as soy sauce, bread, and beer [22], [23], [24], [25]. Studies show that ergosterol at micromolar levels is cytotoxic against human breast cancer cell lines and various other tumor cell lines [26], [27]. In vivo studies identified ergosterol as a protective agent against the promotion of carcinogen-induced bladder tumors in rats and tumor in mice [28], [29]. Mimosine is an alkaloid, nonproteinogenic amino acid found exclusively in tropical legumes of the Mimosa and Leucaena genera [30]. Mimosine is an iron chelator that inhibits DNA synthesis and causes cell cycle arrest in the late G1 phase [30]. Micromolar levels of mimosine inhibit proliferation of breast, lung, and ovarian cancer cell lines, and mimosine is effective in reducing xenograft development of human lung and pancreatic carcinoma cells by increasing the apoptotic index of cancer cells [31], [32], [33], [34], [35]. Although much research has focused on the use of high-dose ergosterol and mimosine to inhibit growth or induce apoptosis of cancer cells, little work has been undertaken to evaluate the ability of these agents, at noncytotoxic levels, to prevent cellular carcinogenesis.

Given the ubiquitous nature of tobacco smoke, carbon exhaust, and high-temperature cooked meats in modern societies, it is important to holistically examine the role of carcinogens, such as NNK, B[α]P, and PhIP, in induction of breast cell carcinogenesis to reveal associated mechanisms and endpoints to be targeted for intervention. Previously, we used our breast cell carcinogenesis model system to demonstrate the ability of NNK at100 pmol/L, B[α]P at 100 pmol/L, and PhIP at 10 nmol/L individually to induce initiation and progression of breast cell carcinogenesis [4], [5], [9]. NNK and B[α]P exhibited comparable abilities to induce cellular carcinogenesis, and combining NNK and B[α]P (NB) additively increased degrees of acquired cancer-associated properties, such as reduced dependence on growth factors and anchorage-independent growth, as well as stem-like cell- and epithelial-to-mesenchymal transition (EMT)-associated properties [6], [7], [8], [9]. The measurable cancer-associated properties acquired by cells after cumulative carcinogen exposures represent constitutive endpoints to reveal stages in the progression of cellular carcinogenesis from noncancerous to premalignant and malignant stages. Using our model, we have also identified endpoints that are transiently induced by a single carcinogen exposure; such endpoints as reactive oxygen species (ROS) elevation and DNA damage play essential roles in induction of carcinogenesis and account for the mechanisms of initiation of cellular carcinogenesis during each carcinogen exposure [6], [7], [9]. Our model system enables us to use a two-step strategy by also using these transient and constitutive endpoints as targets to identify preventive agents capable of blocking cellular carcinogenesis [5], [6], [7], [8], [9].

Herein, we describe use of our model system to elucidate the ability, and associated mechanisms, of NB to enhance PhIP-induced cellular carcinogenesis. We also used our model as a target system to demonstrate, for the first time, the ability, and associated mechanisms, of ergosterol and mimosine individually and in combination to suppress cellular carcinogenesis induced by combined NNK, B[α]P, and PhIP.

Section snippets

Cell cultures and reagents

MCF10A, CF12A (American Type Culture Collection [ATCC], Rockville, MD, USA), and derived cell lines were maintained in complete medium (CM) (1:1 DMEM/HAM׳s F12, 100 ng/mL cholera enterotoxin, 10 µg/mL insulin, 0.5 µg/mL hydrocortisol, 20 ng/mL epidermal growth factor, and 5% horse serum) [4], [5], [6], [7], [8], [9]. All cultures were supplemented with 100 U/mL penicillin and 100 µg/mL streptomycin and maintained in 5% CO2 at 37 °C. Stock solutions of NNK (Chemsyn, Lenexa, KS, USA), B[α]P (Aldrich,

Exposure-dependent enhancement of cancer-associated properties by combined carcinogens

To investigate the ability of combined NNK and B[α]P (NB) to enhance PhIP-induced breast cell carcinogenesis, we repeatedly exposed MCF10A cells to PhIP in the absence and presence of NB for 5, 10, 15, and 20 cycles. Then, we measured the degrees of two acquired cancer-associated properties to assess the progression of cellular carcinogenesis. Growth factors and cellular adhesion to the extracellular matrix are required for normal epithelial cell survival; in contrast, cancerous cells acquire a

Discussion

In this paper, we demonstrated that cumulative coexposures to physiologically achievable levels of multiple environmental and dietary carcinogens (nanomolar PhIP and picomolar NNK and B[α]P [10], [14], [19]) resulted in enhanced progression of ROS-mediated breast cell carcinogenesis. Our model system permits a highly relevant approach and uses various transient and constitutive endpoints to study mechanisms involved in the development of sporadic breast cancer associated with long-term exposure

Acknowledgments

This work was supported by a grant from the University of Tennessee Center of Excellence in Livestock Diseases and Human Health to H-C.R. W. This sponsor had no role in the study design, or in the collection, analysis, and interpretation of data. We are grateful to Ms M. Bailey for textual editing of the manuscript. We thank Ms DJ Trent for technical support in flow cytometric analysis. The authors declare that they have no conflict of interests.

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