Normal breast epithelial MCF-10A cells were purchased from the American Type Culture Collection (Manassas, VA, USA). BaP and CAR were purchased from Sigma; Merck KGaA (Darmstadt, Germany). These agents were prepared as 50 µM stock solutions and were stored at 4°C in double-distilled water. Phenol red-free Dulbecco's modified Eagle's medium (DMEM)/Ham's F12, horse serum, penicillin/streptomycin, antibiotic/antimycotic, epidermal growth factor, human insulin (Novolin R), trypsin-EDTA (10X), Hanks' balanced salt solution and PBS were purchased from Invitrogen; Thermo Fisher Scientific, Inc. (Waltham, MA, USA). Cholera enterotoxin was obtained from Enzo Life Sciences, Inc. (Farmingdale, NY, USA). AKT inhibitor VIII and AKT phosphorylation activator SC79 were purchased from MedChemExpress (Monmouth Junction, NJ, USA). The human 8-hydroxy-2-deoxyguanosine (8-OH-dG) ELISA kit was purchased from Cusabio Technology LLC (Houston, TX, USA). All other chemicals were purchased from Sigma-Aldrich; Merck KGaA.

MCF10A cells were maintained in complete medium (1:1 mixture of DMEM and Ham's F12), supplemented with mitogenic additives including 5% horse serum, 10 µM insulin, 0.5 ng/ml hydrocortisol, 20 ng/ml epidermal growth factor and 100 ng/ml cholera enterotoxin. The medium was also supplemented with 100 U/ml penicillin and 100 U/ml streptomycin. All cultures were maintained at 37°C under 5% CO₂.

BaP and CAR were initially dissolved in dimethylsulfoxide and subsequently in cell culture medium. All treatments were prepared and administered under low light conditions, prior to incubation at 37°C in a 5% CO₂ humidified incubator. MCF-10A cells were divided into three groups, namely BaP treatment, CAR/BaP co-treatment and control. The BaP group was treated with 1 µmol/l BaP for 6, 12, 24 or 36 h. The CAR/BaP group was treated with 1 µmol/l BaP in combination with 0.5, 1.0, 5.0 or 10 µmol/l CAR. The control group was cultured without CAR or BaP. To determine the effect of H₂O₂, MCF-10A cells were cultured with H₂O₂ (200 µmol/l) and CAR (5 µmol/l) for 36 h. The cells were harvested by trypsinization at 6, 12, 24 or 36 h, and were suspended in PBS without Mg²⁺ or Ca^2+. The cells were snap-frozen at −196°C in liquid N₂ until further use.

The cultures were labeled with 5 µmol/l dichlorodihydrofluorescein diacetate for 1 h. ROS levels were detected using flow cytometry (BD Biosciences, Franklin Lakes, NJ, USA) and fluorescence microscopy (Nikon Corporation, Tokyo, Japan). The cells were trypsinized from the cultures and resuspended in PBS for analysis of ROS, using a 15 mW air-cooled argon laser at an excitation wavelength of 488 nm. Dichlorofluorescein fluorescence emission was determined using a 529 nm band pass filter. The mean fluorescence intensity of 2×10⁴ cells was quantified using Multicycle AV software (version 3.0; Phoenix Flow Systems, San Diego, CA, USA).

MCF-10A cells were exposed to BaP/CAR for 48 h (one cycle) and/or exposed for between 5 and 20 cycles to induce cellular carcinogenesis (10). Cultured cells were harvested as aforementioned. The comet assay was performed according to a previously published protocol (9), and tail moment was the indicator of DNA damage. Tail moment is defined as the product of the tail length and the fraction of total DNA in the tail, which can be calculated using commercially available and public domain software (9). Data analysis was performed using Komet software (version 5.5; Andor Technology, PLC, Belfast, Northern Ireland, UK), and Kinetic Imaging (version 5.0; Kinetic Imaging Ltd., Wirral, UK).

The three groups of cells, namely BaP (1 µM) treatment, CAR (5 µM)/BaP (1 µM) co-treatment and control, were plated in 6-well dishes at a density of 1×10⁶ cells/dish cultured for 24 h. The cells were trypsinized and resuspended in PBS. Subsequently, two freeze-thaw cycles were performed and the homogenates were centrifuged at 5,000 × g for 5 min at 4°C. The supernatant was removed and the 8-OH-dG levels were determined using the 8-OH-dG ELISA kit, according to the manufacturer's protocol. Absorbance was determined at 450 nm using Curve Expert Professional software (version 2.3.0; win.cutephp.com/curveexpert_professional_1996267) to create a standard curve. Results are expressed in ng/ml. Samples were assayed in a blind manner.

Cell lysates were prepared by washing cells with PBS and were incubated for 10 min at 4°C in modified radioimmunoprecipitation assay lysis buffer [50 mmol/l Tris/HCl, 150 mmol/l NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 25 µg/ml leupeptin, 10 µg/ml aprotinin, 2 mmol/l EDTA and 1 mmol/l sodium orthovanadate (Sigma; Merck KGaA)]. The cells were scraped from the plates and centrifuged at 5,000 × g for 20 min at 4°C, and the supernatants were collected. The protein concentration levels were determined using a Bicinchoninic Acid assay kit (Pierce; Thermo Fisher Scientific, Inc.), and 40 µg whole cell lysates were separated by SDS-PAGE (10% gels). The samples were transferred onto a nitrocellulose membrane by wet blotting. The membrane was blocked with 5% fat-free dry milk in PBS for 1 h at room temperature, and incubated with primary antibodies overnight at 4°C. The antibodies used were anti-phospho (p)-AKT (Thr³⁰⁸) (1:400; cat. no. 558316; BD Biosciences), anti-murine double minute 2 (MDM2; 1:400; cat. no. 556353; BD Biosciences), anti-p-p53 (Ser¹⁵/Ser²⁰) (1:100; cat. no. sc-10176; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) and anti-β-arrestin (1:100; cat. no. sc-21872; Santa Cruz Biotechnology, Inc.). Following three washes with PBS, the membrane was incubated with goat anti-mouse immunoglobulin G (IgG) (1:8,000; cat. no. 31430; Thermo Fisher Scientific, Inc.) and goat anti-rabbit IgG (1:8,000; cat. no. 31460; Thermo Fisher Scientific, Inc.) secondary antibodies and visualized with SuperSignal West Dura Extended Duration Substrate (Thermo Fisher Scientific, Inc.).

Unless indicated otherwise, each assay was conducted three times. Results are presented as the mean ± standard error of the mean. Results were analyzed by one-way analysis of variance or Student's t-test; multiple comparisons between the groups were performed using the Student-Newman-Keuls method. P<0.05 was considered to indicate a statistically significant difference.

BaP induces DNA damage through the generation of ROS, which initiate oxidative damage to nucleic acids (2). In addition to the β-blocker effect, CAR acts as an antioxidant and a ROS scavenger (11). MCF-10A cells were used in order to investigate the effects of CAR on BaP-mediated ROS production. In the absence of BaP, MCF-10A cells generated ROS spontaneously, and CAR had the potential to decrease ROS production in cells that were not treated with BaP (Fig. 1). BaP increased ROS production in MCF-10A cells; however, CAR, at concentrations ≥1 µM, attenuated BaP-induced ROS production in a dose-dependent manner (Fig. 2, upper panel). Short-term exposure (6 h) of MCF-10A cells to 1 µM BaP induced ROS, and ROS production reached a peak at 24 h (Fig. 2, lower panel). BaP induced a significant increase in ROS levels in a time-dependent manner (between 6 and 24 h), although ROS levels decreased at 36 h, indicating endogenous adjustment (Fig. 2). Co-treatment of CAR and BaP decreased BaP-induced ROS production at all time points investigated in a dose-dependent manner (CAR concentration ≥1 µM) (Figs. 1 and 2).

BaP induced significant DNA damage as demonstrated using the alkaline comet assay in MCF-10A cells. Exposure of the cells to BaP for 5 cycles was sufficient to induce DNA strand breaks in MCF-10A cells, although no DNA damage was observed in the 15–20 exposure cycles using the alkaline comet assay. Treatment of the cells with 1 µM BaP caused DNA damage at 12, 24 and 36 h of culture, compared with control cells; however, CAR attenuated DNA strand breaks that were induced by BaP (Fig. 3A). A concentration-dependent effect was observed using CAR. Treatment of the cells with 10 µM CAR and BaP for 24 h attenuated 68% of BaP-induced DNA damage (data not shown), which corresponded nearly to the levels of the control group (Fig. 3A). As a marker of DNA oxidative damage, 8-OH-dG levels increased in MCF-10A cells treated with BaP determined using the 8-OH-dG ELISA kit, although CAR reversed the effect of BaP (Fig. 3B). Cells cultured with 1 µM BaP for 24 h exhibited apparent DNA damage, and CAR and AKT inhibitor VIII eliminated DNA damage caused by BaP (Fig. 4). These results indicated that CAR has the ability to inhibit BaP-induced DNA damage.

Certain carcinogens induce intracellular ROS accumulation, and increase the expression of PI3K and AKT proteins. ROS also directly activate PI3K, and activated PI3K further phosphorylates its downstream target AKT. The alterations in the PI3K/AKT signaling pathway have been revealed to promote cell survival and proliferation (12,13). In order to investigate the mechanism of CAR suppression in BaP-induced MCF-10A cell carcinogenesis, the level of expression and phosphorylation of target proteins associated with the PI3K/AKT signaling pathway was determined. Western blot analysis using an antibody against p-(activated) AKT^Thr308 indicated that, compared with untreated cells, p-AKT^Thr308 expression was increased significantly in MCF-10A cells that were treated with BaP. When cells were incubated with CAR (5 µM) and BaP simultaneously for various durations, p-AKT^Thr308 expression decreased in all cases (Fig. 5A). These results suggested that AKT phosphorylation induced by BaP could be inhibited by CAR in MCF-10A cells. However, AKT (non-phosphorylated) expression was not influenced by treatment with BaP and CAR for 24 h (Fig. 5B). However, in the absence of BaP, MCF-10A cells were co-cultured with H₂O₂ and CAR, and p-AKT^Thr308 expression increased markedly (data not shown). These results indicated that CAR inhibited BaP-induced PI3K/AKT activation via the inhibition of ROS generation, but not ROS scavenging.

MDM2 is an important AKT substrate. Phosphorylation of MDM2 by AKT can target the tumor suppressor p53 for degradation through ubiquitination. p53 modulates cells that are under oxidative stress and aids the adaptation of the cell's oxidative mechanism to the remodeled redox balance, thus preventing oxidative damage to DNA and proteins (14,15). Therefore, the effect of CAR on the expression of p53^Ser15, p53^Ser20 and MDM2 in BaP-induced cells was investigated by western blotting. As CAR suppresses BaP-induced neoplastic activity and associated protein function at concentrations between 1 and 10 µM, 5 µM CAR was used as the optimal concentration. It was identified that MDM2 expression was upregulated in BaP-treated cells, although CAR was able to eliminate the effects of BaP (Fig. 5C). In addition, the expression of p53^Ser15 was downregulated in BaP-treated cells, although CAR could prevent the influence of BaP, and eliminate these changes caused by BaP at the time points investigated (Fig. 6A). However, p53^Ser20 expression did not alter in BaP-treated cells (data not shown). Furthermore, p53 (non-phosphorylated) expression was not influenced when cells were treated with BaP and CAR for 24 h (Fig. 6B). As effectors of the PI3K/AKT signaling pathway, the MDM2 and p53^Ser15 proteins serve important functions in the effects of BaP/CAR on MCF-10A cell transformation.

When MCF-10A cells were co-cultured with BaP and the AKT inhibitor VIII, BaP had no influence on MDM2 expression and p53 degradation (Figs. 5C and 6A). Furthermore, BaP-induced DNA damage was counteracted by the AKT inhibitor VIII as demonstrated using the alkaline comet assay (Fig. 4). In contrast with the inhibition of AKT, when cells were treated with CAR and SC79 (an AKT phosphorylation activator), enhanced MDM2 expression and p53 degradation were evident from western blot analysis (Fig. 6C and D). CAR was not able to inhibit the effects of SC79, which indicated that CAR acts upstream of the PI3K/AKT signaling pathway. These data indicated that BaP-induced ROS production activated PI3K/AKT, which in turn upregulated MDM2, thus facilitating p53 degradation. CAR prevents cellular carcinogenesis induced by BaP via the suppression of PI3K/AKT and MDM2 activity, which increases p53 levels in MCF-10A cells.