Didymin was obtained from Extrasynthese (Genay, France), and was dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich, St. Louis, MO, USA) and stored at −20°C. Control cultures received the carrier solvent (0.1% DMSO). All other chemicals utilized were in the purest form available commercially.

Human breast adenocarcinoma MDA-MB-231 cells (American Type Culture Collection, Manassas, VA, USA) were cultured in α-minimum essential medium (α-MEM; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with non-essential amino acids, 0.1 mmol/l sodium pyruvate, 1% antibiotic/anti-mycotic solution and 10% fetal bovine serum (FBS) (all Thermo Fisher Scientific, Inc.). In order to obtain the various conditioned media (CM), the MDA-MB-231 cells (2×10⁶/100-mm dish) were treated with or without BBP, DBP or DEHP (all Sigma-Aldrich) at identical concentrations of 1 µM for 6 h. Following washing and culturing for 24 h, the CM of phthalate ester-treated MDA-MB-231 cells (BBP-, DBP- or DEHP-MDA-CM) were harvested (Fig. 1A).

Monocytes were purified from peripheral blood mononuclear cells obtained from healthy consenting donors. Mononuclear cells were isolated from the blood by Ficoll-Hypaque gradient (GE Healthcare Life Sciences, Chalfont, UK). CD14⁺ monocytes were purified with CD14⁺ monoclonal antibody-conjugated magnetic beads (MACS MicroBeads; Miltenyi Biotec GmbH, Bergisch Gladbach, Germany), according to the manufacturer's protocols. mdDCs were generated by culturing CD14⁺ monocytes in α-MEM containing FBS and 20 ng/ml granulocyte-macrophage colony-stimulating factor (GM-CSF) and 10 ng/ml interleukin (IL)4 (R&D Systems, Inc., Minneapolis, MN, USA) for 5 days. The medium was replaced with fresh medium containing GM-CSF and IL4 on day 3. For the maturation of DCs, immature mdDCs were stimulated with lipopolysaccharide (100 ng/ml; Sigma-Aldrich) following priming with interferon-γ (EMD Millipore, Billerica, MA, USA) for 3 h. MDA-MB-231 tumor-associated mdDCs (MDA-TADCs), BBP-MDA-TADCs, DBP-MDA-TADCs or DEHP-MDA-TADCs were generated by culturing CD14⁺ monocytes in α-MEM medium containing FBS, IL4 and GM-CSF in 20% MDA-CM, BBP-MDA-CM, DBP-MDA-CM or DEHP-MDA-CM, and subsequently stimulated as aforementioned. Following washing, the supernatants were collected and identified as MDA-MB-231-TADC-CM, BBP-MDA-TADC-CM, DBP-MDA-TADC-CM or DEHP-MDA-TADC-CM (Fig. 1B). The Institutional Review Board (IRB) of Kaohsiung Medical University Hospital (Kaohsiung, Taiwan) approved the present study protocol and all participants provided written informed consent in accordance with the Declaration of Helsinki (IRB numbers: KMUH-IRB-990174 and KMUH-IRB-20120362).

TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) was utilized for RNA isolation, while complementary (c)DNA was prepared using an oligo(dT) primer and reverse transcriptase (Takara Bio, Inc., Otsu, Japan) following standard protocols (29). RT-qPCR was performed using SYBR Green on the ABI 7500 Real-Time PCR System (Applied Biosystems; Thermo Fisher Scientific, Inc.). Each PCR mixture contained 200 nM of each primer, 10 µl of 2X SYBR Green PCR Master Mix (Applied Biosystems; Thermo Fisher Scientific, Inc.), and 5 µl of cDNA and RNase-free water, in a total volume of 20 µl. The RT-qPCR was performed with a denaturation step at 95°C for 10 min, then for 40 cycles at 95°C for 15 sec and 60°C for 1 min. All PCRs were performed in triplicate and normalized to internal control glyceraldehyde-3-phosphate dehydrogenase mRNA. The relative expression level was presented using the 2^−ΔΔCq method. The primer sequences of target genes in the present study were as follows: RANTES, F 5′-cgc tgt cat cct cat tgc ta-3′ and R 5′-aca cac ttg gcg gtt ctt tc-3′; and GAPDH F 5′-GAGTCAACGGATTTGGTCGT-3′ and R 5′-TTGATTTTGGAGGGATCTCG-3′.

RANTES levels were determined using an ELISA-based kit (R&D Systems Europe Ltd., Abingdon, UK). ELISA was performed according to the manufacturer's protocols. Depletion of RANTES from various CMs was performed using a mouse monoclonal anti-RANTES antibodies (2 µg/ml; Abcam, Cmabridge, UK) and Sepharose™ Protein A/G beads (PAG50-00-0002, Rockland Immunochemicals Inc., Gilbertsville, PA, USA) following standard immunoprecipitation techniques (30). Cytokine depletion was additionally confirmed using an ELISA-based kit.

The cells were plated in 96-well culture plates. Following 24 h of incubation, the cells were treated with vehicle mdDC-CM or specific CM for 72 h. At the conclusion of the assay period, cell proliferation was measured using a water-soluble tetrazolium salts (WST)-1 assay. Cell proliferation was determined using Premixed WST-1 Cell Proliferation Reagent (Clontech Laboratories, Inc., Mountainview, CA, USA) in accordance with the manufacturer's protocols.

Cell migration and invasion assays were performed using QCM™ 24-well Cell Migration and Invasion Assay kits (EMD Millipore). Briefly, the cells were seeded into the migration chamber and mdDC-CM or various CM were added to the lower wells for 24 h as a chemoattractant. At the conclusion of the treatment, the cells were stained using CyQuant GR dye (cat no. 90131) in cell lysis buffer for 15 min at room temperature. The fluorescence of the migrated and invaded cells was subsequently measured using a fluorescence plate reader at excitation/emission wavelengths of 485/520 nm.

Data are expressed as the mean ± standard deviation. Statistical comparisons of the results were performed using an analysis of variance. Significant differences between the means of the test groups were analyzed using Student's t-test. P<0.05 was considered to indicate a statistically significant difference.

In order to understand whether phthalate esters exacerbate cancer progression in the breast cancer tumor microenvironment, the effects of BBP-MDA-TADC-CM, DBP-MDA-TADC-CM and DEHP-MDA-TADC-CM on breast cancer cell proliferation, migration and invasion were investigated. MDA-TADC-CM (20%) increased breast cancer cell proliferation (P=0.01), and this stimulatory effect was additionally enhanced when MDA-MB-231 cells were pretreated with BBP, DBP or DEHP (Fig. 2A) (P=0.001, 0.0001 and 0.0007 for BBP, DBP and DEHP, respectively). In addition, MDA-TADC-CM (20%) induced breast cancer cell migration and invasion (P=0.002 and 0.008 for migration and invasion analysis), and this reinforceable effect was worsened when breast cancer cells were cultured with BBP-MDA-TADC-CM (20%), DBP-MDA-TADC-CM (20%) or DEHP-MDA-TADC-CM (20%) (Fig. 2B and C).

In order to determine the primary factors contributing to MDA-TADC, BBP-MDA-TADC, DBP-MDA-TADC and DEHP-MDA-TADC-mediated breast cancer progression, RT-qPCR analysis revealed that RANTES mRNA levels were increased by 6-, 12-, 17- and 11-fold in MDA-TADCs, BBP-MDA-TADCs, DBP-MDA-TADCs and DEHP-MDA-TADCs, respectively (Fig. 3A). As demonstrated by ELISA, the protein levels of RANTES were enhanced in MDA-TADC-CM (P=0.01), BBP-MDA-TADC-CM (P=0.005), DBP-MDA-TADC-CM (P=0.002) and DEHP-MDA-TADC-CM (Fig. 3B) (P=0.003). The effect of DBP-MDA-TADC-CM on the induction of breast cancer cell proliferation, migration and invasion, and the induction of RANTES was greater than that of BBP-MDA-TADC-CM or DEHP-MDA-TADC-CM (Figs. 2 and 3). As DBP demonstrated the greatest effect in these circumstances, it was selected as the model for investigation of the detailed effects of phthalate ester-associated cancer aggravation in the breast cancer tumor microenvironment. In order to understand its role, RANTES was depleted in MDA-TADC-CM and DBP-MDA-TADC-CM. The effect of MDA-TADC-CM and DBP-MDA-TADC-CM on cancer progression was subsequently assessed. The successful depletion of RANTES from MDA-TADC-CM and DBP-MDA-TADC-CM was confirmed using RANTES ELISA kits (data not shown). As demonstrated in Fig. 4, RANTES depletion inhibited the stimulatory effects of MDA-TADC-CM on breast cancer cell proliferation, migration and invasion (P=0.00002 for cell proliferation, P<0.0001 for migration and invasion), as well as blocking the intensified stimulatory effects of DBP-MDA-TADC-CM on breast cancer cell proliferation, migration and invasion.

The tumor microenvironment has a significant role in the development of chemoresistance to anticancer drugs and tumor progression (7). TADC has been demonstrated to promote the progression of cancer by modulating a number of components in the cancer niche, thereby creating a supportive and permissive microenvironment for tumor survival, proliferation and metastasis (11,31). As phthalate esters stimulate the ability of breast cancer cells to affect mdDCs and thereby intensified breast cancer cell proliferation, migration and invasion, the search for a possible antidote in the fight against phthalate esters-induced cancer aggravation in the breast cancer tumor microenvironment has become a matter of importance. The present study therefore assessed the effect of didymin, a dietary flavonoid glycoside derived from citrus fruits, on DBP-induced cancer progression. As shown in Fig. 5A, the MDA-TADC-CM-induced breast cancer cell proliferation effect was inhibited when cells were pretreated with didymin, and DBP-MDA-TADC-CM-induced breast cancer cell proliferation was additionally reversed when the cells were pretreated with didymin. Similarly, enhancement of breast cancer cell migration and invasion triggered by DBP-MDA-TADC-CM was abrogated upon didymin pretreatment (Fig. 5B and C).