Biochanin A, dimethyl sulfoxide (DMSO), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide reagent for MTT assay, antibodies for β-actin (cat. no. A5441), E-Cadherin (cat. no. 07-697) and Snail (cat. no. SAB1306281), TNF-α (cat. no. T0813) and IL-6 (cat.no. MABF41) were acquired from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). Cell culture medium, including Dulbecco's modified Eagle medium (DMEM) and RPMI-1640 medium, and the other supplements required for cell culture methods, including fetal bovine serum, penicillin and streptomycin, were purchased from Thermo Fisher Scientific, Inc. (Waltham, MA, USA).

Human lung adenocarcinoma cell line (A427) and human monocytic leukemia cell line (AML-193) were obtained from American Type Culture Collection (Manassas, VA, USA). A427 cell lines were cultured in DMEM, whereas AML-193 cells were cultured in RPMI medium. Both the media were supplemented with 10% fetal bovine serum, 100 U/ml penicillin and 100 mg/ml streptomycin under a humidified atmosphere at 37°C with 5% CO₂. Biochanin A was dissolved using dimethyl sulfoxide to prepare a stock solution and was stored at 4°C for subsequent experiments.

The sub-lethal dosage of biochanin A on A427 cells was examined to assess cell viability using an MTT assay. In order to conduct this experiment, cells at a density of 3×10³ cells/well were seeded into 96-well plates and incubated for 24 h. Following culturing, various concentrations of biochanin A (5, 20 and 40 µM) were added by dissolving the compound in DMSO and incubating a further 24 h. After this, the media were removed and 0.1 mg/ml MTT solution was added followed by 6-h incubation in the dark. This was followed by the addition of DMSO of equal volume to solubilize the formazan crystals and their absorbance at 560 nm was recorded using a microplate reader (Sunrise; Tecan, Männedorf, Switzerland). Cells treated without biochanin A in DMSO acted as vehicle controls and the experiment was performed in triplicate.

A427 human lung cancer cell lines were subjected to coculture with human monocytic leukemic AML-193 cells. Ratios (1:1, 1:10, 1:15, 1:25 and 1:50) were chosen to illustrate the role of biochanin A in a potential pro-inflammation coculture method. In brief, A427 cells at a density of 3×10³ cells/well were seeded into 96-well plates and maintained overnight in DMEM supplemented with fetal bovine serum (10%) in order to grow the cells. This was followed by the addition of various biochanin A sub-lethal concentrations (5, 20 and 40 µM) and incubation for 24 h. Following incubation, PBS solution was used to wash the cells to remove any minor amount of biochanin A. Consequently, A427 cells were cocultured with AML-193 cells in different ratios using low protein serum-free medium for 8 h at 37°C. A427 cells cocultured with AML-193 and A427 cells cocultured with AML-193 in dimethyl sulfoxide served as the control and vehicle control, respectively. Conditioned medium was collected after 8 h of coculture and the dead cells were removed by centrifugation at 3,000 × g for 30 min and maintained at −80°C for further use. Conditioned media with and without biochanin A treatment were represented as TCM and NTCM, respectively.

Supernatants of the culture media were used to quantify the amount of TNF-α (cat. no. 550610) and IL-6 (cat. no. 550799) levels using commercially purchased ELISA kits (BD Biosciences Franklin Lakes, NJ, USA) by following the instructions supplied by the manufacturer. Measurements were carried out in triplicate for all assays.

A427 cell lines were seeded in 6-well plates until 95% confluency in DMEM. After removing the media, the cell monolayer was wounded using a sterile needle and was subsequently washed using PBS to remove the debris. Images were captured (0 h). Cell lines were subjected to treatment with both the treated and non-treated conditioned media (TCM and NTCM) and incubated for a further 24 h. Images were captured after the wound formation (24 h). Three independent assays were performed and a percentage were calculated from wound closures with the help of a formula as previously outlined (30).

Inserts of poly-carbonate filters (pore size, 8 mm) were pre-polished using Matrigel and pre-incubated in DMEM for ~3 h prior to plating the cells. The upper compartment was loaded with 250 ml TCM/NTCM cocultured conditioned media of A427 cells at a density of 4×10⁵, whereas the lower compartment was loaded with DMEM (500 ml), which can function as a chemotactic element, and incubated for 24 h. Following this, cotton buds were used to remove the non-migrated cells from the upper compartment and the migrated cells were then attached to the filter on the other side, fixed using formaldehyde (3.5%) for 30 min and stained with crystal violet. Every five fields were calculated and quantified by recording absorbance at 570 nm for each elution of crystal violet using acetic acid (5%). Data were represented as the mean of triplicate measurements.

Initially, the A427 pretreated cells were washed three times with ice-cold PBS solution. Cells were then lysed using radioimmunoprecipitation lysis buffer containing Tris-HCl (10 mM, pH 7.4), EDTA (1 mM), 1% sodium deoxycholate, sodium chloride (100 mM) and NP-40 along with 1X Roche protease inhibitor composed of aprotinin and leupeptin (both 5 mg/ml). Centrifugation was performed at 12,000 × g for 10 min at 4°C to obtain the cell lysates and protein concentrations were estimated using Bio-Rad Protein Assay reagent (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Proteins were subsequently separated by SDS-PAGE and electrophoretically transferred onto polyvinylidene difluoride membranes followed by blocking with bovine serum albumin (1%) for ~3 h at room temperature. Membranes were subsequently incubated at 4°C overnight with primary antibodies against E-Cadherin, Snail and β-actin at dilution ratios of 1:1,000, 1:1,000 and 1:1,500, respectively. Blots obtained after washing were treated with infra-red (IRDye 800 CW Goat anti-Human IgG)-associated secondary antibodies (cat. no. 926-32232; LI-COR Biotechnology, Lincoln, NE, USA) at a dilution ratio of 1:10,000 for ~2 h at room temperature. The resultant blots were scanned using a LAS 4000 Image Quant gel documentation system (GE Healthcare, Chicago, IL, USA).

One-way analysis of variance (ANOVA) was used for all statistical calculations. Tukey's honest significant difference was used as a post hoc test method following ANOVA. P<0.05 was considered to indicate a statistically significant difference. All data were expressed as the mean ± standard deviation of triplicate measurements.

To assess the potential of the pro-inflammatory co-culture method in simulating the original cancer micro-environment background, co-culture with different ratios (1:1, 1:10, 1:15, 1:25 and 1:50) of A427 human lung cancer cell line with AML-193 human leukemic monocytic cell lines were performed for ~6 h. Once the cocultured conditioned medium was attained, we estimated the levels of cytokines (TNF-α and IL-6) using enzyme-associated immunoassays. Results of coculture indicated the formation of a significant amount of TNF-α and IL-6 and the high response was monitored at a 1:15 cocultured ratio (Fig. 1). These types of cancer and myeloid cells (A427/AML-193) are generally identified only in solid tumors but not in liquid medium (31). In addition, generation of cytokines (TNF-α and IL-6) was not observed when using either A427 or AML-193 cultures alone. These results indicate the possible liberation of a stimulus from A427 cells that induces pro-inflammatory characteristics in AML-193 cells.

In order to evaluate the role of biochanin A on cell viability and to examine the operative concentration that is able to produce negligible cytotoxicity to A427 cells, an MTT assay was conducted with sub-toxic dosages of biochanin A (5–40 µM). Results of MTT demonstrated good cell viability, and thus no cytotoxicity, at all the measured dosages of biochanin A (Fig. 2).

In order to elucidate the effects of biochanin A on the pro-inflammatory response via a cocultured method, we performed pretreatment of A427 cells with biochanin A at sub-lethal concentrations of 5–40 µM for ~24 h prior to coculture with AML-193 cells. Cytokine levels (TNF-α and IL-6) were quantified from the supernatant solutions of cell culture. Results demonstrated the significant suppression of TNF-α and IL-6 liberation from AML-193 cells when compared to that of non-treated (control) or dimethyl sulfoxide treated cocultures (Fig. 3). It is known that pro-inflammatory cytokines have a crucial role in cancer metastasis (12) and the use of biochanin A results in the inhibition of the pro-inflammatory response from A427 cells, which might alter the cancer cell micro-environment.

Pro-inflammatory cytokines, such as TNF-α and IL-6, occurring in the cancer microenvironment governs the migration and invasive characteristics to the tumor cells (32). Since the results demonstrated that A427 induced AML-193 cells to produce TNF-α and IL-6 cytokines, we examined whether biochanin A was able to alter the metastatic properties of A427 cells. Migration and invasion studies were conducted with conditioned medium from biochanin A added cocultures of A427 and AML-193 cells. A427 cell lines were cultured to minimal confluency, wounded and further cultured in biochanin A-treated (TCM) or non-treated (NTCM) cocultured conditioned medium, respectively. The data revealed that biochanin A markedly reduced the transition of A427 cells as it reduced the wound healing process (Fig. 4). In addition to these results, A427 cells were cultured in NTCM or TCM (40 µM biochanin A) cocultured conditioned medium to further explore the effects using different in vitro cell line models. Data indicated that biochanin A strongly repressed the coculture-stimulated invasion of A427 cells (Fig. 5); a 19-fold reduction in the number of invasive cells was noted for TCM, as compared with that of NTCM.

Strong correlations have been identified between EMT and pro-inflammatory cancer cell microenvironments (33,34), which helps to activate cancer metastasis. It has previously been demonstrated that biochanin A is able to reduce pro-inflammatory activities and thereby hinder cancer metastasis (35); however, whether biochanin A can alter epithelial-mesenchymal transition still remains to be established. To analyze this, A427 cell lines were developed in non-treated or biochanin A-treated cocultured medium. A significant reduction in Snail expression and a simultaneous depression in E-cadherin was determined from western blot analysis data (Fig. 6), which is indicative of EMT as these are EMT bio-markers (32).