The Effects of Petroselinum Crispum on Estrogen Receptor-positive Benign and Malignant Mammary Cells (MCF12A/MCF7)

Materials and Methods

Origin of the PCE. The parsley root (Petroselinum crispum subsp. tuberosum) was grown, harvested and obtained commercially in 2011 in Mecklenburg-Western Pomerania, at vegetable farming Hofer Neubrandenburg.

Preparation of the PCE. The lignan-isolations were prepared as previously described (17-18). Deep frozen (−20°C) parsley root (40 g) was cut into pieces of 5 mm. The pieces were ground in a mortar with liquid nitrogen and extracted under reflux with 360 ml methanol (methanol 99.5%; Roth, Karlsruhe, Germany) in a water bath at 75°C. The extract was suspended in 10 ml of distilled water and partitioned with 16 ml ethyl acetate (five times) to give on drying an ethyl acetate soluble residue. After cooling the sample, the larger components were removed twice by using a folded filter (diameter: 185 mm; Schleicher und Schuell GmbH, Dassel, Germany). The solution was then evaporated in a 100 ml round bottom flask using a rotational damper (VV2011, Heidolph Instruments GmbH & Co.KG, Schwabach, Germany) under uniform reduction of pressure from 350 mbar to approximately 100 mbar. Adhering extract to the wall of the round bottomed flask was removed with 10 ml distilled water and then dissolved in an ultrasonic bath (Ultrasonics (300/H); Omnilab, Bremen, Germany) and extracted 5 times with 16 ml ethyl acetate (99.5%; Roth, Karlsruhe, Germany) in a 50 ml Falcon tube. This process leads to significant phase separation into a lower, denser phase (water phase), and an upper phase (lipid phase, containing lignans). The upper phase was carefully aspirated by a pasteur pipette and transferred to a 50 ml round bottomed flask. The sample was again evaporated in a rotational evaporator at 45°C, under a uniform decrease in pressure from 400 mbar to about 250 mbar. The evaporated extract was dried overnight using a desiccator (with dried silica). To determine the net weight of the extract, the difference of the tare of the round bottom flask and the weight of the flask with dried extract was made (MC1 Analytic scale AC120S; Sartorius GmbH, Göttingen, Germany). The extract was stored protected from light in a refrigerator at 4°C.

In order to verify the previously reported increased phytoestrogen concentration in PCE the molecular–chemical composition of the extract was further analyzed by pyrolysis-field ionization mass spectrometry by using a LCQ-Advantage (Thermo Finnigan's city & state). The peaks were identified by ion trap technology on ESI mode. The source voltage was set at 4.5 kV while the mass detection range was 150-2000 amu.

Creation of different PCE concentrations. After determining the weight of the extract it was dissolved with 96% ethanol (Roth, Karlsruhe, Germany) in an ultrasonic bath (Ultrasonics (300/H); Omnilab, Bremen, Germany) and the concentration of 100 mg/ml was determined (stock solution). For the following test series, 7 dilutions (excl. the stock solution) were set at the concentrations of 50,000 - 10,000 - 5,000 - 1,000 - 100 - 10 - 1 μg/ml. The extract was then further diluted, 1:100 giving the final concentrations 500 - 100 - 50 - 10 - 1 - 0.1 to 0.01 μg/ml. The dilutions were frozen at −85°C.

Cell lines. The ER-positive, malignant breast cancer cell line MCF7 and the ER-positive, benign breast cell line MCF12A were used. Both cell lines were obtained commercially from the American Type Culture Collection (ATCC) and for further use in 1.5 ml culture medium (PAA, Germany) + 10% DMSO + 20% fetal calf serum (fetal bovine serum, Biochrom, Germany) stored at −180°C.

Cell culture. After slow thawing, the cryopreserved cells were washed by 5 ml cell type-specific medium. For MCF7 cells, DMEM, high Glucose 4.5g (PAA, Germany), for MCF12A, Ham's F12 medium (PAA Germany) were used. For this, the cell suspension was carefully pipetted into 15 ml tubes and diluted with 5 ml culture medium. After vortexing and subsequent centrifugation for 5 min at 1000 rpm (centrifuge Universal 320R Hettich Zentrifugen, Gemany), the supernatant was discarded. The cells were re-suspended in 5 ml culture medium, transferred to 25-cm² cell culture flasks (Biochrom, Germany) and incubated at 37°C, 5% CO₂ for 2-3 days. At a confluence rate of 80%, the medium was changed and the cells were splitted in a larger culture flask (75 cm², 150 cm², Biochrom, Germany). The cells were rinsed with phosphate buffered saline (PbS: Dulbecco's PbS powdered buffer, without Ca & Mg, PAA, Germany) to remove the upper layer of protein on the cells which would prevent an effective engagement of the trypsin before incubating them with trypsin (10%, trypsin EDTA, PAA, Germany) for 5 min at 37°C. The reaction was stopped by adding DMEM (DMEM High Glucose (4,5g) +L-glutamin; without phenol red, PAA, Germany). The complete detachment of the cells was monitored by light microscopy. Following centrifugation for 5 min at 1000 rpm, the supernatant was discharged and cells were re-suspended in 5 ml cell-specific medium. For MCF7, 10% FKS, (PAA, Germany), for MCF12A, 10% Horseserum, (SIGMA, Germany) were used. Penicillin/Streptomycin 0.2% (PAA, Germany) and Amphotericin B 0.5% (PAA, Germany) were added to both cell lines.

Cell counting. Despite the fact that cell proliferation depends on the cell concentration and that cells need contact with neighboring cells in order to grow, when cell count is elevated nutrient shortages occur that may lead to apoptosis. The optimal cell concentration, determined by previous studies was set at 5×10⁵ cells/ml (19). Most cell vitality assays that allow cell count determination are based on the change in membrane permeability of dead cells. Vital cells are impermeable for dyes such as trypan blue. Therefore, only the colorless cells are counted in phase contrast microscopy. To cell count, 20 μl of the cell suspension were diluted with 340 μl of cell culture medium and then combined with 20 μl trypan blue (corresponding dilution 1:20). To avoid trypan blue staining of intact cells, prompt counting was done. For this, about 20 μl of the probe was added to both sides of a Bürker cell counting chamber. Counting was done under a phase contrast microscope at a magnification of 100×.

General and statistical considerations. In all tests, a positive control (17β-estradiol, E2) and a negative control (tamoxifen, TAM) were carried along with the extract in different concentrations levels. Preliminary tests done using the same workgroup yielded optimal concentrations for E2: 10⁻⁹ mol/l and TAM: 10⁻⁴ mol/l. All calculations of the results were performed using Microsoft Excel considering the standard deviation. Statistical analysis was performed using the t-test. Each observation with p<0.05 was considered as statistically significant.

Measurement of cytotoxicity by LDH assay. The LDH assay (Roche, Mannheim, Germany) was performed in accordance with the manufacturer's instructions. Best results were achieved after incubation of treated cells (5×10⁵ cells/ml) for 24 h in the absence (controls: TAM, E2) or the presence of PCE at different concentrations. Maximum LDH release (high control) was determined by incubating cells with Triton X-100 at 1% final concentration (Ferak, Berlin, Germany). Untreated cells were used to determine spontaneous LDH release (low control). After incubation, cells were gently centrifuged for 5 min 1,000 rpm. Subsequently, cell-free supernatants were carefully removed and transferred into a new 96-well microplate. Supernatants were mixed 1:1 with freshly prepared reaction mixture and incubated protected from light for 30 min at room temperature. The principle of the assay is based on a LDH/diaphorase coupled reaction with creation of a red colored formazan. Absorbance of the color was measured at 490 nm (620 nm wave length was used as a reference).

Proliferation measurement: MTT assay. Cell proliferation was analyzed using an MTT-kit according to the instructions of the manufacturer (Roche, Germany). After incubation of treated cells (5×10⁵ cells/ml) for 24 h in the absence (controls: TAM, E2, N2) or presence of PCE at different concentrations, MTT labelled reagent was added to each well in a final concentration of 0.5 mg/ml. Subsequently, cells were incubated under culture conditions for 4 h. During this time the metabolic active cells transformed the yellow tetrazolium salt MTT to purple colored formazan crystals. After addition of the solubilization solution the plates were incubated overnight in a humidified atmosphere at 37°C. With a microplate reader (Model 680, Bio-Rad, Hercules, CA, USA) the color intensity was measured at 570 nm using a reference wave length of 650 nm.

BrdU proliferation-assay. Cell proliferation was analyzed with a 5-bromo-2’-deoxy-uridine (BrdU) labelling and detection kit (Roche, Germany) according to the instructions of the manufacturer. MCF7 and MCF12A cells (5×10⁵ cells/ml) were grown in 96-well tissue culture plates for 24 h in the absence (controls: TAM, E2, N2) or presence of PCE at different concentrations. After labelling with BrdU for 3 h, the cells were fixed and BrdU incorporation into DNA was measured by an ELISA technique. Cellular proliferation inhibition is expressed in relation to controls (100%)±SD.

Immunohistochemical staining for the ERα, ERβ and PR. For immuno-detection of the steroid receptors ERα, ERβ and PR, the Vectastain R Elite ABC-kit (Vector Laboratories, USA) was used according to manufacturer's protocol. The slides were first air dried, rinsed in PBS for 5 min and then incubated with the ABC normal serum for 60 min in a humidified environment. The slides were then washed again and incubated with the respective primary antibodies. Salient features of the antibodies used are presented in Table I. The slides were then incubated with the diluted biotinylated secondary antibody (30 min), followed by incubation with the ABC reagent (30 min) and the ABC substrate (15 min). A single wash (PBS, 5 min) was applied between steps. Finally, the slides were counterstained with Mayer's acidic hematoxylin (30 sec), rinsed with water and covered with Aquatex.