1. Introduction
At present, the influence of dietary habits and food quality, in terms of substance content, on the development of cancer is being increasingly studied [
1]. There are a number of studies pointing to the positive effects of fruit and vegetables, prevalently due to compounds in their contents, such as phenols, flavonoids, vitamins and mineral substances [
2,
3]. On the other hand, spices have been involved in the human diet for plenty of years, and used as essential additional ingredients for much cooking, and as seasoning. Could they be considered to be one of the major sources of anticarcinogenic compounds because they contain antioxidants and other biologically active molecules? Thus, it is not surprising that numerous species have been studied in the context of their effects on human health. Antibacterial effects are demonstrated for sweet peppers, peppers and caraway seeds [
4,
5,
6]. Marjoram, cinnamon and caraway seeds are also reported to have anti-inflammatory effects [
6,
7,
8]. Cinnamon and caraway seeds are shown to have anticarcinogenic effects [
6,
7]. Thyme, pepper and oregano are used as antifungals [
5,
9,
10]. The anticarcinogenic effects of spices are attributed to them containing phenolic compounds [
11].
Therefore, we focused on the effects of the most prevalent polyphenols on prostate cells, with regards to the fact that the urogenital tract is the most exposed tissue upon which the effect of the chemical substances present in fluids passing through it should have the greatest impact [
12]. To date, a number of studies show the anticarcinogenic effects of piperine [
13], capsaicin [
14] and curcumin [
15,
16,
17] on prostate cancer cells. Of the eight kinds of spice tested in this work, studies on the anticarcinogenic effect on prostate cancer cells have been performed on black pepper only [
13,
18]. For oregano, marjoram, anise, thyme, sweet pepper, cinnamon and caraway seeds, no studies have yet been published on their effects on prostate cells [
7,
11,
19,
20,
21,
22,
23,
24].
For our experiments, we directed our attention to phenolic (neochlorogenic acid and 3,4-dihydroxybenzaldehyde) and flavonoid (apigenin and naringenin chalcone) compounds. Apigenin is a flavonoid compound that is abundantly present in fruits and vegetables. Apigenin reduces low density lipoprotein and cholesterol levels; stimulates PPAR-γ; augments the endogenous antioxidants; regulates the death-signaling of reactive oxygen species [
25]; regulates inflammatory mediators, including IL-1β and TNF-α [
26]; inhibits tumor growth and angiogenesis induced by different cancer cells; and has antiproliferative and antitumor properties in the colon, pancreas and prostate cancer cells [
27]. In addition, it was revealed that apigenin can disrupt cancer cell motility by suppressing the focal adhesion kinase/Src signaling [
28], which is a key step in the development of tumors and, ultimately, metastasis. Naringenin chalcone is flavonoid compound and its inhibitory effects are demonstrated in U87MG cells [
29]. Neochlorogenic acid is a phenolic acid, which exhibits antioxidant and chemopreventive activity in colon and breast cancer, and in U937 leukemia cells; it protects cells from oxidative stress by scavenging reactive oxidative stress (ROS), and suppressing the proliferation of breast and colon cancer cells [
30]. The study [
31] showed strong inhibition of growth on a breast cancer cell line (MDA-MB-435) and low toxic effect on a normal breast cell line (MCF-10A). The phenolic compound 3,4-dihydroxybenzaldehyde has antioxidant and anti-inflammatory effects: it decreases the proliferation of human breast cancer, and induces apoptosis with inhibition of casein kinase II activity in leukemia cells [
32]. However, the potential anticancer mechanisms of phenolic (neochlorogenic acid and 3,4-dihydroxybenzaldehyde) and flavonoid (apigenin and naringenin chalcone) compounds have not been elucidated, and the effects of prostate cancer cells have not been tested so far.
The aim of this experiment was to determine the effect of eight selected spice species on three prostate cell lines. The profile representation of the phenolic and flavonoid compounds of the selected spices was performed by liquid chromatography/mass spectrometry (LC/MS). The most representative phenolic and flavonoid compounds were used for the rest of the final evaluation using an MTT assay, a scratch test and a clonogenic assay.
3. Materials and Methods
3.1. Chemicals
The chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA) in ACS purity, unless noted otherwise. Apigenin standard and neochlorogenic acid standard were purchased from Extrasynthese (Genay, France). Naringenin chalcone standard was purchased from Phytolab (Vestenbergsgreuth, Germany).
3.2. Cells
The PNT1A (immortalization of normal, adult, prostatic, epithelial cells), PC3 (androgen-independent) and 22RV1 (androgen-dependent) prostatic cancer cell lines were obtained from the American Type Culture Collection (ATCC) (Manassas, VA, USA). The cells were cultured in a complete RPMI-1640 medium (Hyclone, Waltham, MA, USA) with 10% fetal bovine serum (FBS) (Hyclone, Waltham, MA, USA), supplemented with penicillin and streptomycin (0.1 mg·mL−1) at 37 °C and 5% CO2 in a humidified incubator.
3.3. Preparation of the Spice Samples for Cell-Line Proliferative Activity Testing (MTT Assay)
In this experiment, eight kinds of spice (marjoram, sweet pepper, black pepper, caraway seeds, anise, thyme, cinnamon and oregano) were used. To determine the phenolic and flavonoid compounds from the spices, extraction with 80% (anise, black pepper and caraway seeds) or 100% (thyme, marjoram, sweet pepper, cinnamon and oregano) methanol was used.
1 g sample was weighed for each of eight kinds of spice (Analytical Weight EP 240A, Precisa, Stare Mesto, Czech Republic). The samples of the eight kinds of spice were homogenized in a friction bowl with 10 mL of 80% or 100% methanol, and 0.05 to 0.1 g of sea sand (until evaporation). The homogenization was repeated once more. After the homogenization, the samples were vortexed (Vortex Mixers, VELP Scientifica, Usmate Velate MB, Italy) for 1–2 min, and centrifuged at 4500 rpm and 16 °C for 10 min (Centrifuge Z326K, Hermle, Gosheim, Germany). Subsequently, each sample was filtered through a filter (LUT Syringe Filters Nylon, LABICOM s.r.o., Olomouc, Czech Republic). Samples of the extracts of eight kinds of spice were pipetted (2 mL) and concentrated by nitrogen evaporation at 60 °C.
3.4. Cell-Line Proliferative Activity Testing (MTT Assay)
For the extracts from eight kinds of spice, and for the phenolic and flavonoid compounds from the spices, the treatment was initiated after the cells reached ~60–80% confluence. The cells were then harvested, washed four times with phosphate-buffered saline (PBS) (pH 7.4), and counted using the Countess II FL Automated Cell Counter (Life Technologies, Carlsbad, CA, USA). The cells’ proliferative activity was estimated using the MTT assay. Briefly, the suspension of 5000 cells in 50 µL medium was added to each well in the microtiter plates (E-plates 96) used, followed by incubation for 24 h at 37 °C with 5% CO2 to ensure cell growth. A volume of 50 µL of the medium containing an extract from eight kinds of spice, and phenolic and flavonoid compounds from the conjugated spices, was added to the cells. To determine the effects on cell proliferative activity, the extract from eight kinds of spice (at a concentration of 0.05–25.00 mg·mL−1), and phenolic and flavonoid compounds from spices (at a concentration of 0.001–1 mmol·L−1) were employed. The treated cells were incubated for 24 h at 37 °C with 5% CO2. In addition, 10 µL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT (5 mg·mL−1 in PBS)) was added to the cells and the mixture was incubated for 4 h at 37 °C. The MTT-containing medium was replaced by 100 µL of 99.9% dimethyl sulfoxide to dissolve MTT–formazan crystals and, after 5 min incubation, the absorbance of the samples was determined at 570 nm (VersaMax Microplate Reader, Molecular Devices, Sunnyvale, CA, USA). The experiments were performed in triplicate.
3.5. Preparation of the Samples of Spices and the Analysis of Spice Extracts Using LC/MS
To determine the phenolic and flavonoid compounds of the spices, extraction with 80% (anise, black pepper and caraway seeds) or 100% (thyme, marjoram, sweet pepper, cinnamon and oregano) methanol was used.
1 g sample was weighed for each of eight kinds of spice (Analytical Weight EP 240A, Precisa, Czech Republic). The samples of the eight kinds of spice were homogenized in a friction bowl with 10 mL of 80% or 100% methanol, and 0.05 to 0.1 g of sea sand (until evaporation). The homogenization was repeated once more. After homogenization, the samples were vortexed (Vortex Mixers, VELP Scientifica, Usmate Velate MB, Italy) for 1–2 min, and centrifuged at 4500 rpm and 16 °C for 10 min (Centrifuge Z326K, Hermle, Gosheim, Germany). Subsequently, each sample was filtered through a filter (LUT Syringe Filters Nylon, LABICOM s.r.o., Olomouc, Czech Republic). Samples of the extracts of the eight kinds of spice were pipetted (400 µL) and analyzed using LC/MS.
To determine the selected phenolic and flavonoid compounds, a high-performance liquid chromatograph (HPLC Agilent 1200 Series) with a diode array detector and a triple quadrupole mass detector (6460 Triple Quad) LC/MS was used. For the separation of the phenolic and flavonoid compounds, a column, Zorbax EC 18 of dimensions 50 mm × 3.0 mm and a particle size of 2.7 μm, was used. The column was held at 45 °C. Mobile phase A consisted of 100% methanol, and mobile phase B was 0.2% acetic acid. The flow rate of the mobile phase was 0.6 mL·min−1. The compounds were eluted with a linear upward gradient: 0.00 min (85% B), 0.17 min (85% B), 0.50 min (75% B), 1.70 min (70% B), 4.00 min (70% B), and 6.00 min (85% B). The triple quadrupole mass detector was operated in negative mode. The gas (nitrogen) temperature was 300 °C, the gas flow rate was set to 12 L·min−1, the pressure nebulizer had a value of 45 psi, the temperature of the focusing gas was 250 °C, the flow rate of the focusing gas was set at 11 L·min−1, and the voltage on the capillary tube amounted to 3500 V.
3.6. Wound-Healing Assay (Scratch Test)
The treatment was initiated after the cells reached ~100% confluence. The cells were then harvested, washed four times with PBS (pH 7.4), and counted using the Countess II FL Automated Cell Counter (Life Technologies, Carlsbad, CA, USA). Briefly, the suspension of 105 cells in the medium was added to each well in the microtiter plates (E-plates 6) used, followed by incubation after reaching 100% confluence at 37 °C with 5% CO2. After 24 h of serum starvation, a wound was made in the cell monolayer using a 200 μL pipette tip. The cells were washed using PBS, and treated with 2 mL of the medium containing the phenolic and flavonoid compounds from the spices. To determine the effects on cell proliferative activity, the phenolic and flavonoid compounds from the conjugated spices were added to the cells, and a concentration of 1 mmol·L−1 was employed. Images at a 400 µm magnification were obtained using an EVOS FL Auto Cell Imaging system (ThermoFisher Scientific, Waltham, MA, USA) at 0, 6, 12 and 24 h of treatment. Within each wound, we analyzed five distance measurements using the EVOS FL Auto software (version 1.7). The experiments were performed in duplicate and each microtiter plate measured five times.
3.7. Clonogenic Assay
The cells were then harvested, washed four times with PBS (pH 7.4), and counted using the Countess II FL Automated Cell Counter (Life Technologies, Carlsbad, CA, USA). Briefly, the suspension of 103 cells in the medium was added to each well in the microtiter plates (E-plates 6) used, followed by incubation for 24 h at 37 °C with 5% CO2. After 24 h, the medium was removed, and the cells treated with 2 mL of the medium containing the phenolic and flavonoid compounds from the spices. To determine the effects on cell proliferative activity, the phenolic and flavonoid compounds from the conjugated spices were added to the cells, and a concentration of 1 mmol·L−1 was employed. This was followed by incubation for 24 h at 37 °C with 5% CO2, then a medium change and incubation for 9–14 days at 37 °C with 5% CO2. The medium was removed and the cells were washed with PBS and fixation was completed with methanol:acetic acid (3:1) for 5 min. After fixation, the cells were colored with 0.5% crystal violet in methanol for 15 min. The cells were washed with Milli-Q water. Images were obtained using a Canon EOS 650D (Canon, Ota, Japan). The experiments were performed in duplicate.