Diet and dietary factors play an important role in preventing and treating chronic diseases including cancer [5, 25]. Depending on their isolation step, plant products have been defined as food, food supplement, functional food and nutraceuticals. Pure extracted phytomolecule is named as nutraceuticals, whereas semipurified plant product is named as functional food.
Micronutrients, polyunsaturated fatty acids, and secondary metabolites such as glucosinolates, flavonoids, polyphenols, phytoestrogens, phytosterols, lignans, terpenes and phytates are components of these plant foods .
Curcumin (turmeric), capsaicin (green chilies), epigallocatechingallate (green tea), gingerol (ginger), genistein (soya beans), resveratrol (grapes), caffeic acid phenyl ester (propolis from honey bee), sulforaphane (cruciferous vegetables), silibinin, indole-3-carbinol (cabbage), apigenin (tea, cabbage, garlic), allicin (garlic), lycopene (tomatoes), quercetin (rhododendron cinnabarium), and β-carotene are some of the phytochemicals that are related to tumor prevention .
Thus, natural and synthetic compounds that can be used in the prevention and/or treatment of cancer are the targets of researchers . The beehive products such as honey, propolis, pollen and royal jelly may be included into functional foods .
Reactive oxygen species (ROS) together with other factors are responsible for cellular aging, cell signalling, stress responses, cell proliferation, and many conditions such as cardiovascular diseases, diabetes, arthritis, Parkinson disease, Alzheimer and cancer development [27, 28].
The antioxidants serve as a defensive factor against free radicals; thereby protect lipids and other compounds during oxidative damage. Propolis has been shown to be capable of scavenging free radicals through their pharmacologically active constituents such as flavonoids . Thus, propolis is thought to improve health and prevent diseases such as inflammation, heart disease, diabetes and cancer by its antioxidant potential . Anti-tumoral activity of propolis might be attributed to a single substance, or to synergistic effects of several compounds, or to potential metabolites .
Aliyazıcıoğlu et al. found that fluorescence positivity decreased (between 3.8% and 11.8%) as concentrations of both dimethyl sulfoxide (DMSO) extracts of propolis and pollen increased for K-562 cell culture, but unchanged (between 20% and 83%) for mononuclear cell (MNC) culture by intracellular dichlorofluorescence (DCFH) test by using flow-cytometric fluorescence analysis. They concluded that DMSO extracts of pollen and propolis inhibited the respiratory burst within cancer cell lines probably by their antioxidant potentials .
It has been reported that various activities of propolis may be attributed to a synergism between phenolic (flavonoids, aromatic acids and esters) and other compounds in the resin [7, 9]. Thus we preferred to use whole DMSO and water extracts of propolis, rather than its constituents for our study.
Dimethyl sulfoxide (DMSO) is an amphipathic molecule and soluble in both aqueous and organic media. Due to its physiochemical properties, DMSO is a very efficient solvent for water-insoluble compounds and has been used successfully in the treatment of dermatological, urinary, pulmonary, rheumatic and renal manifestations of amyloidosis. Basically through its anti-inflammatory and reactive oxygen species scavenger actions, its use has been purposed in several gastrointestinal diseases .
Banskota et al. showed that methyl alcohol (MeOH) extract of the Netherlands propolis had anti-proliferative activity toward highly liver-metastatic murine colon 26-L5 carcinoma (EC50, 3.5 mg/mL). They also showed that the compounds isolated from the MeOH extract (benzyl, phenethyl and cinnamylcaffeates) possessed potent anti-proliferative activities with EC50 values of 0.288, 1.76 and 0.114 mM, toward colon 26-L5 carcinoma. Thus, they concluded that anti-oxidative activity of these caffeates may play an important role in their anti-proliferative activities .
Russo et al. found that compounds obtained in ethanolic extract of Chilean propolis such as galangin, caffeicacid, p-cumaric acid, ferulic acid and caffeic acid phenethyl ester (CAPE) by HPLC analysis exhibited DPPH (2,2-diphenyl-1-picrylhydrazyl) free radical scavenging and superoxide scavenging activity in a dose-dependent manner. It was showed that Chilean propolis exhibited anti-proliferative activity toward KB cells, Caco-2 and DU-145 cells. The cell viability was found in the propolis extract treated-KB, -Caco-2 and -DU145 cells as 9%, 45% and 23%, respectively. They suggested that the anti-proliferative activity of ethanolic extract of Chilean propolis might be mediated by its ability to modulate intracellular reactive oxygen species levels .
In the study of Li et al. the effects of ethanolic extracts of Brazilian propolis group l2 and bud resins of botanical origin (B. dracunculifolia), and propolis group 3 on proliferation of metastasis (DU145 and PC-3) and primary malignant tumor (RC58T/h/SA#4)-derived from human prostate cancer cells were evaluated. They found that the strongest inhibition was observed in propolis group 3 (sample #3) extracts whereas moderate growth inhibition was observed in human prostate epithelial cells. They also found that propolis group 3 (sample #3) induced growth inhibition (the cells died at 20 μg/mL treated cells) that was associated with G2 arrest and showed induction of p21 expression but no inhibition of cyclin D1, CDK4 (Cyclin-dependent kinase 4) and cyclin B1 expression. In the RC58T/h/SA#4 cells, they found that resins of botanical origin of propolis group 12 (sample #1) and propolis group 12 (sample #2) induced growth inhibition that was associated with S phase arrest and showed inhibition of cyclin D1, CDK4 and cyclin B1 expression .
Carballo et al. found that Cuban propolis shows cytotoxicity in the range of 5-23 μg/mL without cross resistance in both wild-type and chemo-resistant human tumor cell lines comprising colon, ovarian, and prostate carcinomas (10 μg/mL and 12.3 μg/mL in PC-3 and LNCap) as well as neuroblastoma. They assumed that plukenetione A which was identified for the first time in Cuban propolis, contributes to the anti-tumoral effect of Cuban propolis mainly by targeting topoisomerase I as well as DNA polymerase. They also observed that some components of propolis were more or less active in the isolated form compared with the whole substance and evidenced that there was a multiple interaction (e.g., synergism, antagonism) between propolis constituents .
Wang et al. demonstrated that inhibition of cell growth and induction of apoptosis in PC-3 cells was significantly greater in the combination group (isoflavone and curcumin) than that could be achieved by either agent alone. They also found that the effects of those compounds were associated with decreased Notch-1 expression and DNA binding activity of nuclear factor kappa B (NF-κB) and its target genes such as Cyclin D1, Bcl-2, and Bcl-xL .
In our study, DMSO (100%) extract of propolis was found to be more rich in polyphenols and flavonoids according to water extract of propolis and the antioxidant potentials of extracts were found in correlation with the amount of total phenolic compounds in them.
In this study, we wanted to assess the effect of DMSO and water extracts of propolis on viability of PC-3 cancer cell line by using MTT test. DMSO extracts of propolis at final concentrations of 5, 10, 20 μg/mL reduced the cell viability more than those of DMSO solutions. It was shown that water extract of propolis at concentration of 20 μg/mL had the most cytotoxic activity against PC-3 cell lines. Our results suggest that anti-proliferative effect of propolis extracts might be mediated by their anti-oxidant potentials. According to cell viability results, 20 μg/mL concentrations of propolis extracts were chosen to study expression difference mapping by SELDI-TOF-MS.
Nair et al. observed that quercetin, flavonoid found in many fruits and vegetables, and also in propolis, significantly inhibited the growth of the highly aggressive PC-3 prostate cancer cell line and the moderately aggressive DU-145 prostate cancer cell line, whereas it did not affect colony formation by the poorly aggressive LNCaP prostate cancer cell line or the normal fibroblast cell line BG-9. They found that quercetin significantly down-regulated the expression of specific oncogenes and genes controlling G1, S, G2, and M phases of the cell cycle and up-regulated the expression of several tumor suppressor genes .
In the study of Cheng et al. anti-tumor mechanism of RhizomaParidis total saponin ( RPTS, a component of herb RhizomaParidis) in HepG2 cells was examined by a proteomic analysis. They found a significant change between control (0.01% DMSO) and RPTS (IC50 approximately 10 μg/mL) treated cells after 48 h. Among twelve proteins that had been identified by MALDI-TOF-MS (Matrix Assisted Laser Desorption Ionisation - Time of Flight), six proteins were down-regulated (dUTPase, hnRNPK, GMPsynthase, etc.) and six proteins were up-regulated (DNasegamma, Nucleosidediphosphate kinase A, Centrin-2, etc.) by RPTS treatment in HepG2 cells .
Lee et al. suggested that caffeic acid phenethyl ester (CAPE), a chemopreventive phytochemical derived from honeybee propolis, suppressed SK-Hep1 cell invasion in a dose-dependent manner by abolishment of matrix metalloproteinases (MM2 and MM9) which are associated with the invasive phenotypes of cancer cells and inhibition of NF-κB DNA-binding activity .
Bottoni et al. investigated ciglitazone [PPARγ (peroxisome proliferators-activated receptor γ) agonist]-induced differentiation of a human hepatocarcinoma HepG2 cell line, by monitoring cellular parameters of cytodifferentiation and modifications of cellular protein profiles through 2-DE (Two dimensional electrophoresis) and MALDI-TOF analysis. They found that ciglitazone is a strong differentiating agent for the HepG2 cell line and the proteins of which expression profiles changed, related to cell antioxidant systems, the cell cycle apparatus, signal transduction pathways, cellular stress and invasiveness .
Lee et al. showed that sulforaphane (SFN) which is an isothiocyanate found in cruciferous vegetables, exerted cytotoxicity and increased TUNEL (Terminal dUTP nick end labeling) positive cells in a concentration-dependent manner in LNCaP prostate cancer cells. In their study, levels of nine proteins including tubulin β-2, phosphoglucomutase-3 (PGM3), melanoma-derived leucine zipper containing extra-nuclear factor, activin A type I receptor precursor, smoothelin-A, KIA0073, hypothetical protein LOC57691 and two unnamed proteins changed over 8 folds in SFN treated LNCaP cells compared to untreated control by using MALDI-TOF .
Szlıszka et al. demonstrated that TRAIL (tumour necrosis factor-related apoptosis-inducing ligand)-resistant prostate cancer cells were sensitized by treatment of ethanolic extract of Brazilian green propolis (EEP) by enhancing the expression of TRAIL-R2 and the activity of NF-κB in LNCaP cells .
In this study, statistically significant discriminatory peaks between control PC-3 cells and DMSO extract of propolis-treated PC-3 cells were found to be the proteomic features at m/z 5143, 8703, 12661, 20184 and 32794, detected by CM10 ProteinChip, and the peak at m/z 3772, detected by Q10 ProteinChip (Table 3). Between control PC-3 cells and water extract of propolis-treated PC-3 cells, statistically significant discriminatory peaks were found to be the proteomic features at m/z 15846, 16052 and 24658, detected by CM10 ProteinChip and the peaks at m/z 10348, 10899 and 11603, detected by Q10 ProteinChip (Table 4). Further study is required to identify what those proteins are.