Effect of Turkish propolis extracts on expression of voltage-gated sodium channel Nav 1.5 and 1.7 α-Isoforms in PC-3 human prostate cancer cells

Purpose: To investigate the effect of dimethyl sulfoxide (DMSO) and water extracts of Turkish propolis (WEP) on mRNA expression of Nav 1.5 and 1.7 α isoforms of Voltage-Gated Sodium Channel (VGSC) proteins in PC-3 human prostate cancer cells. Methods: DMSO and WEP (20 µg/mL each) were incubated for 24 h with PC-3 cells and total RNA was extracted using a commercial kit. Real-time polymerase chain reaction (RTPCR) assay was used to determine mRNA levels of the isoforms of VGSC. Expressions of VGSC were assumed to be 100 % in PC-3 cells incubated without extract. Results: Both extracts decreased the expression of VGSC isoforms to varying extents. Expressions of Nav 1.5 and 1.7 was 61.43 ± 4.92 and 58.17 ± 2.88 %, respectively for DMSO; while for WEP, the values were 83.54 ± 15.96 and 80.40 ± 13.87 20 µg/mL, respectively. Conclusion: This results suggest that DMSO and water extracts of Turkish propolis may have antimetastatic activity in PC-3 cells due to down-regulation of expressions mRNA of VGSC α-isoforms.


INTRODUCTION
Propolis is a bee product which has been used in folk medicine since ancient times [1]. It is composed of 50 % resin, 30 % wax, 10 % essential and aromatic oils, 5 % pollen and 5 % various other substances, including organic debris. Propolis contains considerable amounts of flavonoids, polyphenolic substances such as quercetin, caffeic acid and caffeic acid phenethyl ester (CAPE), pinocembrin, galangin, p-coumaric acid.
Prostate cancer is one of the most commonly diagnosed cancers in men and the first leading cause of cancer-related deaths in the United States of America and the European Union [3][4][5]. MR/IR (mortality rate to incidence rate ratio) of prostate cancer is as high as 40 % in Asia compared to 18 % in Europe, 10 % in Northern America and 25 % worldwide [6]. The etiology of prostate cancer is multifactorial, and a number of dietary and lifestyle factors have been implicated in the development and progression of the disease [5]. In addition, prostate cancer cells usually metastasize mainly to bones, lymph nodes and lungs body [3].
VGSCs are integral membrane proteins which usually mediate fast transport of Na + in and out of cells depending on electrochemical gradient. They are functionally up-regulated in metastatic prostate cancer cells like breast cancer, smallcell lung cancer, non-small-cell lung cancer, mesothelioma, melanoma and cervical cancer cells [7]. VGSCs contain α subunit and one or more β subunits. The VGSC α subunit gene family consists of 10 members (Nav 1.1-1.9 and Navx) and β subunits consist of 4 members (β1-β4) and one splice variant of β1A, which have distinct tissue specificities and developmental expression profiles. VGSC Navα 1.5 mRNA was detected in most human tissues assayed and showed the strongest expression in the heart, and high level in adult and fetal brain, the testes and the prostate. VGSC Nav 1.7 mRNA was also present in most tissues assayed, and the highest expression was found in the testes, adult and fetal whole brain, and placenta [8].
According to the CELEX hypothesis, phytochemicals like resveratrol, curcumin, capsaicin, genistein and ginseng, omega-3 polyunsaturated fatty acids and minerals have anti-cancer effects through down-regulating of VGSC expression, and partly up-regulated VGKC (Voltage-Gated Potassium Channel Protein) expression [9].
In this study, we investigated the effects of propolis extracts on mRNA expression of VGSC Nav 1.5 and Nav 1.7 α-isoforms in PC-3 cells.
Analysis of the phenolic compounds (flavonoids) of Turkish propolis was previously carried out in our laboratory. According to HPLC analysis, the main flavonoids of DMSO extracts of propolis were found to be galangin, naringenin, chrysin, kaempferol, quercetin and cinnamic acid derivatives, while caffeic and caffeoyl quinic acids were present in the water extracts [11].

Preparation of DMSO and water extracts of Turkish propolis
Each natural propolis samples were ground (Retsch, ZM 200) then frozen at -80 o C. The ground propolis samples were mixed and bottled in 5 g-portions. The portions were dissolved in 20 mL of DMSO (100 % w/v) or deionized water by continuous shaking in an incubator (Shelleb/Sheldon Mod: 514, USA) at 150 rpm and 60 o C for 24 h. Extracts of 250 mg/mL concentrations were obtained by centrifuging at 4000 rpm for 10 min. Collected supernatants were mixed and stored at 4 °C in the dark. Working solutions at concentration of 20 µg aqueous extract/mL and µg DMSO extract/mL were prepared by diluting stock WEP and DMSO extracts with deionized water and DMSO respectively. For total RNA extraction from PC-3 cells (Treated and not treated with propolis extracts), QIAGEN (GmbH, Hilden, Germany) OIAamp RNA Blood Mini Kit (Cat.No. 52304) was used. The protocol recommended by the kit manufacturer for RNA extraction from cells samples was precisely followed. cDNA was synthesized from 5 µL of total RNA in a final volume of 20 µL reaction using the AMV(Avian Myeloblastosis Virus) Reverse Transcriptase (Roche Diagnostics, GmbH, Mannhein, Germany) with Nav 1.5, Nav 1.7 and β-Actin primers at 2.5 µL from 10 pmol following the manufacturer's protocol. The smallest dilution of cDNA Standard was given the relative value 100 and, following the same reason of dilution, other three points were 10, 1 and 0.1. The results were expressed as percentage of differences relative to normal controls (relative expression) using (Software 4.05).

Data analysis
All data were expressed as arithmetic mean ± standard deviation (SD).

Expression of Nav 1.5 and Nav 1.7 α isoforms of VGSC
As shown in Table 1, both extracts decreased expressions of VGSC Nav 1.5 and Nav 1.7 to different extents. Lower expressions of VGSC Nav 1.5 and Nav 1.7 were obtained in PC-3 cells incubated with 20 µg/mL DMSO extract of Turkish propolis (61.43 ± 4.92 and 58.17 ± 2.88 %, respectively) but higher expressions of VGSC Nav 1.5 and Nav 1.7 were obtained in PC-3 cells incubated with 20 µg/mL aqueous extract of Turkish propolis (83.54 ± 15.96 and 80.40 ± 13.87 %, respectively).

DISCUSSION
The poplar origin of Turkish propolis has been well documented [12,13]. Propolis samples collected in various regions of Turkey have been investigated for antibacterial, antifungal, antioxidant and anti-carcinogenic activities [13,15].
Cinnamic acid and cinnamic acid derivatives, pinocembrin, pinobanksin, chrysin, galangin, vanillin, p-cumaric acid, ferulic acid, caffeic acid, naringenin, CAPE have been detected in Turkish propolis [11,13,14]. In addition, it has been reported that propolis collected from different countries contain phenolic compounds like genistein [16], catechin and resveratrol [17]. In previous study, it was reported that aqueous and DMSO extracts of propolis had antioxidant properties to different extent [18]. In MTT assay results, aqueous and DMSO extracts showed cytotoxic effects on PC-3 cells depending on the concentration [18]. The results of MTT assay for propolis in PC-3 cells are in agreement with those obtained for Turkish propolis by Turan et al [15] and Cuban propolis by Carballo et al [19]. Based on results from cytotoxic assays, 20 µg/mL propolis was chosen for Real-Time PCR assay of VGSC Nav 1.5 and 1.7 α-isoforms.
In this study, expression levels of both VGSC Nav 1.5 and 1.7 α-isoforms were reduced by DMSO and WEP in PC-3 cells when compared with control PC-3 cells which were not incubated with extracts. The highest reduction in expression of VGSC Nav 1.5 and 1.7 α isoforms was obtained in PC-3 cells incubated with 20 µg/mL DMSO extract of propolis. This result may be related to synergistic effect of some antioxidant compounds in propolis, especially in the DMSO extract. This study is the first in literature on investigation of expression of VGSCs isoforms in PC-3 cells incubated with extracts of propolis.
Experimental data on VGSC Nav 1.5 and Nav 1.7 α-isoform expressions are in consonance with CELEX hypothesis. The effects of these extracts may be related to flavonoids, polyphenolics, minerals and fatty acid content of Turkish propolis. Previous studies have shown that propolis contains resveratrol, genistein as well as polyphenolic compounds, Zn + other minerals and fatty acids. Fraser et al [20] reported that resveratrol dose-dependently inhibited the activity of VGSC and significantly suppressed lateral motility, transverse motility and invasion in strongly metastatic MAT Ly-Lu prostate cancer cells [20].
Since propolis is natural and non-toxic to humans at low concentrations, researchers may find this natural product a suitable candidate antimetastatic substance a viable substitute for toxic tetrodotoxin in blocking VGSC activity in cancer cells.

CONCLUSION
The findings show that both aqueous and DMSO extracts of Turkish propolis decrease the expression of VGSC Nav 1.5 and 1.7 α-isoforms in PC-3 cells. Further studies to investigate the effects of propolis extracts with different concentrations on expression of all isoforms of VGSC, function of VGSC and protein level in PC-3 and the other cancer cells are required to ascertain contribution of antitumoral and antimetastatic activities of propolis.