Growth Inhibition and Apoptosis Induction of Essential Oils and Extracts of Nepeta cataria L. on Human Prostatic and Breast Cancer Cell Lines

Nepeta is a genus belonging to the family of Lamiaceae, including approximately 250 species. There are 79 species of this genus found in Iran (Jamzad, 2012) of which 38 species are endemic to the country (Emami & Aghazari, 2011). Nepeta cataria L. (catnip), is a short herbaceous perennial, growing 27–100 cm tall and wide. It has the square stem with brown-green foliage. The coarse-toothed leaves are triangular to ovate. The species has white flowers with fine purple spots and a strong aromatic odor. This plant is grown in Asia and Southeast Europe. It is distributed in north, northwest, northeast, west and center of Iran (Jamzad, 2012). Nepeta cataria is antipyretic, antispasmodic, sedative, diuretic and diaphoretic (Duke, 2001). Previous studies reported that therapeutic properties of Nepeta species are due to the presence of essential oil (EO) and flavonoids in the plant. Nepeta lactones and its isomers are the main constituents Abstract

Apoptosis or programmed cell death is the hemostatic process that controls cell population in normal tissues and in many disease conditions such as ischemic damage, autoimmune disorders and in different types of cancer. Apoptotic cells display some modification such as DNA breakdown, cleavage in proteins and over express of caspases that trigger proteolytic cascade and lead to cleavage of several main proteins that are essential for cell survival (Elmore, 2007). PARP is one of the caspase targets and in response to cellular stresses it detects and repairs DNA damage (Chaitanya et al., 2010).
Prostate and breast cancer are the most common and hormone-dependent malignancies in men and women, respectively (Bener et al., 2008;Safaei-Ghomi et al., 2009). The purpose of this study was to evaluate the effect of N. cataria on apoptosis process and its cytotoxic activity in both breast and prostate cancer cell lines that could be promising for treatment of these cancers in future. Cytotoxic activity of different extracts of the aerial parts of the plant (N. cataria) including methanol extracts, n-hexan, methylene chloride (CH2Cl2), ethyl acetate (EtOAc), n-butanol (n-BuOH), and H2O fractions and the essential oil were compared on MCF-7, DU-145 and PC3 cells. Moreover, PI staining of treated cells and western blot analysis of proteins involved in the apoptosis cascade were studied to reveal the mechanism of cell death induced by the plant.

Chemicals
AlamarBlue® (resazurin) from Sigma (Saint Louis, MO, USA); RPMI-1640 and FBS were obtained from Gibco. All antibodies were bought from Cell Signaling technology (Boston, USA); ECL Western blotting detection reagent was purchased from Bio-RaD (USA); the fluorescent probe propidium iodide (PI), protease inhibitor cocktail (Sigma P8340), sodium citrate, Triton X-100, phenyl methyl sulfonyl fluoride (PMSF) and Quanti Pro BCA Assay Kit were purchased from Sigma (Steinheim, Germany); all solvents as analytical grade used in this work were purchased from Merck, Germany.

Plant Materials
Aerial parts of N. cataria were collected in June 2013 from 1900 meters of altitude in a village called Reen, (km southwest of Bojnoord), North Khorasan province, northeast of Iran, and were identified by Souzani from the Herbarium of School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran where the voucher specimen (No:12055) of the plant was deposited. The extraction method was done according to previously reported protocol (Tayarani-Najaran et al., 2013) ( Figure 1). The essential oil (EO) was prepared, using a Cleavenger type apparatus after 3 h hydrodistillation of the aerial parts (100 g) of N. cataria. The oil was passed over anhydrous sodium sulfate to obtain the dried slightly yellow colored oil.
Extracts and the EO were subjected to cytotoxic assay and the essential oil of the plant was subjected to GC and GC-MS analysis.
The GC-MS analyses were conducted using a Agilent 5,975 apparatus with a HP-5ms column (30 m × 0.25 mm i.d., 0.25 µm film thickness) interfaced with a quadruple mass detector and a computer equipped with Wiley 7n.l library; device setting were as follows :oven temperature 50 0 C (5 min), 50 0 C-250 0 C (3 o C/min), 250 0 C (10 min); injector temperature 250°C; volume injection, 0.1 Table 1. Chemical Composition of Essential Oil Obtained by Hydro-Distillation from Aerial Parts of N. Cataria µL; split ration, 1:50; carrier gas Helium at 1.1 ml min; ionization potential, 70 eV; ionization current, 150 µA; ion source temperature, 250°C; mass range, 35-465 mui. The constituents of the oils were identified by calculation of their retention indices under temperature programmed conditions for n-alkanes (C8-C20) and the oil on a CP-Sil 8CB column. Identification of individual compounds was made by comparison of their mass spectra and retention indices (RI) with those authentic samples and those given in the literature. Quantification of the relative  PC3 cells were seeded in a 24-well plate (105 cell per well) after 24h treated with the fractions (EtOAc). Floating and adherent cells were collected and incubated at 4°C overnight in the dark with 500 µl of a hypotonic buffer (50 µg/ml PI in 0.1% sodium citrate plus 0.1% Triton X-100).Flow cytometric analysis was performed by using a FACScan flow cytometer (Becton Dickinson). Around 10,000 events were counted with FACS (Nicoletti, 1991) Western Blotting Analysis PC3 cells were treated with different concentrations including 62.5,125 and 250 µg/ml of the EtoAc extract of N. catariafor 48 h. after that cells rinsed with cold PBS. The cell pellet was resuspended in a lysis buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% triton X-100, 1 mM EDTA, 0.2% SDS, 1% protease inhibitor cocktail and 1 mM phenylmethylsulfonyl fluoride and then put on ice for 30 min. centrifugation at 14000 rpm for 20 min at 4°Cwas done (Hettich Universal 320R, Germany). Supernatant pull and protein content were measured by BCA protein assay kit (BioRad) with BSA as standard. Western blot analysis was performed on protein extracts from the pc3 cells for PARP, Caspase and Bax. Equal amounts of proteins were loaded to 12.5% SDS-PAGE (w/v).
After electrophoresis, proteins were transferred to a polyvinylidene fluoride (PVDF) membrane.
Protein bands were detected using an enhanced chemiluminescence (Pierce ECL western blotting substrate) and Alliance gel doc. (Alliance.Gel doc, UK).  Sample Preparation 500 mg/ml essential oil and 50 mg/ml extracts stock solutions were dissolved in DMSO. Then cells were treated with different concentrations (15-500 µg/ml) of stock after diluting the stock solutions in (RPMI-1640) medium.

Cell Viability
AlamarBlue® assay was performed to measure DU-145, PC3 and MCF-7 and cells viability. To screen cell viabilities, DU-145, PC3 and MCF7 (104 cells per well) were seeded in 96-well plate and were treated by various concentrations of essential oil and each fraction of N. cataria. After 48 h incubation, 1% of total well content alamarBlue® was added to each well and the absorbance was measured after 4 h of incubation, at 570 nm and 600 nm using Synergy H4 Hybrid Multi-Mode Microplate Reader (BioTek, Winooski, USA). The cytotoxicity of essential oil and extracts were determined as IC50, using Graph Pad Software (Graph Pad Prism 5 software) and presented as mean ±SEM of three independent experiments with three replicates for each concentration of N. cataria extract.

Apoptosis PI Staining
In order to detect apoptotic cells propedium iodide, a florescent DNA binding dye, was utilized. First, apoptotic cells become permeable with triton-X 100 so DNA fragments and are moved from apoptotic cells to the hypotonic buffer. Apoptotic cells exhibit low fluorescent intensity and appear before G1 peak in the flow cytometry histogram in comparison with the control unaffected cells (Nicoletti et al., 1991).

Figure 1. Schematic Partitioning of N. Cataria Between Different Fractions and Essential Oil Preparation
Images were analyzed and quantified using the Gel-Pro Analyzer v. 6. 0 Gel Analysis Software.

Statistical Analysis
One way analysis of variance (ANOVA) and Bonferroni's posthoc were used for data analysis. All results were expressed as mean ± SEM and p values below 0.05 were considered statistically significant.

Essential Oil Composition
The 30 components were identified as volatile, representing 99.7% of EO composition after GC-MS analysis of the oil obtained from aerial parts of the N. cataria by hydro-distillation (Table 1). The grouped contents of the essential oil were determined as monoterpene hydrocarbons 0.3%, oxygenated monoterpenes 98.1%, sesquiterpene hydrocarbons 0.9%, oxygenated sesquiterpens 0.2%, and the miscellaneous 0.3%. The major oil components of the essential oil were nepetalactone stereoisomers.

Cytotoxicity of the Various Extracts of the N. cataria
The cell viability of cancerous cells treated with different concentrations of MeOH, n-hexane, CH2Cl2, EtOAc, n-BuOH and H2O extracts and the essential oil of N. cataria (15-500 µg/ml) were monitored after 48 h. Only EtOAc extract of the plant could significantly decreased cell viability in PC3 cells, as a concentration dependent manner (Fig. 2). Doses inducing 50% cell growth inhibition (IC50) against PC3 cells are presented in Table 2. doxorubicine (1 µM) was used as a positive control.

Role of Apoptosis PI Staining
Apoptosis was measured with PI staining following treatment with EtOAc extract of N. cataria to detect the sub-G1 peak resulting from DNA breakdown. Sub-G1 peak as an indicative of apoptotic cells was induced in EtOAc extract of N. cataria treated cells, but not in the control cells. EtOAc extract of N. cataria treated cells showed a sub-G1 peak in PC3 cells in a concentration dependent manner that indicates the involvement of an    DOI:http://dx.doi.org/10.7314/APJCP.2016.17.S3.125 Growth Inhibition and Apoptosis Induction of Essential Oils and Extracts of Nepeta Cataria L N. cataria on PC3, DU-145 and MCF-7 cells showed the potent cytotoxic activity of EtOAc extract on PC3 cells. After that apoptosis induction was verified in treated cells following observation of sub G1 peak in flow cytometery histograms and increased in pro-apoptotic Bax protein and cleavage of PARP. Lack of the protective effect of PARP promotes apoptosis of cells which was reported previously for anticancer drugs (Chaitanya et al., 2010 ).
It has been reported some botanical families have isoflavones that mimic the function of estrogenic compounds and contain steroidal structure (Kim et al., 2008). In this regard, N. cataria L. contain β-sitosterol which is similar to human steroids has been used to relieve the complications related postmenopausal syndrome (Dweck, 2006). Several studies suggested estrogens and their receptors (ERs) involved in progression and control cell growth in prostate and breast cancers (De Mayo et al., 2002;Kuiper et al., 1996;Prins et al., 2001). Since the expression level of the ERα and ERβ are higher in PC3 compare to DU145 cells (Linja et al., 2003), so it is expected that PC3 cells, as estrogen sensitive cell, show greater response to the phytoestrogens of the plant extracts. As expected in our study, more cytotoxic effect was observed in PC3 cells. Both DU-145 and PC3 cells with same androgen receptors profile (AR¯) introduced as castration-resistant prostate but there was some evidence reported that over-passaged PC3 cells can change its negative androgen receptors profile to positive (PC3AR+) (Buchanan et al., 2004). Therefore, we hypothesize more cytotoxic activity of N. cataria on PC3 cells in comparison with DU145 AR¯ cells might be due to different response of cells to sex hormones.
It is found that apoptosis is the preferred mechanism of cell death against chemotherapy agents (Hannun, 1997). In this study, EtOAc extract of N. cataria could enhance the expression level of Bax protein and cleavage of PARP on PC3 cell line. In apoptotic cells due to the presence of fragmented DNA, lower fluorescence intensity of PI stained cells were absorbed and appeared as a sub-G1 peak in the histogram of flow cytometry. This effect was dose-dependent; so by increasing the percentage of apoptotic cells, the AUC of sub G1 peak increased. Since cells treated with increasing concentration of EtOAc fraction of N. cataria showed sub G1 peak, apoptosis was determined as the key mechanism of cell death in cytotoxicity of this plant. In conclusion, the semi polar EtOAc extract of N. cataria induces apoptosis in pc3 cells compare to DU-145 cells and further analytical assays are suggested to identify the active phytochemicals possess cytotoxic activity.

Acknowledgement
The authors would like to thank Mr. M. Malaekeh for his assistance in flow cytometry. This work was supported by grants (No. 921271) from Research Affairs of Mashhad University of Medical Sciences as a part of a Pharm. D. thesis . The authors have declared no conflict of interest. apoptotic process in EtOAc extract -induced cell death (Figure 3).

Effect of N. cataria on apoptotic proteins (PARP, Bax and caspase 3)
Cleavage of 116 kDa PARP to 89 and 24 kDa fragments consider as the sign of apoptosis (Elmore, 2007). Western blotting analysis showed that in PC3 cells treated with EtOAc (48 h) caspase 3 and PARP were cleaved to active form (Figure 4). The level of Bax protein has an important role in the induction of apoptosis via the mitochondrial pathway. Protein expression of Bax enhanced in PC3 cells treated with EtOAc extract of N. cataria (Figure 4).

Discussion
This study was conducted to verify the cytotoxic effect of methanol extracts, n-hexan, CH2Cl2, EtOAc, n-BuOH, and H2O extracts and EO obtained from the aerial parts of the N. catariaon on PC3, DU-145 (prostate cancer) and MCF-7 cells (breast cancer).
Obtained EO from N. cataria contains 32 compounds among which 30 of the total oil mixtures were identified and listed in Table 1. Nepetalacton with 97.7 percent was identified as the major component.
There are several reports on cytotoxic activity of some genus of Nepeta. Baloch et al. reported crude methanol extract and crude acetone fraction of N. praetervisa have maximum cytotoxic effect on brine shrimp with ED50 values of 0.60 and 0.56 µg/ml respectively (Baloch et al., 2013). In another study, ethyl acetate extract of N. suavis Stapf. showed high cytotoxic effect against brine shrimp larvae with LD50 of 41.3 μg/ml (Rehman et al., 2015).
EtOAc extract of the N. cataria showed the lowest IC50 value on PC3. Extraction with different solvent might remove compounds of the opposite polarity in the obtained extract. Since EtOAc is a semi-polar solvent it seems semi-polar compounds are responsible for the cytotoxic activity.
Shakeri et al. studied the cytotoxic effect of EO obtained from N. ucrainica ssp. kopetdaghensis on human ovarian carcinoma A2780 and breast adenocarcinoma MCF-7 cell lines and reported the IC50 values less than 50 μg/ml for both cells. The most abundant component of essential oil in this species was germacrene D (53.0%) (Shakeri et al., 2014).
Cytotoxic evaluations of essential oils of N. glomerata against the renal adenocarcinoma cell line revealed 48% of inhibition of proliferation at 100 µg/ml. The main substances of the essential oil of N. glomerata were α-pinene, spathulenol and carvacrol. The EO obtained from N. cataria with active antimicrobial properties has been analyzed previously and nepetalactone was reported as the main component (Zomorodian et al., 2012;Zomorodian et al., 2013). According to the studies mentioned above, it is clear that nepetalactone form the major components in the essential oil of N. cataria.
The cytotoxic evaluations of methanol extract, n-hexane, CH2Cl2, EtOAc, n-BuOH, and H2O fractions and the essential oil obtained from the aerial parts of the