Antitumoral Effects of Melissa officinalis on Breast Cancer in Vitro and in Vivo

The plant kingdom provides an enormous potential for discovery of new drugs for the treatment and prevention of diseases. Some of the plant species, including Taxus baccata, Podophyllum peltatum, Camptotecha accuminata, and Vinca rosea have well-recognized anticancer activity in breast cancer, and several isolated pure compounds and their semisynthetic derivatives have been evaluated in clinical trials and marketed (Mukherjee et al., 2001). Lemon balm, M. officinalis L. is one of the most used medicinal plants in Europe and the Mediterranean region,as a herbal tea for its aromatic, digestive and antispasmodic properties in nervous disturbance of sleep and functionalgastrointestinal disorders (Bisset et al., 1994). The leaves emit a distinct fragrant lemon odour when bruised. The chemical composition is essential oil, polyphenolic compounds: caffeic acid derivatives in large proportions, such as rosmarinic acid, trimericcompounds and also some flavonoids such as luteolin-7-0-glucoside. Some pharmacological properties have been attributed to the principal constituents (De Sousa et al., 2004). Enriched extracts containing rosmarinic acid are used as a virostatic


Introduction
The plant kingdom provides an enormous potential for discovery of new drugs for the treatment and prevention of diseases.Some of the plant species, including Taxus baccata, Podophyllum peltatum, Camptotecha accuminata, and Vinca rosea have well-recognized anticancer activity in breast cancer, and several isolated pure compounds and their semisynthetic derivatives have been evaluated in clinical trials and marketed (Mukherjee et al., 2001).
Lemon balm, M. officinalis L. is one of the most used medicinal plants in Europe and the Mediterranean region,as a herbal tea for its aromatic, digestive and antispasmodic properties in nervous disturbance of sleep and functionalgastrointestinal disorders (Bisset et al., 1994).The leaves emit a distinct fragrant lemon odour when bruised.The chemical composition is essential oil, polyphenolic compounds: caffeic acid derivatives in large proportions, such as rosmarinic acid, trimericcompounds and also some flavonoids such as luteolin-7-0-glucoside.Some pharmacological properties have been attributed to the principal constituents (De Sousa et al., 2004).Enriched extracts containing rosmarinic acid are used as a virostatic
According to the in-vitro cytotoxicity analysis, using MTT and NR assays, this plant found as very effective against human colon cancer cell line (HCT-116) (Encalada et al., 2011).M. officinalis is a plant, which has shown several types of biological activity.Essential oil from M. officinalis has a great economical importance due to its use in industry, especially the pharmaceutical industry.de Sousa et al, demonstrated that M. officinalis essential oil inhibited the viability of several tumour cell lines in a concentration-dependent manner.In this study, we investigated the antitumoral effects of Melissa officinalis on breast cancer.

Collection of the plant material
The herbal parts of O. acutidens was collected from Ziyarettepe district, Ulas, Sivas-Turkey, in 18.07.2009.The taxonomic identification of plant materials was confirmed by a senior plant taxonomist, Dr. H. Askin AKPULAT, in Department of Biology, Cumhuriyet University, Sivas, Turkey.The voucher specimens have been deposited at the Herbarium of the Department of Biology, Cumhuriyet University, Sivas-Turkey (AA 4402).

Preparation of the M. officinalis extracts
Extracts of air-dried and ground plant materials were prepared by using water as solvent.A portion (100 g) of dried plant material from MO was extracted with deionized water (yield; 5.72% w/w), in a Soxhlet apparatus during 6 h.After this period, solvent-extract mixture was filtrated and filtrate was freeze-dried under vacuum.The extract obtained as powder was kept at room temperature until tested.It was dissolved in deionized water at 1 mg/ mL concentration and further dilutions were made in Dulbecco's Modified Eagle Medium (DMEM) (Sigma, St Louis, MO, USA).

Reagents and drugs
Dulbeccos modified Eagles medium (DMEM) , Fetal bovine serum (FBS), and 2.5%.trypsin were purchased from Sigma-Aldrich St. Louis, MO, USA.All reagents were free of endotoxins as determined using the Limulus amebocyte lysate assay (sensitivity limit, 0.025 ng/ml), which was purchased from Sigma (St Louis, MO).FITC Annexin V Apoptosis Detection Kit I, were purchased from BD Pharmingen (San Diego, CA).

Cytotoxicity assay
The level of cytotoxicity MO extracts on MCF-7, MDA-MB-468 and MDA-MB-231 cells .was determined using the trypan blue dye exclusion test (Roomi et al., 2011) and 3-(4,5-dimethylthiazol-2-yl)-2, diphenyltetrazolium bromide (MTT) proliferation assay.For MTT assay; first, tumor cells were seeded into 96-well tissue culture plates.After an attachment period of 6 h at 37 ºC in a 5% CO 2 humidified incubator, the cells were treated with fresh medium alone or medium containing MO extracts for 48 h.During the last 2 h of incubation, 40 mL MTT (2.5 mg/ mL; Sigma) was added into each well (0.42 mg/mL).At the end of the incubation, the MTT was removed and the cells were lysed with dimethylsulfoxide.Metabolically viable cells were monitored for conversion of MTT to formazan using a Multiskan FC 96-well microtiter plate reader at 570 nm (Thermo Scientific, MA, USA).The level of cytotoxicity was calculated using the following formula: cytotoxicity (%) = (A-B/ A) x 100, in which A is the 570-nm absorbance of cells treated with medium alone and B is the 570-nm absorbance of cells treated with MO extracts.
Every experiment also included one set of positive control (paclitaxel).All experiments were performed in triplicate and repeated at least twice.MO extracts induced apoptosis of MCF-7, MDA-MB-468 and MDA-MB-231 cells was studied by flow cytometry using annexin-V staining.In early stages of apoptosis, phosphatidyl serine is exposed at the external surface of the cell and can be detected by annexin-V.Late apoptotic cells and necrotic cells will also show Propidium Iodide (PI) positivity.Living cells, however, will show neither annexin-V nor PI positivity.

Apoptosis assay of in vitro studies of
Briefly, MCF-7, MDA-MB-468 and MDA-MB-231 cells were untreated or treated for 24 h with MO extracts at different dilutions.As a positive control, MCF-7, MDA-MB-468 and MDA-MB-231 cells were incubated in the presence of paclitaxel.
After incubation, cells were washed twice with cold PBS and then resuspended in 1X Binding Buffer (10 mM Hepes, 140 mM NaCl, 2.5 mM CaCl 2 , pH [7.4]) at a concentration of 1 x 10 6 cells/mL.Then 100 μl of the solution (1 x 10 5 cells) transferred to a 5 ml culture tube and 5 μl of annexin V-FITC and 5 μl Propidium Iodide added and incubated for 15 min at room temperature (25°C) in the dark.Later 400 μl of 1X Binding Buffer was added to each tube and analyzed by flow cytometry (Becton Dickinson FACS Calibur, Heidelberg, Germany) within 1 hr.All analyses were performed with CellQuest software (Becton Dickinson).

In Vivo tumor volume Studies
A total of 21 female albino Wistar rats (160±10 g body weight) 50-59 days old maintained in Animal Laboratory of Cumhuriyet Universty Faculty of Medicine, at temperature 24±2 ºC with a 12 hour light/dark cycle and 60%±5% humidity.They were provided with standard pellet diet and water ad libitum.The experiment was carried out as per the guidelines of Ethical Committee for the Purpose of Control and Supervision of Experiments on Animals, of Cumhuriyet Universty Faculty of Medicine.
All rats were divided into three groups of 7 rats each.Rats in Groups I and II were induced mammary carcinogenesis by providing single subcutaneous injection in right pectoral area. of 25 mg DMBA in 1 mL emulsion of sunflower oil (0.75 mL) and physiological saline (0.25 mL) to each rat (Chidambaram et al., 1996).During the experimental period, animals were weighed weekly.Animals were observed daily to assess their general health.
After DMBA administration, right pectoral area of all rats were followed up for the tumoral development.Palpation of mammary tumors began 4 week after animals received DMBA.The volume of every tumor was measured weekly using calipers.Tumor volume was calculated using the formula: Tumor volume (cc) (Dx d 2 x π) / 6 (D= big diameter, d= small diameter).The results are expressed as the mean±Standart Error.
When the nodule reached to a mean volume of 250±4.3mm 3 (± S. E.), fine needle aspiration biopsy was performed from the nodules.Nodules reached that size in a mean of 8±2 weeks.Histopathological examination was performed from that biopsy for each nodule.
After histopathological examination revealed the breast cancer, treatments with MO extract was started in group II (Treatment Group).In Group I: control animals received no drug.In Groups II and III, rats were given MO extract (100 mg/kg, body weight) daily through an oral gavage.Groups III rats did not had DMBA induced mammary carcinogenesis, they had been used as control group for the side effects of MO extract treatment.
After 4 weeks of treatment, rats were sacrificed and tumors were removed from the animals of Groups I and II.The tumor volume inhibition ratio (%) was calculated by the following formula: Inhibition Ratio (%) = [(A -B) / A] x 100, where A is the average tumor volume of the control group, and B is the tumor volume of the treated group.

Histopathology and morphological observations
Each tumor tissues were sampled macroscopically and fixed in 10% formaldehyde solution for 24 hours.Tumor tissues then processed in autotechnicon device later embedded in parafin blocks and cut sections with 3-5 μm thickness were obtained and stained with Haemotoxylen-Eosin stain for routine histopathological examination by light microscopy.

Immunohistochemistry
Caspase 7: Tumor sections were deparaffinized with xylene and dehydrated with ethanol.The slides were then immersed in water for 10 min.For antigen retrieval, the slides were boiled in EDTA buffer, pH 8.2, for 15 min.in a microwave oven and subsequently cooled for 20 min.Next, the slides were washed in PBS and endogenous peroxidase was blocked with 0.3% hydrogen peroxide for 15 min.After washing with Phosphate Buffer Solution (PBS), the sections were incubated at 27 ˚C for one and a half hour for Caspase 7 antibody.After antibody reaction, the slides were washed and incubated with a multilink antibody for 20 min., washed in PBS and incubated for 20 min with the avidin-biotin-peroxidase complex.After washing with PBS, the slides were incubated for 3 min with AEC chromogen, and finally counter-stained with hematoxylin prior to mounting.
Ki -67: For immunohistochemical staining, the deparaffinized and redehydrated tissue sections were inactivated the endogenous peroxidase by an incubation with 3% H 2 O 2 for 10 minutes.To recover antigen, these sections were put into EDTA solution (pH 8.5) and heated in the microwave oven twice.The slides were then washed with PBS (pH 7.2-7.6)twice.Non-specific binding sites were blocked with Ultra V Block (ScyTek Laboratories, Logan, Utah, USA) for 20 minutes.After the redundant liquid was discarded, the sections were incubated with primary antibody Ki-67 (ScyTek Laboratories, Logan, Utah, USA) at room temperature for 1,5 hour and washed with PBS.Then the slides were incubated with biotinylated secondary antibody (ScyTek Laboratories, Logan, Utah, USA) for 20 minutes and washed with PBS, followed by incubation with streptavidin-HRP (ScyTek Laboratories, Logan, Utah, USA) for 20 minutes and washed with PBS.The antibody binding sites were visualized by incubation with a AEC kromogen (ScyTek Laboratories, Logan, Utah, USA) solution.The slides were counterstained for 1 min.with hematoxylin and then dehydrated with sequential ethanol for sealing and microscope observation.
The percentage of proliferating neoplastic cells was evaluated directly by light microscopy.Quantification of the proliferation was performed by counting, Ki-67 positive cells in 4-6 random fields per slide.Caspase-7 activity was evaluated semi quantativelly in the cytoplasm of the tumor cells either in living or in the necrotic tumor cell areas.

In vivo apoptosis assay
Apoptosis was evaluated by using terminal deoxynucleotydil transferase dUTP nick and labelling (TUNEL) method.In Situ Cell Death Detection Kit, POD (Roche, Germany) is used for apoptosis.Tumor sections were deparaffinized and dehydrated according to standard protocols.Tissue sections were incubated with Proteinase K working solution for 30 min at 21-37 0 C. The slides were then washed with PBS (pH 7.2-7.6)twice.Positive control was incubated with DNase I recombinant for 10 min at 15-25 0 C. Negative control was incubated with Label solution (without terminal transferase) instead of TUNEL reaction mixture.The slides were then washed with PBS (pH 7.2-7.6)three times.Converter-POD was added on slides and incubated in a humidified chamber for 30 min at 37 0 C. The slides were then washed with PBS (pH 7.2-7.6)three times.DAB Substrate was added on slides and incubated for 10 min at 15-25 0 C. The slides were then washed with PBS (pH 7.2-7.6)three times.The slides were mounted and analysed by fluorescence microscope (Olympus DP 70, Melville, NY, USA).

Statistical analysis
Results are reported as mean±standard error (SE).We tested the data from the experiments for statistical significance using the Mann-Whitney U Test.A p-value less than 0.05 was considered significant.

Effect of Melissa officinalis on human breast cancer cells in vitro
These results show that MO induces a significant reduction in the viability of MCF-7, MDA-MB-468, MDA-MB-231 cells in a dose-dependent manner (Figure 1).Crude extracts of MO showed cytotoxicity to three cancer cell lines with IC 50 values 18±2.0 μg/mL, 17±1.4  μg/mL, 19±1.8 μg/mL, respectively (Table 1 and Figure 1).
Percentage of Annexin-positive cells detected in treated cell lines were 26±2.1% in MCF-7 cells; 25±2% in MDA-MB-468 cells; 24±1.9% in MDA-MB-231 cells, compared with 12±1.4%, 13±1.2%,10±1.0%, of Annexinpositive cells detected in untreated cells respectively (Table 2).Results are representative of at least three independent experiments, all of which had similar results.Results were analyzed using the CELL Quest software program.When these values were compared with each other (Annexin-positive cells detected in treated cell lines versus annexin-positive cells detected in untreated cells) , results were statistically significant with each other.These results showed that MO treatment have apoptosis inducing effect on MCF-7, MDA-MB-468 and MDA-MB-231 cells .

Immunohistochemistry Results
Immunohistochemistry experiments of this study showed that Ki-67 activity was most intensive in Untreated Control Group; (Figure 3-a).The expression of Ki-67 in MO treated Group was lower than the Untreated Control Group (Figure 3-b).
When the semiquantitative scoring system results of immunohistochemistry studies were compared, the difference was statistically significant (Table 3) (p< 0.05).
In-vivo immunohistochemistry experiments of this study showed that the MO extract treatment induced the activation of Caspase-7 (Figure 2, 3).When the semiquantitative scoring system results of immunohistochemistry studies were compared, the difference was statistically significant (Table 3) (p< 0.05).
At the result of the Terminal Transferase dUTP Nick

Discussion
The ingestion of plant extracts with medicinal properties represents an alternative for the treatment of different pathological states in economically unprivileged countries.
However, in the absence of a scientific basis, such practices may generate serious adverse effects.The analysis of the pharmacological activity of plant extracts may therefore make possible the design of less expensive therapies to be used in developing regions.
M. officinalis showed cytotoxicity to three cancer cell lines with IC 50 values 18±2.0 μg/Ml, 17±1.4 μg/mL and 19±1.8 μg/ mL respectively.In this study, those IC 50 values were considered as "good" activity on breast cancer cell lines.The antitumour properties of several antioxidants have been described (Keum et al., 2000).Antioxidant activity of Melissa tea and hydro-alcoholic extracts have been described previously (Lamiason et al., 1991).Thus our data indicated that the antioxidant properties of M. Officinalis essential oil might contribute to its antitumoral activity.
Apoptosis plays an important role in the fight against cancer.The format (Annexin V-FITC) retains its high affinity for PS and thus serves as a sensitive probe for flow cytometric analysis of cells that are undergoing apoptosis (Vermes et al., 1995).In this study Annexin-positive cells were detected by flow cytometry in MO treated cell lines at 26±2.1% in MCF-7 cells; 25±2% in MDA-MB-468 cells; 24±1.9% in MDA-MB-231 cells (Table 2), compared with 12%, 13%, 10%, of annexin-positive cells detected in untreated cells respectively.So, results of this assay showed that MO extracts treatment induced apoptosis in breast cancer cells.
During apoptosis a group of proteases are activated which cause DNA fragmentation, cytoplasmic shrinkage and membrane blebbing.Here, we therefore examined apoptosis in tumoral tissues by using terminal deoxynucleotydil transferase dUTP nick and labelling (TUNEL) method .TUNEL Assay results of our in-vivo studies demonstrated that MO extracts treatment increased the apoptotic cells in tumoral tissues of MO treated rats.
Antigen KI-67 is a protein that in humans is encoded by the MKI67 gene (Bullwinkel et al., 2006).It is a nuclear protein and associated with cell proliferation and it is an excellent marker to determine the growth fraction of a given cell population (Scholzen et al., 2000).In this study, we studied the Ki-67 levels of the DMBA induced tumoral tissues to determine the MO effect on tumoral growth.In-vivo immunohistochemistry experiments of this study showed that the MO extract treatment decreased the Ki-67 activity in tumoral tissues of rats.
Caspase-7 is a caspase protein that plays a key role in programmed cell death, or apoptosis.Caspase-7 is activated in the apoptotic cell both by extrinsic (death End Labeling (TUNEL) Assay of this study while TUNEL positive cells has been detected rarely in the Untreated Control group, numerous TUNEL positive cells were intensively observed in the Allium Sivasicum treated group (Figure 4).When the semiquantitative scoring system results of TUNEL Assay studies were compared, the difference was statistically significant (Table 3) (p< 0.05).
ligand) and intrinsic (mitochondrial) pathways (Shin et al., 2001).In caspase cascade pathway, mature caspases-3 and -7 cleave a large set of substrates, ultimately resulting in the characteristic morphological and biochemical hallmarks of apoptosis.In-vivo immunohistochemistry experiments of this study showed that the MO extract treatment induced the activation of caspase-7.These results may suggest that MO induced the apoptosis in breast cancer cells via the caspase cascade pathway.
In DMBA induced rat mammary tumor volume experiments, treatment with MO showed a 40% tumor volume inhibition.This difference is statistically significant (p < 0.05).This result showed the tumor growth inhibition potential of MO crude extract.
In conclusion, these results showed that natural Melissa officinalis extract may constitute a potential antitumor compound against breast cancer.Experiments are necessary to identify which of the components are responsible for these activities.

Figure 1 .Figure 2 .
Figure 1.Cytotoxicity of Melissa officinalis (MO) against MCF-7, (circles), MDA-MB-468 (triangles), MDA-MB-231 (squares) cells.Target cells were incubated with OA for 48 h at different concentrations (μg/mL).Cytotoxicity was then determined using the trypan blue dye exclusion test and MTT proliferation assay.Triplicate wells were used for each group.Each point represents the mean±SE.One of at least three independent experiments .(p=0.002) a) b) c) d) DOI:http://dx.doi.org/10.7314/APJCP.2012org/10.7314/APJCP..13.6.2765Antitumoral Effects of Melissa officinalis on Breast Cancer in Vitro and in Vivo toxicity (weight loss, ruffled fur and behavioral changes) were observed in any of the MO treated rats (Group II and III).

Figure 3 .Figure 4 .
Figure 3. Representative Immunohistochemistry Photomicrographs of TUNEL-Positive Apoptotic Cells in DMBA Induced Rat Mammary Tumor Tissue of Rats.a) Untreated Control Group; b) Melissa treated Group.