ANTIPROLIFERATIVE AND ANTIOXIDANT EFFECTS OF ERUCA SATIVA (JARJEER) LEAVES EXTRACT ON CARCINOMA OF WOMEN’S BREAST

Objective: This work aims to investigate the influence of Eruca sativa leaves extract on the cell viability of the breast carcinoma cell line (MCF-7). Methods: In vitro , breast cancer cell line (MCF-7) treated by E. sativa leaves extract for 48 h. The cell viability, proliferation, and apoptosis were assessed using colorimetric (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, flow cytometric technique, and antioxidant enzymes (superoxide dismutase [SOD] and catalase [CAT]) measurement. Results: This study demonstrated that the incubation of MGF-7 cells with E. sativa for 48 h caused a significative reduction in cell viability and proliferation of MGF-7 cell line. In parallel, E. sativa treatment induces a significant increase in apoptosis of MGF-7 cells compared to control. Moreover, flow cytometry analysis demonstrated that the inhibition of MGF-7 cell proliferation existed at the G2 and M phase in the cell-division cycle. Finally, the intracellular antioxidant enzymes SOD and CAT activities were significantly increased in the administered cells compared with unadministered MCF-7 cells. Conclusions: Taken together,


INTRODUCTION
Carcinoma of the breast is the second overriding reason for mortality in womenfolk. Breast cancer incidence rates were 32.0% followed by hepatocellular carcinoma (13.5%) among females in Egypt [1]. Medicinal herbs are regarded as the key sources of bioactive compounds that can be utilized for the medical management of varied diseases such as cancer. However, among the 250,000-500,000 herb sorts on land, only 1-10% has been examined by chemical and pharmaceutical experts for their potentiality and medicative worth, particularly for chemotherapy impact [2]. Eruca sativa or rocket is an "endemic" sort of the Cruciferae and productized chiefly in lands that surround the Mediterranean Sea [3]. The rocket contains great quantities of ascorbic acid and antioxidant compounds [4]. The previous studies reported that rocket species exhibit many medicinal possessions. Consistently, arugula salad types have now been proven a valuable resource for antioxidant complexes, as a rich source of three essential vitamins such as vitamin C, tetraterpenoids, and polyphenols [5]. Importantly, several studies suggested a relationship between the potential to reduce the chance of some specific types of carcinoma and augmented consumption of Cruciferae vegetables, composing their rich sources [6]. The present study aims to evaluate the in vitro antitumor activity of alcoholic extract of E. sativa leaves on the cell viability of the breast carcinoma cell line (MCF-7). As well as antioxidant, activities of E. sativa leaf extracts were evaluated.

Preparation of the alcoholic extract of E. sativa leaves
One hundred and ten gram powder of shade dried leaves of E. sativa was extracted with 75 ml of 95% ethanol. After that, the mixture was shacked, using a magnetic stirrer for 3 h/day and allowed to stand for 21 h for 3 days. Then, the mixture was filtered on Whatman filter paper 0.45 µm, and the extract was dissolved in 450 ml 95% ethanol and filtered again. The dissoluble ethanol extract was condensed to dryness at 60°C under low pressure. Dissolvent eradication of the resultant ethanol extract was weighed 13.5 g. The percent yield was computed utilizing the following form: (weight of extract/original weight×100 giving 12.27% yields) of green fatty crude E. sativa leaf ethanol extract. The herbal extract was kept in the fridge and was suspended in distilled water before administration [7].
Cell line MCF-7 mammary bust carcinoma cell-division line (ER+) got acquired from initial malignant tumor of a case, but cell line was purchased from the Research and Development of Medical Experimental Research Center, Kafrelsheikh University. Cells were expanded and raised in an adherent culture in minimum essential medium supplemented with 10% (v/v) fetal bovine serum, 1 mM Pyruvic Acid Sodium Salt, 10 µg/ml beef insulin, and 1% (v/v) antibiotic-antimycotic agent. Cells were raised until 37°C confluence in a moistened climate of 5% CO 2 in the ambient ether and were passed out once a week with 0.25% trypsin.
Cell lysate preparation MCF-7 cells got placed into 12-well dishes at 5×106 cells/well and permitted attachment for 48 h. MCF-7 human breast carcinoma cells were incubated for 48 h with the 50% inhibitory concentration (IC 50 ) level of treated cells while comparison cells were incubated with vehicle only. After incubation, trypsin was utilized to assemble cells, flushed 3 times with phosphate-buffered saline (PBS) (pH 7.4) and the pieces were transmitted into extract solution that comprised 20 mM potassium phosphate buffer (pH 7) and a protease inhibitor cocktail. The cells were homogenized and centrifuged at 10,000 rpm at 4°C for 10 min. The resulting supernatant will be used for the measurement of antioxidant enzymes activity.

(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay
The inhibition of MCF-7 cell proliferation on medical treatment with test sample was set by the MTT assay according to the method of Oka et al. [8] using (Sigma Aldrich Chemical Company, UK) MTT assay kit. The rationale of this way is built upon cellular reduction of MTT to an azure formazan result by dehydrogenases of mitochondria of living cells. The blue color intensity made by this process is proportionate to the number of living cells. Trypan Blue (0.4%) melted in PBS was utilized to count cells. 0.2 ml trypsin-suspended cell was placed into 0.3 ml medium and 0.5 ml 0.4% Trypan Blue. Cells were reckoned with a hemocytometer after 5 min. Cells were seeded down into a 96well dish with density 5×103 cells/well and incubated (37°C and 5% CO 2 . After 24 h, media was substituted by new media encompassing variant concentrations of test samples and incubated for another 24 h comparison cells administered with intermediary only were placed to the same dish. Ten microliters of 5 mg/ml MTT solution were added for each one and these dishes were enwrapped with the tin sheet and incubated for 3 h. Then, the intermediary was taken away and the remnant cells were melted in dimethyl sulfoxide. The absorbency was gauged at 595 nm. The mean values for the absorbance of the ones incubated with test specimens were compared to the mean worth for the absorbance of the comparison cells to compute the per cent of living cells (% of comparison cells) and when subtracted from 100 give the % of cell death (inhibition). The outcomes were given as the average±SE of three independent experiments; each repeated 3 times.

Assay for apoptosis and DNA damage
Programmed cell death (apoptosis) and damaged DNA were observed by utilizing an Annexin V-Fluorescein isothiocyanate (V-FITC) apoptosis detection kit (BD Pharmingen TM , USA). Administered and comparison cells were flushed two occasions with PBS and re-suspended in a 1-fold binding buffer at a concentration determined to be 1×106 cells/ml. Five microliters of Annexin VFITC Conjugate and 10 µL of propidium iodide (PI) solution were added to 500 µL of each cell suspension, incubated for 10 min at ambient temperature and then analyzed by flow cytometry instrument (FAC Star caliber, Becton Dickinson, USA) utilizing excitation at 488 nm and a 515 nm bandpass filter for FITC detection and a filter over 600 nm for PI detection. The percentage of cells in each stage was computed with the software Cell Quest [9].

Assays of superoxide dismutase (SOD) and catalase (CAT)
Determination of SOD activity was done using kit commercially available (Bio diagnostic, Cairo, Egypt). The SOD activity depends on its ability to inhibit the phenazine methosulfate-mediated reduction of nitro blue tetrazolium dye [10]. CAT activity was also determined by a ready-for-use colorimetric kit (Bio diagnostic, Cairo, Egypt). The CAT activity relies on the ability of the enzyme to react with a known quantity of H 2 O 2 . The reaction is stopped after exactly 1 min with CAT inhibitor. In the presence of peroxidase (horseradish peroxidase), remaining H 2 O 2 reacts with 3, 5-Dichloro -2-hydroxybenzenesulfonic acid and 4-aminophenazone to form a chromophore with color intensity inversely proportional to the amount of CAT in the original sample [11]. These assays were done agreeable to the instructions provided by the manufacturer. SOD and CAT activities in each specimen were expressed as U/ml.

Statistical analysis
Data were analyzed utilizing IBM SPSS statistics 21 (SPSS Inc., Chicago, Illinois, USA). They were shown as mean±standard division. Independent sample t-test was utilized to measure variations between groups. Level of significance for probability value was <0.05 as well as <0.01 and p<0.001 signified a highly significant result.

RESULTS
Our study assessed the cytotoxicity of E. sativa affected MGF-7 cells by MTT assay. Forty-eight hours after treatment with E. sativa, the incubation of MGF-7 cells with E. sativa for 48 h brought about a reduction in the viable cell, with approximate IC 50 of 41.13 µg/mL. The IC 50 was 41.13±0.03 µg/mL. In parallel, E. sativa dose-dependently impeded cell line proliferation MGF-7 (Fig. 1).
Herein, the ability of E. sativa to induce apoptosis was examined using Annexin V-FITC apoptosis kit. Our analysis of flow cytometry of Annexin V-FITC showed that showed a significant increase in apoptosis of MGF-7 cells (55.5±3.6)% treated by the E. sativa alcoholic extract for 48 h in compared with untreated MGF-7 cells as control (8.5±0.5)% (Table 1 and Fig. 2).
To find out extra info on the machinery behind the growth inhibition effects of E. sativa cell on MGF-7 cell-cycle distribution, we used flow cytometry techniques. Forty-eight hours after treated with E. sativa, our analysis of flow cytometry demonstrated that the inhibition of MGF-7 cell proliferation existed at the G2 and M phase cell-division cycle, as reflected with a larger number of accumulated cells (Table 1 and Fig. 3). Specifically,   Table 2).   cell lines to 50% (IC 50 ). We found that MCF7 cell line treatment with E. sativa increased cytotoxic activities as reflected with inhibition of cell proliferation in a time-dependent manner with an IC 50 of 41.13 µg/ml. This may suggest its anticancer potential. These findings confirm with the past study by Michael et al. [12] showed that IC 50 (µg/ml) of 70% aqueous ethanolic extract of E. sativa on MCF7 cell line was 21.7. While, Azarenko et al. [13] indicated that the main isothiocyanate Erucin existed in arugula species (cabbage, cauliflower, kale, arugula, E. sativa) were inhibited MCF7 cell proliferation in a time-dependent manner with 50% inhibition the IC 50 that happens at a concentration of 28 mM. Pawlik et al. [14] found that MCF7 cells after 96 h of treatment with E. sativa IC50 were 9.7 µM. This may suggest the anticancer potential of E. sativa.

Carcinoma of Women
The arrest of cell-division cycle progression is recognized as a stopping point in the cell-division cycle transition such as G0, G1, S, and G2/M stages for cell reduplication and subdivision. In the current study, E. sativa effectively induced G2/M arrest. Hence, cell cycle growthinhibitory at G2M by E. sativa was accompanied by the arrest in mitosis.
Since both non-cancerous cell and cancerous cell proliferation is contingent on the cell-division cycle in well-balanced, many composites that act against cancer, brought about an extended mitotic arrest through induced cell G2/M arrest [15].
It illustrated that E. sativa might be a suitable candidate as described previously for breast cancer therapy as long as it induced conditions of oxidative stress in MCF-7 cells. There was an enhancement in the actions of SOD and CAT. Our investigation exhibited that E. sativa augmented the action of cellular SOD and CAT in 48 h. This may be due to an increase in free radicals contribute to augment in action and plane of enzymatic antioxidant. The antioxidants activity of E. sativa is due to the presence of polyphenols contents [5]. The present findings agree with the past study by Alam et al. [16] demonstrated that E. sativa seeds have antioxidant action and do a preventive impact on Mercury (II) chloride-induced nephrotoxicity. Consistently, Hussein et al. [17] reported that the extracts of E. sativa in ethanol increasing/preserving the plane of antioxidant molecules and enzymatic antioxidant in rats with an ethanol-induced liver injury.

CONCLUSION
In summary, the incubation of MGF-7 cells with E. sativa for 48 h significantly showed reduced cell viability and proliferation. Furthermore, E. sativa treatment of MGF-7 cells increased G2/M accumulation increased the actions of intracellular enzymatic antioxidant SOD and CAT. In general, our results imply that E. sativa brings about apoptosis and G2/M arrest concomitant with reduced cell viability and proliferation in breast cancer MGF-7 cells line this may help in clinical pretreatment and protection for breast cancer.