Antioxidant and Anticancer Efficacies of Anethum graveolens against Human Breast Carcinoma Cells through Oxidative Stress and Caspase Dependency

Anethum graveolens, belonging to the family Apiaceae, has been extensively used for medicinal and therapeutic purposes since long. Plants encompass rich number of effective constituents with less toxicity. Thus, nowadays, the attempts are being made to search plant constituents that can prevent and reverse the chronic diseases, such as cancer. In this study, an in vitro antioxidant and anticancer efficacies of Anethum graveolens (AG-ME) were studied on human breast (MCF-7), lung (A-549), and cervical (HeLa) carcinoma cell lines. The antioxidant efficacies of AG-ME were evaluated by total antioxidant, DPPH radical scavenging, H2O2 scavenging, and ferrous reducing antioxidant assays. Further, the anticancer potential of AG-ME was also determined against different cancer cell lines. The AG-ME exhibited strong antioxidant activities as observed by antioxidant assays. AG-ME also showed a dose-dependent anticancer/cytotoxic potential against MCF-7, A-549, and HeLa cell lines. The AG-ME-induced reduction in GSH and increase in SOD activities indicates the role of oxidative stress in AG-ME-induced MCF-7 cell death. The results also exhibited that AG-ME triggered ROS production and significantly reduced MMP level. Moreover, a dose-dependent increase in caspase-3 and caspase-9 activities suggests that the AG-ME-induced MCF-7 cell death is caspase-dependent. Together, the present study provides reasoning and reassurance for the uses of A. graveleons for medical purposes as an antioxidant and anticancer agent. Additional investigations are required to examine biological and anticancer activities under an in vivo system to discover a possible beneficial use of AG-ME against diseases.


Introduction
Free radicals and ROS are produced by physiological and biochemical processes in the human body [1]. A number of chemically reactive radicals, i.e., superoxide, hydroxyl radicals, and H 2 O 2 , are derived from oxygen during ROS generation [2]. It is well documented that overproduction of such free radicals can cause serious damage to biomolecules such as DNA, RNA, proteins, and lipids, which leads to pathogenic conditions to human [2]. ROS play the most important character in the pathogenesis of diverse physiological conditions, such as cellular damage, neurodegenerative, diabetes, hepatic, and cardiovascular diseases [3]. Endogenous antioxidant enzymes, e.g., glutathione peroxidase, superoxide dismutase, and catalase are accomplished of neutralizing free radicals, and hence, sustaining optimum cellular activities [4]. Nonetheless, under high ROS production and oxidative stress, these endogenous antioxidant enzymes may not be adequate to maintain optimum cellular activities. Thus, the dietary antioxidants may be essential. Natural products or plants have always been used to treat and cure various diseases since ages [5,6]. Plants produced various secondary metabolites, for example alkaloids, terpenoids, flavonoids, and vitamins, that take part in the neutralization of free radicals and delay chronic ailments caused by ROS and oxidative stress [7][8][9]. Natural antioxidants from plant materials play a vigorous part in the production against the action of free radicals [10,11]. Numerous studies have shown that consumption of plant seeds and leaves with strong antioxidant potential and are capable to lower the causation of different chronic diseases like cancer [12][13][14]. Anethum graveolens (family: Apiaceae) is known for medicinal and therapeutic purposes since long in the conventional system of medicine [15]. A. graveolens has been described for its biological potential and pharmacological potential, e.g., antioxidant [16], anti-inflammatory [17], antidiabetic [18], antimicrobial [19], and anticancer [20] activities. Ethanolic and acetonic extracts of A. graveolens flowers and leaf [21,22] and essential oil of A. graveolens [16] have been shown to possess high antioxidant activities. Cancer is the abnormal growth of cells which forms a tumor. These cells are different than normal cells and are not accountable for normal growth controlling mechanisms [23]. The cytotoxic/antiproliferative potential of plants and plant-derived components can be measured by different cytotoxic endpoints, such as MTT, neutral red uptake, and cellular morphological analyses, using various cancer cell lines [24][25][26]. The available treatment for cancer is chemotherapy and radiotherapy, but the main disadvantage of current drugs is their side effects [25]. Plants encompass rich number of effective constituents with less toxicity [27]. Thus, nowadays, the attempts are being made to search an anticancer agent from plant origin which can prevent and reverse the development of cancer [28,29]. Hence, this study was aimed at investigating the antioxidant and anticancer efficacies of A. graveolens (AG-ME) against different human cancer cells (MCF-7, A-549, and HeLa).

Materials and Methods
2.1. Chemicals. All chemicals used were obtained from Sigma Aldrich. Cell culture medium, trypsin, and antibiotic solution were purchased from Invitrogen. Plastic wares and consumables were obtained from Nunc, Denmark. All the reagents and solvents used were of analytical grade.

Sample Collection.
A. graveolens seeds were purchased from Riyadh, Saudi Arabia. A taxonomist in the Botany Department, KSU, has identified the seeds, and a voucher specimen number has been submitted in the herbarium.
2.3. Preparation of Extract. The A. graveolens seeds were screened manually. Seeds were ground to a coarse powder. About 500 g of grounded seeds were soaked in 1.5-liter methanol for 7 days with regular agitation. The extract was filtered using Whatman #1 filter paper and funnel. The filtrate was then concentrated to dryness in a rotary evaporator at 40°C under reduced pressure. The obtained methanolic extract of A. graveolens seeds, named as AG-ME, was stored at 4°C for further use.

In Vitro Antioxidant Assays
2.4.1. Total Antioxidant Power. The total antioxidant power of AG-ME was measured using the phosphomolybdate assay following the method reported [30]. In brief, 500 μl of seed extracts (50-1000 μg/ml) was added to 3 ml of reaction mixture (0.6 M sulphuric acid, 28 mM sodium phosphate, and 1% ammonium molybdate). The mixer was then incubated at 95°C for 10 min to complete the reaction. After cooling, the reaction mixer was read at 695 nm. The percent antioxi-dant power was calculated by the formula:  [32]. In brief, extract solution (50-1000 μg/ml; 2 ml) was mixed with 4 ml of 20 mM H 2 O 2 solution in phosphate buffer (pH = 7:4). Following 10 min incubation, the absorbance was measured at 230 nm wavelength. The scavenging activity of H 2 O 2 was calculated using the following formula: where A 0 is the O.D. of control and A 1 is the O.D. of the extract.

Ferric
Reducing Antioxidative Capacity. The ferric reducing capacity, which imitate AG-ME antioxidant efficacy, was estimated by Fe +3 to Fe +2 method [33]. In brief, 200 μl of each concentrations of AG-ME was mixed with 2.5 ml of 0.2 M of sodium phosphate buffer and potassium ferricyanide solution (1% w/v). Then, the solution was mixed by vertex and incubated for 20 min at 50°C. Further, 2.5 ml of trichloroacetic acid (10% w/v) was added in the mixer. After centrifugation at 3000 rpm, a supernatant (2.5 ml) was added with equal volume of deionized water and 0.5 ml ferric chloride (0.1% w/v). The developed color was read at 700 nm.
2.5.2. Anticancer Activity (MTT Assay). The anticancer potential AG-ME was examined against MCF-7, A-549, and HeLa cells using 3-(4,5-dimethylthiozol-2-yl)-2,5-diphenyltetrazolium bromide salt, MTT assay [34]. The dried extract (AG-ME) was firstly dissolved in DMSO then diluted in a cell culture medium to reach the final concentrations of 10-1000 μg/ml. The final concentration of the DMSO solvent used for cytotoxicity assessments was not more than 0.04% in culture medium. In brief, MCF-7, A-549, and HeLa cells were allowed to grow in 96-well plates (1 × 10 4 cells/well) for overnight. The cells were then exposed to 10-1000 μg/ml concentrations of AG-ME for 24 h. After incubation, 10 μl MTT solution (5 mg/ml) was added in wells and incubated for 4 h. The developed formazan crystals were dissolved in 200 μl DMSO, and absorbance was read at 550 nm.
2.5.3. Anticancer Activity (NRU) Assay. The anticancer efficacy of AG-ME was also assessed by NRU assay using the method as described previously [35]. Briefly, MCF-7, A-549, and HeLa cells were plated in a 96-well and were allowed to grow overnight. Afterwards, cells were treated to 10-1000 μg/ml AG-ME for 24 h. Following treatment, the cell culture medium was aspirated and replaced with medium containing 50 μg/ml neutral red dye. Following 3 h incubation, the dye was extracted using destaining solution (50% ethanol, 49% water, and 1% acetic acid) and the plate was read at 550 nm. Considering 100% cell viability in control sets, viability of treated groups was calculated using the following formula: HeLa cells were plated in 24-wells and were allowed to grow for overnight in a CO 2 incubator. Thereafter, cells were treated for 24 h with 10-1000 μg/ml of AG-ME for 24 h. The morphological changes were analyzed under light microscope at 20x magnification power.
2.5.5. SOD and GSH Activities. Role of oxidative stress in the cell death induced by AG-ME was assessed using commercially purchased kits (Cayman Chemicals) for glutathione (GSH) and superoxide dismutase (SOD) assays. Briefly, MCF-7 cells were seeded in 6-well culture plates and incubated for overnight. After exposing the cells at 250-1000 μg/ml of AG-ME, the cells were harvested and homogenate was prepared by sonication. Following the centrifugation, the supernatant of the control and exposed cells were collected and assay was done as per the protocol given with kits.
2.6. Quantitative and Qualitative ROS Assays. For measuring the quantitative and qualitative ROS generation, MCF-7 cells were treated with 250-1000 μg/ml of AG-ME. Subsequently, cells were incubated in DCF-DA fluorescent dye for 1 h in dark. Then, the fluorescence strength of qualitative DCF-DA probe was visualized under a fluorescence microscope and quantitative intensity of the fluorescence was obtained by using a fluorescence microplate reader at 485 and 528 nm excitation and emission, respectively.

MMP Measurement.
Loss of MMP in MCF-7 cells exposed to AG-ME was evaluated by using the Rh-123 fluorescent dye. In brief, MCF-7 cells were exposed to AG-ME at 250-1000 μg/ml for 24 h. Then, cells were incubated with Rh-123 dye (10 μg/ml). Afterwards, fluorescence intensity was observed as previously described [35], using a fluorescence microscope and measuring the plate using fluorescence microplate reader.

Measurement of Caspase-3 and Caspase-9 Activities.
For the assessment of caspase activation, MCF-7 cells were treated with 250-1000 μg/mL of AG-ME. The activities of caspase-3 and caspase-9 were measured by using caspase colorimetric assay kits according to the manufacturer's recommended protocol (BioVision Inc., USA). The changes in caspase-3 and caspase-9 activities were determined by comparing the results with untreated control sets.
2.9. Statistical Analysis. The assay was performed in triplicates (n = 3) and were repeated in three independent experiments. ANOVA was used for statistical analysis, and values with p < 0:05 were considered statistically significant.

Total Antioxidant Power.
The results of total antioxidant efficacy of A. graveolens seed extract (AG-ME) at different concentrations are shown in Figure 1(a). Since, the phosphomolybdate method is quantitative; therefore, the total AG-ME antioxidant power is presented corresponding to ascorbic acid. AG-ME showed a concentration-dependent antioxidant activity at 50-1000 μg/ml. At 100 μg/ml, the antioxidant efficacy of AG-ME was 15.1%, while at 250, 500, and 1000 μg/ml, the antioxidant capacity of AG-ME was 36.3%, 58.6%, and 85.6%, respectively. The IC 50 value (344.3 μg/ml) of AG-ME showed considerable antioxidant capacity compared to ascorbic acid (247.0 μg/ml) ( Table 1).

3.3.
Hydrogen Peroxide Scavenging Activity. The results of scavenging potential of AG-ME on hydrogen peroxide are shown in Figure 2(a). As shown in the figure, a concentration-dependent (50-1000 μg/ml) strong hydrogen peroxide scavenging activity of AG-ME was observed. At the concentration of 500 μg/ml, the H 2 O 2 scavenging activity of AG-ME was 78.65%; however, at the same concentration, ascorbic acid was 82.43%. The H 2 O 2 scavenging activity of AG-ME closely resembled to the ascorbic acid. The IC 50 of AG-ME and ascorbic acid were 126.3 μg/ml and 100.5 μg/ml, respectively (Table 1).

Morphological Change Assessment.
The changes in cellular morphology of MCF-7, A-549, and HeLa cells are shown in Figure 5. As shown in the figure, obvious morphological changes were observed at 24 h treatment with AG-ME at 1000 μg/ml. Compared to untreated control, the MCF-7, A-549, and HeLa cells exposed to AG-ME at 1000 μg/ml lost their original shape, become less in number, rounded, and shrunken with apoptotic bodies ( Figure 5).

SOD and GSH Activities.
To study the oxidative stressmediated cytotoxicity of AG-ME in MCF-7 cells, SOD and GSH activities were measured by exposing the cells with various concentrations of AG-ME (250, 500, and 1000 μg/mL) for 24 h. MCF-7 cells treated with AG-ME exhibited a concentration-dependent increase in SOD and a reduction in GSH activities. The reduction in the GSH activity was observed as 14%, 39%, and 60% at 250, 500, and 1000 μg/mL, respectively, (Figure 6(a)). However, MCF-7 cells treated with 250, 500, and 1000 μg/mL of AG-ME showed an increase of 10%, 28%, and 42%, respectively, in SOD activity ( Figure 6(b)).      the early biochemical markers of apoptosis progression. To determine the depletion in MMP level, we used Rh-123 dye which indicates potential-dependent accretion in the mitochondria. As depicted in Figures 7(a) and 7(b), a significant dose-dependent decline of MMP occurred in MCF-7 cells exposed to AG-ME. The results exhibited a 14%, 32%, and 49% of loss in MMP at 250, 500, and 1000 μg/mL, respectively.

Discussion
The involvement of oxidative stress (OS) in neurodegenerative diseases, diabetes, cardiovascular diseases, ageing, and cancer is well documented [36,37]. Natural antioxidants from plant materials may provide protection against OS by inhibiting free radicals [38]. Therefore, in this study, the antioxidant and anticancer efficacies of A. graveolens, a known ayurvedic medicine used in traditional medicine system, were studied. The antioxidant activity is a composite process generally happening via various mechanisms and is affected by many other reasons that cannot be completely distinct by a single method. Therefore, it is necessary to measure by more than one type of assays, to take into elucidation of different mechanisms of antioxidant efficacies [33,[39][40][41].
Taking into account, in this study, four corresponding assays were performed to assess the antioxidant efficacies of AG-ME. Total antioxidant, DPPH, H 2 O 2 scavenging, and ferrous reducing antioxidative capacity assays were achieved. The methods used in this study have different mechanism of reaction. Total antioxidant efficacy and DPPH assays are based on both single-electron transfer that produced violet solution [42] and hydrogen atom transfer reaction [43]. OHis one of the most reactive free radicals; thus, it is required to deliberate the H 2 O 2 scavenging activity of natural antioxidants. It can be generated by Fenton reaction between H 2 O 2 and ferrous ion [44]. However, the mechanism of ferrous reducing antioxidative capacity is based on electron transfer [43]. All these assays significantly revealed the antioxidant and antiradical potential of AG-ME. Our results showed that the AG-ME possess significant total antioxidant capacity corresponding to 85.6% as compared to ascorbic acid (98.6%) at highest concentration, i.e., 1000 μg/ml. These data suggest the potential equivalent antioxidant ingredients of AG-ME since ascorbic acid is used as a reference standard to compare the potential antioxidant efficacy of AG-ME [45]. With increasing concentrations of AG-ME, there was an increase also found in DPPH activity with IC 50 value of 225 μg/ml. This indicates an increased ability of AG-ME to donate hydrogen ions ensuing in a brighter solution, which is relative to the number of gained electrons [46]. Thus, it may be suggested that AG-ME has potential DPPH activity by reducing the radicals to equivalent hydrazine as a result of its hydrogen ion donating ability. These results are in agreement with [47], who has reported that A. graveolens showed radical scavenging activity. The hydrogen peroxide scavenging capacity of AG-ME was also evaluated. As shown in Figure 2(a), a dose dependent radical scavenging potential of AG-ME was examined with the IC 50 of 126.3 μg/ml compared to ascorbic acid (100.5 μg/ml). The abnormal gathering of hydrogen peroxide is accountable for OS which are associated with many chronic diseases [48,49]. Consequently, regulation of H 2 O 2 production by plant antioxidants is of high interest in biological research [50]. The ferrous reducing antioxidative capacity measures the reducing capacity of an antioxidant and its properties that are related to presence of compounds which exert their action by breaking free radical chain via donating a hydrogen atom [51]. As shown in Figure 2(b), the absorbance of AG-ME was increased with the increasing concentrations because of formation of Fe ++ complex, as it was also observed in ascorbic acid. As observed in this investigation, the ferrous reducing activity of AG-ME might be due to the hydrogen donating by AG-ME, which is accountable for its reducing capacity [52]. All these antioxidant assays clearly exhibited strong antioxidant activities, but slightly lesser than standard ascorbic acid. These results are accordance to other reports exhibiting antioxidant activity of A. graveolens essential oil and extracts because of the existence of the hydroxyl group, which are liable for antioxidant efficacies [16,21,22]. We have further evaluated the anticancer efficacies of AG-ME against MCF-7, A-549, and HeLa cell lines. The findings of MTT and NRU assays clearly showed that AG-ME significantly inhibited MCF-7, A-549, and HeLa cell viability with the increasing doses. There are various methods available for measuring the cytotoxicity of materials under in vitro condition [53]. In this study, we used 3-(4,5-dimethylthiozol-2-yl)-2,5-diphenyltetrazolium bromide salt assay (MTT) and neutral red uptake (NRU) assays, since they are wellknown and widely used methods for cytotoxic/antiproliferative efficacies of plant extracts. These assays are based on colorimetric measurements of viable cells after incubation with test materials. The MTT assay measures the functional state of mitochondria which indicate cell viability [54], and NRU assay is based on incorporation of NR dye into lysosomes of viable cells [55]. The AG-ME was found to inhibit MCF-7, A-549, and HeLa cells by 90%, 79%, and 81%,

MCF-7 cells A-549 cells HeLa cells
Control 1000 g/ml Figure 5: Morphological analysis of cytotoxicity of AG-ME for MCF-7, A-549, and HeLa cell lines exposed for 24 h at 1000 μg/ml concentration. Images were grabbed at 20x magnification power using phase contrast microscope (Olympus, CKX41, Japan). 7 BioMed Research International respectively, at 1000 μg/ml of AG-ME by MTT assay and 86%, 79%, and 71%, respectively, by NRU assay. The MCF-7 cells were found to be more sensitive towards AG-ME followed by A-459 and HeLa cells. The IC 50 values obtained for MCF-7, A-549, and HeLa cells were 104 μg/ml, 122 μg/ml, and 156 μg/ml, respectively, by MTT assay and 109 μg/ml, 250 μg/ml, and 312 μg/ml, respectively, by NRU assay. The cytotoxic activity of AG-ME was also validated by changes observed in the cellular morphology. The MCF-7, A-549, and HeLa cells exposed to AG-ME at 1000 μg/ml lost their original shape, become less in number, rounded, and shrunken with apoptotic bodies. These results are corresponding to other reports which exhibited cytotoxic/ inhibitory activities of A. graveolens essential oil on MCF-7, HeLa, and Caco-2 cell lines with IC 50 values of 67 μg/ml, 93 μg/ml, and 216 μg/ml, respectively, as measured by MTT assay [56]. In another study, it has also been demonstrated that the essential oil of A. graveolens revealed a dosedependent anticancer/antiproliferative activity against human hepatocellular carcinoma cells with IC 50 of 59.6 μg/ml [20]. The methanolic extract of A. graveolens has also been reported to exhibit cytotoxic/anticancer activities against mouse leukemia L1210 cells [57]. Further to explore the mechanism(s) of cytotoxicity induced by AG-ME, we have selected most sensitive cell line, MCF-7. Herein, we explored the effects of AG-ME on the GSH and SOD activities in MCF-7 cells treated with 250-1000 μg/ml of AG-ME for 24 h. GSH and SOD are known to play a significant role in maintaining cellular redox balance or oxidative stress through its antioxidant mechanism. Our results showed that the GSH activity was decreased and SOD activity was increased with the increasing concentrations of AG-ME. These results are accordance to the previously published reports suggesting that the exposure of plant extracts could decrease the GSH [58] and increase the SOD activities [59] in MCF-7 (human breast carcinoma). The lowering of GSH activity and an increase in SOD activity indicated that oxidative stress is involved in MCF-7 cell death-induced by AG-ME. Oxidative stress tempted by the accumulation of ROS increases the sensitivity of cancer cells and is capable of inducing apoptotic cell death [60]. Based on the results obtained by DCF-DA assay, AG-ME was found to increase ROS production in MCF-7 cells, suggested that AG-ME induced oxidative stress by ROS generation. Excessive ROS generation is known to cause damage to cellular DNA, lipids, proteins, and biomembranes which leads to cell death [61]. Studies showed that excessive ROS generation can facilitate mitochondrial permeabilization [62]. The decrease of MMP level is one of the early biochemical markers of apoptosis progression. The findings of the study also displayed that AG-ME significantly decreased MMP level in MCF-7 cells. These findings provided a strong evidence towards the role of ROS mediated loss of MMP in MCF-7 cell death. To further elucidate the pathways of cell  BioMed Research International death induced by AG-ME, caspase-3 and caspase-9 enzyme activities were evaluated. Caspase-3 is one of the caspases involved in the final implementation of dying cells whereas caspase-9 is an initiator involved in the intrinsic pathway [63]. Thus, to understand the mechanisms of action induced by AG-ME, caspase-3 and caspase-9 enzyme activities were measured. The results exhibited that AG-ME induced a dose-dependent increase in caspase-3 and caspase-9 activities in MCF-7 cells. These results suggest that the AG-MEinduced MCF-7 cell death is caspase-dependent. The caspase-dependent anticancer activity of plant extract has also been previously reported in MCF-7 [64]. A large number of phytoconstituents present in methanolic extracts of A. graveolens have already been reported [65]; therefore, we assume that the anticancer efficacies of AG-ME observed in present investigation might be due to the existence of phytocompounds in it. Herein, the reported results also supported the relationship of cytotoxicity with antioxidant activities. Consequently, the antioxidant activities of AG-ME might contribute to its cytotoxicity/anticancer activities.

Conclusion
The present study provided a reasoning and reassurance for the uses of A. graveolens for medical purposes. The methanolic extract of A. graveolens (AG-ME) showed significant antioxidant efficacies as observed by total antioxidant, DPPH radical scavenging, H 2 O 2 , and ferrous reducing capacities. As it can be seen from this investigation, A. graveolens may be used as a good supply of natural antioxidants through probable nutrition supplement. AG-ME also showed a 9 BioMed Research International dose-dependent strong anticancer/cytotoxic potential against MCF-7, A-549, and HeLa cell lines. It further exhibited oxidative stress-mediated ROS production and loss of MMP in MCF-7 cells. A dose-dependent increase in the activities of caspase-3 and caspase-9 suggests that the AG-ME-induced MCF-7 cell death is caspase-dependent. Additional investigation is required to examine biological and anticancer activities under in vivo system to discover possible beneficial use of AG-ME against diseases.

Data Availability
The data used to support the findings of this study are included within the article.

Conflicts of Interest
The authors declare that they have no conflicts of interest.