Traganum nudatum Possesses Anticancer Efficacy against Breast Cancer MDA-MB-231 Cells

Background: Medicinal plants are effective sources for both traditional and modern medicines; herbal medicine has been shown to have genuine utility. 80% of rural population depends on natural products as primary health care. Traganum nudatum (T. nudatum) is a medicinal plant widely used in alternative medicine. T. nudatum has not been greatly investigated for its anticancer potential. Aim: This study aimed to evaluate the in vitro anticancer efficacy of T. nudatum against breast cancer cell line; MDA-MB-231. Materials and Methods: Extracts of T. nudatum were evaluated to assess their in vitro cytotoxicity against the MDA-MB-231 breast cancer cell line. Leaves, flowers, and stems of T. nudatum were extracted by using 70 % ethanol. the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase (LDH) assays were used to evaluate the in vitro cytotoxicity of these extracts against MDA-MB-231 cell line. The apoptotic cellular morphological changes were investigated using inverted and fluorescence microscopes. Results: The results showed that T. nudatum extracts exhibited anticancer activity with a halfmaximal inhibitory concentration (IC50) of 150 μg/mL against MDA-MB-231 cell line. Loss of cell integrity, shrinkage of the cytoplasm, and cell detachment were observed in the extract-treated MDA-MB-231 cells. Conclusion: T. nudatum extracts showed cytotoxic effects against MDA-MB-231 cell line. It may be used as a future medication to cure breast cancer. Original Research Article Barnawi; JPRI, 33(47A): 831-838, 2021; Article no.JPRI.70438 832


INTRODUCTION
Cancer is the second cause of death worldwide. In women, breast, lung, and colorectal are the most common cancer types, they account for an approximate 50% of all new cancer diagnosed in women in 2020. Worldwide, 19.3 million new cancer cases and 9.6 million deaths were estimated in 2020 [1]. Particularly, in Saudi Arabia, 17,522 cases (8,296 males and 9,226 females) were diagnosed as cancer patients, and the cancer death rate was 9,134 across all ages and gender [2]. The rate of cancer death is higher in female than male, it ranges from 0.1-9.1 in females and 0.1-7.3 in males [3]. The Saudi Cancer Registry (SCR) reported that breast cancer as the first encountered cancer among Saudis of all ages (15%) and ranked it first in newly diagnosed female patients (27.4%) [4]. For cancer treatment, surgical removal, chemotherapy, immunotherapy, gene-therapy, and nano-therapy were applied in preclinical and clinical settings. Even though, the conventional chemotherapy still the best and potential approach for cancer therapy, upon the use of chemotherapy, several side effects were reported [5]. To overcome side effects and chemo-resistance post chemotherapy treatments, different protocols using new natural anticancer agents are in need [6][7][8].
Phytochemical examination of several medicinal and wild plants was reported as of possible anticancer compounds. Today, an estimate 60% of drugs used for cancer treatment were produced from natural products [9]. Approximately, 60% of anticancer chemotherapeutic agents were originally isolated from natural sources such as plants and animals resources [9,10].
Traganum nudatum (T. nudatum) is a plant belonging to Chenopodiaceae family, which is a small shrub whose flowering takes place in the autumn. T. nudatum is a native halophytic shrub in arid zones of the Mediterranean basin, it grows at high temperatures and can tolerate increased salinity and aridity [11]. Moreover, T. nudatum can be used in traditional therapy to treat diabetes, diarrhea, rheumatism, otitis, skin diseases, and wound healing due to its antioxidant potentials [12][13][14]. In addition, it shows high antimicrobial activity against many species of bacteria and fungi. For instance, its extracts possess promising antibacterial activities against Bacillus subtilis, Staphylococcus aureus, Enterococcus faecalis, Listeria monocytogenes, Proteus mirabilis [15]. The aim of this study was to investigate the effects of different T. nudatum extracts on cell proliferation, migration, and apoptosis in MDA-MB-231 breast cancer cell line.

Collection of Plant Material
Fresh aerial parts of T. nudatum were collected and identified from Al-Madinah Al-Munawara, Saudi Arabia. At room temperature, the plant was left to air dry partially for 2-3 days. Then the plant was chopped and grounded by using an electrical grinder until it obtained powder. Each 50g of T. nudatum was extracted by an ethanolwater mixture (70/30 V/V) for 48 h. Then this step was repeated three times. After that, the filtrate was refluxed in (2L) 70 % ethanol at 50 °C using a rotary evaporator for 36 h in a continuous extraction (Soxhlet) apparatus (S.P. Verma -Popular Science Apparatus Workshops PVT LTD-India). Pooled and concentrated ethanol extract was filtered and re-concentrated under reduced vacuum pressure, keeping a constant temperature of less than 50 °C. The extract yield was kept at room temperature for later use.

Assessment of Cell Viability
Cell viability was assessed by an MTT assay [16]. Briefly, MDA-MB-231 and HepG-2 cells were seeded at 5 × 10 4 cells/well in 24-well plates and left to attach overnight. Different T. nudatum extract was applied on cancer cell lines and incubated for 48 h in CO2 incubator. Then, 100 μL MTT solution (5 mg/mL in saline) was added to each well and incubated for 2 h at 37 °C. The culture media were aspirated and then 1mL methanol was added to dissolve the formazan crystals. The optical density (O.D.) value was read at 490 nm using a microplate reader (Thermo Fisher Scientific Inc., Waltham, MA, USA). The cell viability (%) was calculated using the following formula: The relative cell viability (%) = treated group/control group × 100%.
The half-maximal inhibitory concentration (IC50) was calculated using origin software. Data were represented as the averages of three independent experiments.

Lactate Dehydrogenase (LDH) Assay
The cytotoxicity of the extract was also assessed by the lactate dehydrogenase (LDH) release assay on MDA-MB-231 cells [17]. Briefly, MDA-MB-231 cell lines were treated with the different extracts for 48 h. Then, 100 μL of the culture medium was collected and 100 μL of LDH reaction solution (Sigma LDH Cytotoxicity Assay Kit, USA) was mixed for 30 min. The developed color was measured at 490 nm using a microplate reader (Thermo Fisher Scientific Inc., Waltham, MA, USA).  [18]. Then, the cells were observed for nuclear changes (chromatin condensation and nuclear fragmentation) and characteristics of apoptosis under a fluorescence microscope attached to an Axiocam 506 color camera (Zeiss, Germany).

Migration Assay
MDA-MB-231 cells (5 × 10 4 ) of were separately seeded per well in 6-well plates in DMEM medium supplemented with 10% serum, until confluence in a 5% humidified CO2 incubator at 37 °C. A wound was created with a 200 μL pipette tip and the debris was removed by a wash with PBS. The cells were incubated with or without extract at the concentration of 40 µg/mL. The results were the average of three independent experiments. The cells were photographed using a phase contrast microscope (Leica, Germany) at three time points (0, 24 and 48 hours). The area of migration was measured using Image J software and the percentage of wounds recovered was calculated.

Annexin-V and Dead Cell Assay
The assay was determined using Muse™ Annexin-V & Dead Cell (7-AAD) kit (Merck Millipore, USA) as per the instructions of the manufacturer. Briefly, 1 × 10 5 cells/ well/1 mL culture media were seeded in a 6-well plate and incubated at 37 °C for 24 h. The cells were then treated with T. nudatum extracts for 24 h. The concentrations of the extracts used for MDA-MB231 were 250 μg/mL, and 500 μg/mL, respectively. After treatment with T. nudatum extracts, the cells were re-suspended in 1% FBS. One hundred microliter of the cells were transferred to 1 mL micro-centrifuge tubes and 100 μL of Muse™ Annexin-V & Dead Cell reagent was added and mixed with the cells, followed by 20 min incubation at room temperature in the dark. The cells were then analyzed using Muse™ cell analyzer. The assay could identify four types of cells: i) non-apoptotic live cells: Annexin-V (−) and 7-AAD (−), ii) early apoptotic cells: Annexin-V (+) and 7-AAD (−), iii) late apoptotic cells: Annexin-V (+) and 7-AAD (+), and iv) nonapoptotic dead cells: Annexin-V (−) and 7-AAD (+).

Statistical Analysis
The criterion for statistical significance was set at p <0.05 or p <0.01. All data are presented as mean ± SD. T test was used to compare all groups against the control group to show the significant effect of treatment.

IC50 of T. nudatum Extract against HepG-2 and MDA-MB-231 Cell Lines Post 48 h.
Different concentrations of T. nudatum extract ranged from 0 to 500 µg/ml were used to determine the IC50 against HepG-2 and MDA-MB-231 cell lines in vitro post 48 h of treatment. The results showed that the IC50 of T. nudatum extract against HepG-2 was 200 µg/ml and against MDA-MB-231 cell lines was 150 µg/ml (Fig. 1).

Treatment with T. nudatum Extract Increased LDH relEase from HepG-2 and MDA-MB-231 Cells in vitro
HepG-2 and MDA-MB-231 cell lines were incubated with T. nudatum extract to determine its cytotoxic effect through releasing LDH in vitro.
The data showed that compared to the untreated HepG-2 cells, the treatment with T. nudatum extract showed significant increase in the release of LDH post 48 h in vitro. Treatment of MDA-MB-231 cells with T. nudatum extract also showed significant increase in the LDH release post 48 h in vitro (Fig. 2).  (Fig. 3 A and B).

DISCUSSION
To find new anticancer agents safe and with little side effects on vital organs when compared to the current chemotherapeutic agents upon the treatments, several studies have been reported to screen and evaluate the anticancer efficacy of thousands natural components extracted, isolated, and purified from several medicinal plants [19].  [20]. This finding agrees with previous report showed that there is a significant positive correlation between various secondary metabolites content such as phenolics, flavonoids, saponins and anthocyanins with the total antioxidant capacity. These findings agreed with previous study reported the anticarcinogenic activity of plants extracts against tumor cell lines [21]. The present study reported that the treatment of MDA-MB-231 cell line in vitro with IC25 of T. nudatum extract decreased the migration distance ratio when compared to the control untreated cancer cells. The morphological changes in the apoptotic cells were also apparent in the Hoechst 33258 staining, which facilitated the detection of cell death caused by apoptosis. Induction of apoptosis has been considered as a hallmark for the identification of anticancer drugs [23]. One of the noteworthy findings of this study was that T. nudatum plants induced apoptosis. Exposure of MDA-MB-231 cell line to 250 or 500 µg/ml of T. nudatum extract for 48 h. increased the percentages of the necrotic and apoptotic cells (early and late apoptosis) in a concentration dependent manner due the potential biological activities of T. nudatum [20].

CONCLUSION
In summary, this study reported that T. nudatum extract showed anticancer effects against human breast cancer cell lines (MDA-MB-231) in vitro.

DISCLAIMER
The products used for this research are commonly and predominantly use products in our area of research and country. There is absolutely no conflict of interest between the authors and producers of the products because we do not intend to use these products as an avenue for any litigation but for the advancement of knowledge. Also, the research was not funded by the producing company rather it was funded by personal efforts of the authors.

CONSENT
It is not applicable.

ETHICAL APPROVAL
We conducted our research after obtaining proper approval.