Anticancer property of hexane extract of Suaeda fruticose plant leaves against different cancer cell lines

Purpose: To evaluate the bioactivity of hexane extract of S. fruticosa leaves against the cancer cell lines HepG2, MCF-7, and HCT-116, and to determine the chemical composition-function relationship. Methods: Using the liquid-liquid extraction method, the nonpolarL constituent compounds were isolated from the leaves. The cytotoxicity of the hexane extract was evaluated using an SRB assay. Mechanism of action was verified by observing the appearance of apoptotic bodies using fluorescence microscopy, while anti-proliferative activity was assayed via flow cytometry. Results: The results revealed that secondary metabolites in the hexane extract demonstrated the highest cytotoxicity, and thus anticancer activity, against HCT-116 cells, with an IC50 of 17.15 ± 0.78 mg/mL. The presence of apoptotic bodies indicate an ability to induce apoptosis. Flow cytometry results suggest that the secondary metabolites stalled the cell cycle at the G0/G1 phase. Conclusion: The results indicate that S. fruticosa hexane extract may be considered a potential new source of the anti-cancer compound, momilactone B.


INTRODUCTION
Cancer is an invasive disease, with global impacts in both developed and developing countries; 23.6 million new cases are expected by 2030 [1]. The cancer cell engages in complicated pathways to ensure its survival inside the body and resistance to different therapies [2]. Understanding these pathways can lead to innovations in the activation of appropriate mechanisms to control cancer cell proliferation and induce programmed cell death [3]. Apoptosis is cited as a favourable pathway and informs many strategies for the development of novel cancer therapies [4]. However, their success is currently hampered by unpleasant side effects.
Natural products have recently received increased attention among those searching for new anticancer therapeutics [5,6]. Plant-derived therapies have had significant success against different cancer types both in vivo and in vitro through the induction of apoptosis [7,8]. Plants produce bioactive compounds known as secondary metabolites that enable them to survive in and adapt to different habitats. Halophyte plants are able to manage stress conditions using antioxidant system mechanisms [9]. Medicinal halophytes are rich in bioactive secondary metabolites such as antioxidants, polyphenols, and flavonoids; these compounds have shown antimicrobial, antiviral, anticancer, and anti-inflammatory activities while remaining nontoxic to normal cells [10][11].
Suaeda fruticosa, a member of the Chenopodiaceae family, is a halophytic medicinal plant and is highly salt-tolerant. Seeds and leaves of the plant have been classified as safe for human consumption or forage, and are used as a phytoremediation tool [12,13]. S. fruticosa is known for being rich in a bioactive compound; a recently isolated polysaccharide from the plant demonstrated antioxidant, anti-filamentary, antinociceptive, hypoglycaemic and antihyperlipidaemic properties in in vitro and ex vivo assays [14,15]. The shoots and leaves of the plant are rich in phenols, flavonoids, tannins, alkaloids, saponins, proanthocyanins, and carotenes, indicating an impressive pharmacological spectrum as compared to other halophytes in the same family, such as Salsola kali [16].
Juice and decoction from the S. fruticosa Leaf have been used to treat fever, flu, skin disease, rheumatism, and helminthiasis livestock diseases [17,18]. Different extracts of the shoots have also been tested against different cancer cell lines, with the most active extract being dichloromethane against colon carcinoma cell lines DLD-1 and HT-29, with IC 50 values of 10 ± 1 and 12 ± 14 μg/mL, respectively. Conversely, root methanolic extract showed less toxicity against human lung carcinoma (LU-1) and hormone-dependent prostate carcinoma (LnCaP), with an IC 50 ≤ 50 μg/mL [16,21]. Despite this plant's richness in bioactive compounds, no studies have been conducted that explored the bioactivity of the plant's leaves against a range of cancer cell lines. The current study aims to investigate the ability of the nonpolar crude extracts from S. fruticosa leaves to activate different mechanisms to prevent cancer cell proliferation or induce cancer cell apoptosis; it will also conduct LC\MS-MS profiling of each of the different extracts.

EXPERIMENTAL Cell lines, chemicals, and biochemicals
Ethanol, methanol, and SRB stain were purchased from Sigma Chemical Co. (St. Louis, MO, USA). All other chemicals were obtained from Gibco/Life Technologies (Carlsbad, CA, USA) unless otherwise indicated. Cell culture vessels were obtained from Nunc A/S (Roskilde, Denmark). Human colon (HCT 116), liver (HepG-2) and breast (MCF-7) cancer cell lines were acquired from Vacsera (Giza, Egypt). Cells were maintained in RPMI 1640 cell culture medium supplemented with 1 mM sodium pyruvate, 2 mM L. glutamine, 100 units/mL penicillin-streptomycin, and 10 % fetal bovine serum. Incubation was in a humidified environment at 5 % CO 2 and 37 ºC.

Plant collection and crude extract preparation
Fresh leaves from S. fruticosa plant were collected from Al-Birk beach located in Geographic coordinates (18 o 12'44.8"N 41 o 32'09.7"E) in the Asser region of Saudi Arabia on 21 July 2017 Plant authentication was carried out by Zouhair Barnoumy, and a voucher specimen was kept in King Khalid University herbarium (voucher no. kku-2017-2526). For crude extract preparation, 400 g of fresh leaves were washed with distilled water and ground with a grinder, then immersed in 300 mL of hexane and left with stirring at room temperature (18 -24 o C) for seven days. The extract was filtered using filter paper and concentrated to dryness under reduced pressure using a rotary evaporator (Ika, Germany) at 40 ºC.
Hexane crude extract was 2.38 g. Following evaporation, 0.01 g of crude extract was diluted in 1 mL of dimethyl sulfoxide (DMSO) to provide a stock solution for bioactivity assay. The crude extract was stored at 4 ºC until used.

Evaluation of cytotoxicity activity of S. fruticosa crude extracts
The cytotoxicity and anticancer properties of prepared S. fruticosa leaves hexane crude extracts were tested against human breast (MCF-7), colon (HCT 116), and liver (HepG-2) cancer cell lines using a SulfoRhodamine B (SRB) assay as described by Skehan et al [19]. Briefly, the different cancer cell lines were exposed to a range of concentrations (0.01 to 100 µg/mL) of hexane crude extract and incubated in a humidified incubator aerated with 5 % CO 2 at 37 °C for 72 h. Doxorubicin was used as a positive control. Treated cells were fixed with TCA (10 %) for 1 h at 4°C. Cells were washed with water several times to remove the TCA, and then a 0.4 % SRB solution was used to stain cells in the dark for 10 min. Stained cells were then washed with 1 % glacial acetic acid. Finally, Tris-HCl was used to dissolve the SRBstained cells. After drying overnight, the colour intensity of the remaining cells was measured at a wavelength of 540 nm with an ELISA plate reader.

Determination of apoptosis activity
For apoptotic body detection, treated cells were washed twice with PBS and then collected using 0.25 % trypsin-EDTA. Cells were then stained using ethidium bromide (EtBr) and Acridine Orange (AO) at a 1:1 concentration and transferred to slides. Stained apoptotic bodies were detected and photographed with a Nikon Fluorescent microscope (Japan).

Assessment of cell cycle distribution using DNA flow-cytometry
Adherent cancer cells were exposed to IC 50 equivalent concentrations of extract solutions for 48 h. Cells were then suspended using 0.25 % trypsin-EDTA, washed with ice-cold PBS, and re-suspended in 0.5 mL of PBS. Cells were then fixed in 70 %, ice-cold ethanol at 4 °C for 1 h before being stored at -20 °C until analysis. Upon analysis, fixed cells were washed with icecold PBS and re-suspended in 1 mL of PBS containing 50 µg/mL RNase A and 10 µg/mL propidium iodide (PI). After a 20 min incubation at 37 C, cells were assayed for DNA content by a FACSVantage TM Flow Cytometer (Becton Dickinson Immunocytometry Systems, San Jose, CA). For each sample, 10,000 events were acquired. Cell cycle distribution was calculated using CELLQuest software (Becton Dickinson Immunocytometry Systems, San Jose, CA) [20].

LC-ESI-QTOF-MS/MS analysis
Analysis performed on SCIEX X500R QTOF system includes UPLC-MS/MS (Woodlands Central Indus. Estate. SINGAPORE). The separation was performed using Phenomenex Kinetex 2.6 μm Phenyl-Hexyl 100 A (50 × 4.6 mm). The mobile phase consists of, phase A (10 mM ammonium formate in water) and phase B (0.05 % formic acid in methanol). A variable gradient flow rate was used, which is described in (table 1). A Positive Non-targeted mode was used for the analytes (Table 1).  Table  3).

Cytotoxicity
Prepared crude extract was tested against the cancer cell lines MCF-7, HCT-116, and HepG2. Results revealed that hexane significant effects (Table 2, Figure 1). Hexane extract IC 50 value was the highest at 17.15 ± 0.78 µg/mL against the HCT-116 cell line. These results were confirmed by cell viability curves (Figures 1a, 1b  and, 1c) respectively.

Fluorescence microscopy analysis of cell viability and apoptosis
Results obtained from fluorescence microscopy showed that hexane extract was able to induce apoptosis. As shown in Figure 2, general morphological characteristics of apoptotic processes such as chromatin condensation, membrane blebbing, and apoptotic bodies were observed. The presence of a necrotic cell death pathway was also detected (Figure 2).

Cell cycle
To identify which phase in the cell cycle was affected by S. fruticosa hexane extract we used flow cytometry for tracking DNA through the cell cycle. Results revealed that the hexane extract exerted similar activity on the different cancer cell types. The hexane extract was able to arrest the cell cycle at the G0-G1 phase (Table 3 and Figure 3).

LC\MS-MS
The non-polar secondary metabolites of the plant were isolated by solid extraction with hexane then qualitatively analysed using LC-ESI-TOF-MS/MS. The result of the analysis revealed the presence of monoterpenes, diterpenes and phenolic compounds beside some fatty alcohols, fatty acids, steroidal and miscellaneous compounds. Table 4, Table 5 and Table 6 show

DISCUSSION
All natural product working with crude extract are suffering from the complexity of the determination of the structure-function relationship of the extracts, generally due to the numerous number of the particles in the extract. Moreover, not all particles have a function or even help to presents the estimated function. Quite the contrary, some ingredients may be inhibiting the work of the active ingredients. On the other hand, the positive view reveals the presence of the cofactors or coenzymes of the secondary metabolites, which may can induce one or more pathways like apoptosis to prevent cancer cell proliferation. Current study took upon itself the clearance of this complicity not only to determine the activity of the hexane crude extract but also to analyse the chemical composition of the extract in order to reduce or eliminate the probabilities of the inactive ingredients by do a correlation between the impact and the composition [7,8].
S. fruticosa is an edible, medicinal, halophytic plant rich in bioactive compounds that used in folk medicine for the treatment of many different diseases [16,21].
In this study, we evaluated the anticancer activity of hexane leaf extract of S. fruticosa against three different cancer cell lines (HCT-116, MCF-7, and HepG2) in a concentration-dependent manner. Recent studies reported that hexane, dichloromethane, water, and methanol extracts of different parts of the S. fruticosa plant can inhibit the growth of certain cancer cell lines in vitro [16,21]. Among these, the dichloromethane and methanol extracts had the most significant effects on colon cancer cell line DLD-1 (IC 50 10 ± 1 and 15 ± 1 µg/mL, respectively). The current study used hexane extract which is more nonpolar than methanol and dichloromethane but yielded almost similar results (17.15 ± 0.78 µg/mL, Table 1).
The effects of the extract on MCF-7 and HepG2 cancer cell lines were less impressive. Interestingly, the extracts have been tested against normal human skin fibroblast cell lines and no significant toxicity was found [16]. This harmony between the previous results and what has been obtained in this study indicates that plant S. fruticosa even different extracts have a good potential to have anti colon cancer prodrugs. Furthermore, the ability of plant chemical composition to induce apoptosis in colon cancer cells (HCT116) (Figure 1) as demonstrated by the morphological cell changes more than other cell line (liver and breast) types, may open a promising specific pathway for colon cancer different types lead to the development of new anticancer therapeutics. These conclusions encouraged us to go through the cell cycle in hope to determine the phase that hexane extract shows its impact. Current study indicated that hexane extract was able to arrest cell cycles at the G0-G1 phase in all the three cancer cell lines ( Table 2).
Many existing anticancer drugs also work by arresting the cell cycle, either at the G0-G1 or G2-M transitions [24]. Halting the cell at G0-G1 may present an avenue for the induction of apoptosis by activating cell cycle exit at this checkpoint via cyclins D or E or other restriction signals [24].
On the other hand, our LC\MS-MS results confirmed the presence of momilactone B compound (Table 5), which had been reported to have anticancer activity (potential chemotherapy) against and promising inhibition in pre-clinical models, not only that but also can inhibit cell cycle in G1 which explain the prevention of cells to enter S phase. [25]. Furthermore, recent studies revealed the ability of momilactone to inhibit the growth of Breast cancer cell line through STAT5b and a caspase-3 dependent pathway [25].

CONCLUSION
The findings of this study reveal that S. fruticosa plant is a new natural source of a potential chemotherapeutic agent, namely, the momilactone B compound. The compound is a non-toxic natural primary source for manufacturing several drugs such as anticancer, antiviral, antifungal, antioxidative, and anticoagulant.