Cell Cycle Arrest in Different Cancer Cell Lines “ Liver , Breast , and Colon ” Induce Apoptosis under the Influence of the Chemical Content of Aeluropus lagopoides Leaves Extracts

Natural product especially secondary metabolites that produced by plants under the stressed condition shown to have a different pharmacological impact. Aeluropus lagopoides is one of the typical halophyte plants survivals under stressed conditions. It has been used for wound healing and as a painkiller. The bioactivity and the chemical composition of this plant have poorly investigated. Consequently, chemical components of A. lagopoides leaves were extracted using hexane (nonpolar), ethyl acetate (semi-polar), n-butanol (polar) to extract the most extensive variety of metabolites. The cytotoxicity and anticancer impact of extracted secondary metabolites evaluated against breast (MCF-7), colon (HCT-116), and liver (HepG2) cancer cell lines using SRB test. The mechanism of action verified by observing the appearance of apoptotic bodies using the fluorescent microscope while their antiproliferative impact had been evaluated using flow cytometer. Results revealed that secondary metabolites extracted using hexane and ethyl acetate were having the highest cytotoxicity and thus anticancer activity effect on HepG2 with IC50 (24.29 ± 0.85, 11.22 ± 0.679 μg/mL) respectively. Where apoptotic bodies observed, flow cytometer results exhibited that secondary metabolites can inhibit cell cycle in G0/G1 phase. Accordingly, A. lagopoides hexane and ethyl acetate extracts may consider as a candidate anti-cancer drug.


Introduction
Cancer, the multistage process in different aspects molecular and morphological become the second cause of death around the world [1].In Saudi Arabia over 15.800 people were diagnosed with cancer [2].The production of anticancer drug facing the challenges of cancer cells complications and resistance against the drug.Consequently, the perception of the pathways inside the cancer cell based on the role of the enzymes and their catalysis is the real way to keep cancer cells under control.Secondary metabolites which produced by different plants and organisms to adapt to their environment may have roles as toxins to initiate or to inhibit different pathways inside targeted cells.On the other hand, apoptosis is one of the critical pathways inside cancer and normal cells.So, it is -like other pathways-controls by some enzymes like caspase or cyclin which can be activated by many catalysts.Whatever, natural products are considered as the best candidate anticancer drugs due to their fewer side effects.Consequently, became the primary source of almost 80% of approved drugs from (FAD) [3].Therefore, the global focus was on the isolation of bioactive compounds, identification and measuring their therapeutic potential in cancer prevention and treatment [4].Plant, especially medicinal plants have used as a source of medicine since in early civilization for treating different diseases [5].Plants have given famous names of drugs which used against cancer as anticancer or chemoprevention drugs.Such as taxol, vinca alkaloids, camptothecin and topoisomerase inhibitors [6][7][8].Halophyte plants cultivated under stressed abiotic conditions [9].Subsequently, they are the most candidate plants to develop different molecular, physiological, and morphological mechanisms for their survival [10].Halophyte plants have investigated as sources of nutrition [11], fuel [12], bioremediation [13].Medical halophytes are rich in bioactive secondary metabolites such as antioxidant, polyphenol, flavonoids compound these components showed biological activity as antimicrobial, antiviral, anticancer, anti-inflammatory where they are often nontoxic to normal cell lines [14][15][16].Aeluropus lagopoides, member of halophyte plants from the family Poaceae.It is a perennial grass that tolerated to salinity, grows in the coastal salt marshes [17].It is a productive plant used as a source of nutrition and livestock [18,19].It has been used for wound healing and as a painkilling in the Arabian countries [20].To our knowledge, this is the first report on the GC\MS profiling and anticancer activity of A.lagopoides crud extracts against cancer cell lines.The current study is aimed to investigate the ability of A. lagopoides non-polar, semi-polar and polar leaves crude extracts to activate different necessary mechanisms to prevent cancer cell proliferation or to induce cancer cells to apoptosis.

Results: 2.1. Cytotoxicity
The prepared crude extracts tested against different cancer cell line MCF-7, HCT116, and HepG2.Results revealed that hexane and ethyl acetate extracts present a significant impact comparing to n-butanol extracts (

Fluorescence Microscopic Analysis of Cell Viability and Apoptosis
The results obtained from the fluorescence microscope revealed that hexane and ethyl acetate extracts have a high ability to induce apoptosis.(Figure .2). Cells exhibited different apoptotic morphological characteristics such as membrane blebbes, chromatin condensation in addition to apoptotic bodies formation.

Cell cycle analysis
In order to identify which phase in the cell cycle was affected by A.lagopoides hexane and ethyl acetate crude extracts, the current study used flow cytometer.Results revealed that the hexane and ethyl acetate extracts have similar impact mechanism on the cancer cell types.Thus, the result manifests that the impact of the hexane and ethyl acetate extracts showed in the G0-G1 phase (Table 2.) (Fig. 3-5).

Qualitative phytochemical analysis
The quantitative phytochemical analysis of A.lagopoides leaves hexane, ethyl acetate, and butanol extracts show the presence of a different chemical compound (Table 3).

Discussion
Control of biological processes within the cell such as cell division or transcription-translation of protein or apoptosis requires activation of many biological pathways performed by a complicated enzyme system, which requires the presence of many cofactors and coenzymes [21].For this reason, predicting the activation of pathways via pure compound may not be accurate; hence the use of the crude extract may enhance synergistically impact between secondary metabolites.Consequently, crude extract had been chosen as a bio-pathways inducer in the current study to induce apoptosis which considered as the most likely pathway for the cancer therapy strategy [22].For that, our study focused on the isolation of active ingredients and secondary metabolites from Aeluropus lagopoides plant leaves.To determine the cytotoxic, apoptotic and antiproliferative active chemical groups the mechanism of action had investigated from different points of view.Consequently, extracts analyzed and connected to their observed effect.Our results showed that hexane and ethyl acetate extracts have the highest cytotoxic activity compared to polar extracts (Table1) indicating that non-polar components of A. lagopoides tend to activate one or more antiproliferative pathways.However, this is directly related to the phase in which the antiproliferative achieved because each phase has a different definition for which mechanism that has been activated by excluding inactive mechanisms.Results revealed that hexane and ethyl acetate extract at equipotent concentrations arrested the cell cycle in G0-G1 phase with high value almost 65-78 % in all cancer cell lines.That indicates that all extracts ingredients are not clastogenic agents because cell cycle will arrest at G2 after exposure to clastogenic agents to repair DNA damages [23].Depending on that, cells arrested in the cell cycle should choose to activate one of the suggested pathways like apoptosis, necrosis or differentiation.Whereas cancer cells have lost their ability to return to differentiation, thus they must go to apoptosis or necrosis.Hence, to suggest the mechanism of action of different extracts components, the cellular morphology changes had been screened by fluorescent dyes mix (Ethidium Bromide: Acridine Orange) to distinguish apoptotic symptoms from necrotic signs (Figure2).In this point of view, both extracts look to induce apoptotic that may activate via cyclin E or D, R point or one of the chick points that present in G0-G1 phase [24,25].On the other hand, the qualitative chemical analysis (Table 3) indicates that both hexane and ethyl acetate extracts contain hydrophobic bioactive compounds such as phytosteroid, terpenoids, cardioglycoside and fixed oil, which had confirmed their antiproliferative activity in previous studies [8,26].In the light of affording mentioned and observed results; the suggested mechanism of action of hydrophobic molecules extracted from A. lagopoides is related to their capability to flow through the phospholipid bilayer membrane of the cell easily, this changes the internal cellular homeostasis, which leads to arrest cell division.However, the interesting point here is all different pathways will induce -in the end-apoptosis which is the preferred mechanism to eliminate cancer cells.

Extraction and crude extracts preparation:
The fresh leaves of Aeluropus lagopoides collected from Al-Hridha beach at (17.7644488,41.9261616),Asser region, Saudi Arabia on 17 July 2017.For the crude extract preparation, 100 grams of fresh leaves were washed with distilled water and ground via grinder with 500 ml of aqueous ethanol 80%.Then immersed in 1L of aqueous ethanol 80% and left for seven days with stirring at room temperature (18-24 0 C).The ethanol extract was filtered using filter paper and concentrated to dryness under reduced pressure using a rotary evaporator at 37 0 c (Ikia -Germany).The concentrated crude extract weight 10g, and the extraction yield (10%).The crude extract was then reconstituted in 400 ml of distilled water and extracted with different solvents according to the polarity (hexane, ethyl acetate, and n-butanol) non-polar, semipolar and polar respectively using the liquid-liquid extraction method.After that, the solvent phase was separated and reevaporated using rotary evaporator.Then, left for complete evaporation of the solvent at room temperature (20-26 0 C) for five days.Hexane crud extract weight 1.3721, and the extraction yield (10% from the ethanol yield), ethyl acetate 0.9816, and the extraction yield (9.8% from the ethanol yield), and n-butanol 3.723 g, and the extraction yield (30% of the ethanol yield) respectively.Further, 0.01 gram of each crude extracted diluted in 1 ml of (DMSO) dimethyl sulfoxide as a stock solution for bioactivity assay.At 4 0 C the crude extracts were stored for further studies.

Cytotoxicity activity of A.lagopoides crude extracts
The cytotoxicity and anticancer activities of prepared A.lagopoides leaves crude extracts were tested against Human breast (MCF-7), Human colon (HCT 116), and Human liver (HepG-2) cancer cell lines using SulphoRhodamine-B (SRB) assay described by Skehan et al. [27].Different cancer cell lines exposed to a range of concentrations (0.01 to 100 µg/ml) of hexane, ethyl acetate, and nbutanol crude extracts then incubated in 5% CO2 humidified incubator at 37 °C for 72 h.Doxorubicin used as a positive control.Treated cells were fixed with TCA (10%) for 1h at 4°C.Subsequently, to remove TCA, cells were washed with water many times, and then 0.4% SRB

Detection activity signals of apoptosis.
For apoptotic bodies detection, treated cells were washed using PBS washing buffer twice and then collected using 0.25% trypsin-EDTA.After that, the cells were stained using Ethidium bromide (EtB) and Acridine Orange (AO) 1:1 concentration cells were transferred to slide.Stained apoptotic bodies were detected and photographed under a Nikon Fluorescent microscope (Japan).

4.5.
Cell cycle using DNA flow-cytometry Adherent cancer cells exposed to IC50 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 transferred to -20 C until required for analysis.Upon analysis, fixed cells were washed with ice-cold PBS and resuspended in 1 ml of PBS containing 50 µg/ml RNase A and 10 µg/ml propidium iodide (PI).After 20 min incubation at 37 C, cells were analyzed for DNA contents by FACSVantageTM (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) [28].

Statistical Analysis
The IC50 calculation was performed using Sigma Plot version 12.0.

Qualitative phytochemical analysis of the different crude extract
The crude hexane, ethyl acetate, and butanol of A.lagopoides leaves investigated for the qualitative presence of alkaloids, flavonoids, phenols, tannins, terpenoids, Phyto steroid, cardio glycoside, saponin, and fats.The presence represented by (+) and the absence with (-) as described in [29,30].

4.8.
GC\MS for plant extract The different crude extracts from the leaves of A.lagopoides were analyzed using a Perkin Elmer GC-MS (Model Perkin Elmer Clarus 500, USA) equipped with a fused silica capillary column (30 m × 0.25 i.d., film thickness 0.25 µm) coupled with a Perkin Elmer Clarus 600C MS as described in [31].Identification of phytochemical compound conducted using the database of National Institute Standard and Technology (NIST) library.

Recommendation
This study considered as the first report investigates the importance Aeluropus lagopoides plant leaves chemical constituents in the view of the impact on cancer cells and thus opened the door widely by reducing the effort and time required for searching of the most effective and influential compounds found in the proposed chemical groups.On the other hand, it concluded that hydrophobic compounds could change the internal homeostasis of cells, thereby stimulating the cell division arrest which encouraged us to suggest further research on the molecules that regulate current phases like cyclin E and D in addition to p53.

Figure 1 .
Figure 1.Dose-response curve of different extracts of A.lagopoides against solid tumor cell line (a).MCF-7, (b).HCT116, and (c).HepG2.Cells were exposed to the extracts for 72h.Cell viability was determined using SRB-U assay and data are expressed as mean±S.D. (n=3).

Figure 3 .
Figure 3.Effect of A.lagopoides extracts on the cell cycle distribution of HCT-116 cells.Cells were exposed to Hexane extract (B); Ethyl acetate extract (C) for 24 h and compared with cell control (A).Cell cycle distribution was determined using DNA cytometry analysis and different cell phases were plotted (D) as a percent of total events (n = 3).

Figure 4 .
Figure 4. Effect of of A.lagopoides extracts on the cell cycle distribution of MCF-7 cells.Cells were exposed to Hexane extract (B); Ethyl acetate (C) for 24 h and compared with cell control (A).Cell cycle distribution was determined using DNA cytometry analysis and different cell phases were plotted (C) as a percent of total events (n = 3).

Figure 5 .
Figure 5.Effect of A.lagopoides extracts on the cell cycle distribution of HepG2 cells.Cells were exposed to Hexane extract (B); Ethyl acetate (C) for 24 h and compared with cell control (A).Cell cycle distribution was determined using DNA cytometry analysis and different cell phases were plotted (C) as a percent of total events (n = 3).

Figure 6 .Figure 7 .Figure 8 .
Figure 6.Typical gas chromatograph of the chemical constituents of hexane extract from the leaves of A.lagopoides

Table 2 .
Effect of Hexane and ethyl acetate extracts of A.lagopoides on the cell cycle distribution of three tumor cell lines for 24 h and compared with control cells.

Table 3 .
Qualitative phytochemical analysis of A.lagopoides leaves different crude extract GC\MS profiling for the different crude extracts indicate the presence of different molecules belonging to a different chemical group (Table 4)

Table 4 .
GC/MS profiling for the chemical composition of different crude extracts from the leaves of A.lagopoides

Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 18 September 2018 doi:10.20944/preprints201809.0347.v1 solution
was used to stain cells in a dark place for 10 min.Stained cells washed with 1% glacial acetic acid.Finally, to dissolve SRB-stained cells, Tris-HCl was used.After drying overnight, the color intensity of remained cells was measured at 540nm by Elisa.