Monitoring ZEO apoptotic potential in 2D and 3D cell cultures and associated spectroscopic evidence on mode of interaction with DNA

Recognizing new anticancer compounds to improve Breast cancer treatment seems crucial. Essential oil of Zataria Multiflora (ZEO) is a secondary metabolite with some biological properties, yet underlying cellular and molecular anticancer properties of ZEO is unclear. GC/MS analysis revealed that carvacrol is the major ingredient of the essential oil. ZEO increasingly suppressed viability in MDA-MB-231, MCF-7 and T47D Breast cancer cells while nontoxic to L929 normal cells in monolayer cell cultures (2D), whereas MDA-MB-231 multicellular spheroids (3D) were more resistant to inhibition. ZEO significantly induced cell apoptosis confirmed by fluorescent staining, flow cytometry analysis and DNA fragmentation in MDA-MB-231 2D and 3D cell cultures. ZEO increased ROS generation and subsequent loss of ΔΨm, caspase 3 activation and DNA damage which consequently caused G1 and G2/M cell cycle arrest in a dose- and time-dependent manner in 2D. S phase arrest occurred in cell spheroids therefore ZEO possible DNA interaction with gDNA was investigated and revealed ZEO binds DNA via intercalation. Altogether, these data corroborate anticancer properties of ZEO and suggest that cell culture format (2D monolayer vs. 3D spheroid) plays a critical role in drug response and provides new insights into the mechanisms underlying ZEO cytotoxicity effect on Breast cancer cells.

Breast cancer is a common type of malignancy among women and has a complex and heterogeneous nature. Due to its various underlying cellular and molecular characteristics this disease is indeed a collection of diseases with variable clinical behaviors and outcomes that makes its treatment quite challengeable with existing therapeutic procedures [1][2][3] . Currently, chemotherapy is the dominant and most effective cancer treatment. The main goal of chemotherapy is to impose death upon cancer cells via inducing apoptosis without triggering inflammatory response and with minimal side effect on normal cells. Although chemotherapy has succeeded to some degrees and responded well in certain types of tumors but in many cases it is unable to remove all cancer cells and may cause collateral damage to normal cells and tissues. Some synthetic compounds can prevent, suppress or even reverse the progression of cancer at the cost of adversely affecting rapidly dividing normal cells, increasing drug resistance and high treatment costs. Hence such drawbacks are necessary to be addressed to raise the success rate of chemotherapy treatment [4][5][6] . In vitro and in vivo studies on phytochemicals, a large group of plant products classified as alkaloids, saponins, glycosides, triterpenes and polyphenols have shown very promising anticancer properties 7 . Vinblastine, Vincristine and Taxol (tubulin-binding agent), teniposide and etoposide (topoisomerase II inhibitor) irinotecan and topotecan (topoisomerase I inhibitor), are traditional examples of plant-derived compounds with diverse applications in cancer therapeutics [6][7][8][9] . Zataria Multiflora Boiss also called Avishan-e-Shirazi in Iran is a thyme-like and aromatic traditional medicinal plant that belongs to the Lamiacea family and grows extensively in southern and central parts of Iran, Afghanistan and Pakistan. Zataria Multiflora essential oil (ZEO) is a hydrophobic concentrate with specific aroma present in the plant as secondary metabolite and due to its hydrophobic nature can facilely cross the membrane to reach inside the cell. Modern pharmacological studies indicate that EO extracted from Z. Multiflora, due its phenolic compounds possesses a wide range of biological properties including innate immune system stimulant 10 , antioxidant, antibacterial [11][12][13] and antifungal activities 14,15 . To the best of our knowledge, only a few reports have been published regarding the cytotoxic effects of ZEO.

MDA-MB-231 cell spheroids generation. Tumor cells are generally less sensitive to chemotherapeutics
in solid tumor models such as cell spheroids than in monolayer cell cultures. Furthermore spheroid's diameter is an important parameter in characterizing drug efficiency, for instance, small spheroids with diameters up to 200 μm are sufficient to reflect in vivo-like, cell-cell and cell-matrix interactions and are frequently used for anticancer drug screening 17 . However, spheroids with diameters of approximately 500 μm have been suggested to be a better choice than small spheroids, because they mimic the pathophysiological conditions of solid tumors, such as the specific hypoxic areas in the center and proliferation gradients more properly 18,19 . For this reason, homogenous multicellular spheroids from MDA-MB-231 cells were generated by using Hanging Drop and subsequent maintenance and increase of spheroids size was achieved by using Liquid Overlay method 20 . In Hanging Drop method spheroids form under the effect of gravity as soft solid structures that can resist small physical impacts during handling. By using this method spheroids were generated in only 2 days and with subsequent combination with liquid overlay, after 4 days, ready to use cell spheroids with uniform size, shape, high cell density and minimum cell damage were generated. This is a simple, fast and low-cost method to generate multiple uniform spheroids with high viability. Dimensions of spheroids and viability of cells were controlled by the density of  initial cells and time of culture. After 4 days, the growth of spheroids was monitored by measuring the volume and surface area of at least 100 spheroids by Image J software, and the average diameter of spheroids were calculated 555 ± 29.71 μm ( Fig. 2A). Proliferation gradient of spheroids around 550 μm in diameter were also observable  after this period: proliferative cells were located in the outer layer and quiescent cells were located near to the center where oxygen and glucose are scarce and cell death and necrosis often occurs( Fig. 2B) 19,21 .    (Fig. 4A). After treatment of spheroids for 24 h with 118 μg/ml (IC50), the percentage of early apoptotic cells were 22.1%, higher compared to control group. In addition to apoptosis specific morphological changes, a representative hallmark of apoptosis is DNA Degradation into oligonucleosome-size fragments in a distinctive event in apoptosis 22 . In the present study, this fragmentation event was observed qualitatively by resolving cellular DNA on agarose gel and quantitatively by TUNEL assay. In TUNEL assay, after treatment of MDA-MB-231 cells with IC50 concentration of ZEO for 4 h, DNA fragmentation was quantified by flow cytometry and Compared to untreated cells up to 45.39% apoptosis was observed (Fig. 4C). DNA fragmentation analysis on agarose gel which is the gold standard to confirm the induction of apoptosis also showed the apoptosis characteristic ladder pattern in MDA-MB-231 cells. It is worth to specify that DNA laddering pattern was more evident in three doses 7, 15 and 30 μg/ml (Fig. 4B).

ZEO induces intracellular ROS generation, loss of ΔΨm and increases caspase-3 activity in MDA-MB-231 cells.
There is increasing evidence that ROS induced by apoptotic stimuli leads to mitochondrial dysfunction and we hypothesized that ZEO may have caused apoptosis in MDA-MB-231 cells via increasing ROS production. It has been shown that ZEO usually causes oxidative stress therefore increase of ROS levels in MDA-MB-231 cells was measured by the ROS-specific fluorescent dye (DCFH-DA). The ROS-specific fluorescent staining assay confirmed that ROS level rapidly increases in MDA-MB-231 cells treated with IC50 concentration of ZEO after 12 h (34.44%) (Fig. 5A). ROS augmentation may oxidize mitochondrial pores and therefore disturb MMP (mitochondrial membrane potential) and cause apoptosis. MMP is a major event in mitochondrial apoptotic pathway 23 . Rhodamine 123, a fluorescent dye with the ability to be selectively absorbed by mitochondria, was used to evaluate the effect of different concentrations of ZEO on MMP in MDA-MB-231 cells. Following ROS damage to the cell, the membrane permeability of mitochondria increases and the MMP decreases 24 . A significant reduction of MMP in a dose-dependent manner after treatment with 3-30 μg/ml of ZEO was observed which indicates that the mitochondrial apoptosis pathway may have involved in apoptosis-induced by ZEO (Fig. 5B1,B2). Caspase-3, a key effector in the process of apoptotic cell death 24 is a cysteine proteases which mediates apoptosis by proteolysis of specific substrates, that are considered the primary executioners of apoptosis 25 . To analyze whether ZEO triggers apoptosis in a caspase dependent manner, the activity of caspase-3 was measured.
Caspase-3 activation capability in MDA-MB-231 cells was evaluated after incubation of cells with 3, 7, 15 and 30 μg/ml of ZEO for 4 h. The activity of caspase3 increased nearly 4.3, 5.6, 6.15 and 6.2 times respectively compared to the negative control (Fig. 5C).

ZEO treatment leads to DNA damage (DNA oxidation and strand break) in MDA-MB-231 cell.
Increased levels of ROS in ZEO treated cells may cause DNA damage. Hence to investigate the effect of ZEO on DNA oxidation, production of 8-oxoguanine (8-oxo) as the most common oxidative base lesion was evaluated 26 . As shown in (Fig. 6B) (Fig. 6A1).
These results indicate that the apoptosis induced by ZEO is tightly correlated with DNA oxidation and strand  (Fig. 7). Additionally, our results indicated that after 24 h treatment by 3, 7 and 15 μg/ml concentrations of ZEO, cells arrest 30%, 25%, and 34% in G2/M phase of cell cycle respectively. While only 10% of untreated controls were in G2/M phase (Fig. 7). On the other hand, the treatment of MDA-MB-231 cell spheroids for 24 h with IC50 concentration of ZEO resulted in a marked accumulation of cells in the sub-G1 phase, suggesting a significant apoptotic, but not necrotic, cell death. It is worth notifying that, the ZEO induced S phase cell cycle arrest in MDA-MB-231 cell spheroids is intrinsically different in nature compare to 2D monolayer (Fig. 7).

UV-Visible, fluorescence and circular dichroism spectroscopy shows ZEO interacts with DNA via intercalation.
DNA is the primary intracellular target for a wide range of anticancer agents and DNA-drug interaction determines the cells fate by altering transcription and/or replication inhibition [27][28][29] . After DNA extraction, its concentration and quality was evaluated. Samples with absorbance ratio (A260/A280) ≥1.8 were accepted as pure genomic DNA. Due to chromophoric groups in purine (adenine and guanine) and pyrimidine (cytosine and thymine) moieties responsible for the electronic transitions DNA molecule has a maximum absorption at 260 nm 28 . To verify extracted DNA integrity electrophoresis was perform on 0.8% agarose gel to ensure high molecular weight DNA (Fig. 8A1,A2). UV-Vis absorption spectroscopy is an effective, simple and most common method to investigate the interaction of small molecules with genomic DNA 28,30 . In this method in order to establish whether there is any interaction between DNA and the drug, DNA spectrum intensity modifications and maximum absorption shifting are inspected from when the ligand is free in solution to when the ligand is bound to DNA. As shown in Fluorescence spectroscopy is a useful technique to study the interaction of a chromophore with other molecules. Since DNA molecule per se is not a chromophore and no fluorescence was observed for the ZEO. Therefore, Ethidium Bromide (EB) was added to DNA solution as an intercalative probe and enhanced DNA fluorescence dramatically at 600 nm 31 . Hence, whenever a second ligand, which competes for the DNA binding sites, is added, it substitutes the EB and fluorescence quenching occurs. The emission spectra of the EB-DNA solutions in the presence of the increasing amounts of ZEO have shown in Fig. 7C which clearly exhibits a decrease in the fluorescence intensity at 600 nm. Quenching mechanism and binding parameters in competitive binding experiment at the presence of ZEO and EB was identified according to Stern-Volmer equation as described previously 32,33 . As it is illustrated in Fig. 7C b&c Stern-Volmer plot is linear and binding parameters are n = 1.1 and K a = 0.37 × 10 4 which indicate that quenching mechanism is dynamic and binding mode is intercalation respectively. Circular dichroism (CD) is a technique to assess conformational changes, binding mode and interaction affinity of small molecules with biomolecules. Inspecting modifications of CD signals at 200-320 nm is informative to detect and follow DNA conformational changes, damage and/or cleavage upon interaction with drugs 34 . Rat hepatocyte genomicDNA displayed the typical spectrum of B-form DNA with positive band at 275 nm and a negative band at 245 nm due to base stacking and right-handed helicity respectively 35 . The CD spectra of DNA in the presence of the ZEO are shown in (Fig. 7D). In the presence of ZEO, DNA CD spectra positive and negative peak intensities increased which indicates the stabilization of the right-handed BDNA.

Discussion
Despite all advances in Breast cancer treatment, due to limited efficacy of chemotherapeutics the disease incidence is growing and efforts to develop novel and effective treatments has been fruitless 4 . Currently, natural compounds and herbal medicines in particular, has become more popular than synthetic drugs and provided an alternative treatment option for patients. Relentless proliferation of cancer cells, place the drugs with the ability of inhibiting DNA synthesis among the best choices to treat cancer 36 . A wide range of anticancer drugs aim DNA as the primary intracellular target and regulate cell functions by modulating transcription or replication inhibition in a way which leads to programmed death of cancer cells [37][38][39] . The anti-proliferative effect of ZEO has been previously reported in cancer cells 40 , however underlying cellular and molecular mechanism for antitumor properties of ZEO has not been elucidated. Therefore in the present study, we initially have evaluated the antiproliferative and anti-apoptotic effects of ZEO, a polyphenolic Essential Oil isolated from Zataria Multiflora in 2D and 3D cell cultures, followed by its possible apoptosis mechanism in MDA-MB-231 cells and Interaction properties with DNA.
According to GC/MS analysis, the major components in hydrodistilled ZEO are oxygenated monoterpenes, monoterpene and sesquiterpene hydrocarbons, oxygenated sesquiterpene and a non-phenolic portion 41 . Although the essential oil's main components remain the same between plants from different geographical regions, but their relative quantities differ in various regions. Some reported carvacrol as the dominant compound, while others have reported thymol "an isomer of carvacrol" as the main constituent of the fresh plant. However carvacrol is the primary constituent in the dried plant. It is clear that geographical variation, cultivar differences, stage of plant growth, preparation process and other factors may influence oil composition both quantitatively and qualitatively 15 . According to our findings, the essential oil of Zataria Multiflora contains substantial amounts of carvacrol, which is renowned for its anti-oxidant, anti-microbial and anti-fungal properties and also has been reported to possess cytotoxic effects on cancer cells in vitro and in vivo 42,43 .
One of the goals of this study was to validate the cytotoxicity effect of ZEO on Breast cancer cells specifically in multicellular spheroids of MDA-MB-231. Traditionally, ex vivo drug screening is performed on conventional 2D monolayer cell cultures. However 3D cultures such as cell spheroids may be more physiologically relevant. Cells growth in multicellular spheroids similar to what observed in tumors in vivo have lower proliferation rate compared to 2D cell cultures. As such, it is reported that multicellular spheroids are superior models for preclinical drug screening. Multiple methods have been implemented so far to generate tumor spheroids in vitro, however these have not been largely applied, as they require specialized equipment, and complicated protocols 21,44 . In this study we have generated spheroids with high size and volume uniformity and high circularity. 5000 cells per spheroid were produced in short culture time (4 days) by combination of liquid overlay and hanging drop methods. ZEO exhibited inhibition against the examined cancer cells (MDA-MB-231, T47D and MCF-7) in a dose-dependent manner and was significantly less toxic to normal fibroblast cells (L929), indicating a favorable selectivity toward cancer cells. The cytotoxic property of ZEO may be due to the presence of phytochemicals such as polyphenolic compounds in the EO. However, our results indicate that spheroids were more resistant to the toxic effects of the ZEO compared to 2D cultures.
The induction of apoptosis is one of the principle mechanisms by which anti-cancer drugs kill cancer cells. MDA-MB-231 cells treated with ZEO exhibited certain apoptotic cell characteristics such as cell rounding, cell shrinkage and cytoplasmic condensation, as well as presence of sub-G1 phase cell population. Externalization of membrane phosphatidylserine is an indication of programmed cell death which is quite detectable by increased binding of FITC conjugated Annexin V, a calcium dependent phospholipid binding protein. The number of early apoptotic cells increased in a concentration-dependent manner in MDA-MB-21 cells, treated with ZEO. A hallmark of apoptosis is the fragmentation of nuclear DNA that was clearly evident in the cells treated with ZEO. Mitochondria play a crucial role in the apoptosis induced by synthetic and natural anti-cancer agents and many of these agents implement their antitumor activity through ROS-dependent apoptotic signaling. ROS are by products of cellular respiration and play a crucial role in apoptosis 45 . Elevated cellular ROS levels leads to the loss of mitochondrial membrane potential, which causes the release of pro-apoptotic factors and the activation of caspase-3. Caspase-3 activation subsequently leads to DNA breakage, nuclear chromatin condensation, and apoptosis 25,46 . Treatment of MDA-MB-231 cells with ZEO resulted in the mitochondrial dysfunction, accompanied with ROS augmentation and ROS level considerably increased after cells were exposed to ZEO, ZEO-induced loss of mitochondrial membrane potential, activity of Caspase-3 enhanced by different concentration of ZEO, resulted in oxidative stress and free radicals attack to DNA, leaving DNA oxidation and single and double strand breaks, which causes apoptosis 47 .
Uncontrolled cell proliferation is the typical characteristic of cancer and cell cycle arrest another major target for cancer therapy, similarly can contribute to the inhibition of cancer cells proliferation. Cell cycle distribution status is often considered as a key parameter in cell survival, growth and proliferation. Our results indicate that ZEO treatment of monolayer MDA-MB-231 cells can induce G1 and G2/M cell cycle arrest, reducing the number of cells in S phase and induce apoptosis. Whereas ZEO treated spheroids showed S phase arrest in MDA-MB-231 cell spheroids followed by increased accumulation of cells in Sub G1 phase. These data together demonstrated ZEO induces cell cycle arrest in 2D and 3D cell cultures at completely different phases, this is because cells in 3D had a rounder morphology and a wider array of cell-cell and cell-ECM interactions and mimic tumor physiological environment more realistically. Since DNA replication is an essential phase of the cell cycle, many of the cytotoxic agents induce their effect via covalent or noncovalent interaction with DNA. Hence ZEO and rat hepatocyte genomic DNA interaction was explored via spectroscopy. Alterations in DNA UV-Vis spectra at 260 nm such as Hyperchromicity and red shift by adding increasing amounts of ZEO indicated that ZEO has induced conformational changes in DNA by forming DNA-ZEO complexes which causes deformation and instability in DNA structure. ZEO in competitive fluorescence substituted ethidium bromide this shows that ZEO can intercalate in DNA. Additionally observing hyperchromicity at 275 nm positive band and 245 nm negative band of CD spectra also confirmed ZEO intercalates in DNA.
In summary, our findings indicate that ZEO by inducing apoptosis via increasing intracellular ROS level and direct genome targeting executes its antiproliferative effect on MDA-MB-231 cells in both 2D and 3D cell cultures without considerable cytotoxicity against normal fibroblast cells. Furthermore, MDA-MB-231 cell spheroids in 3D cell culture demonstrated more resistance to ZEO cytotoxic effects Compared to 2D monolayer cell culture.

Methods
Plant collection and extract preparation. The leaves and flowers of Zataria Multiflora (Lamiacea) were collected in May month from mountainous areas of Shiraz in Iran. Consequently ZEO was extracted from the air-dried through hydrodistillation using the all-glass Clevenger-type apparatus. The density of ZEO calculated by digital balance (Acculab, Sartorius group, Germany) and the average was reported 996 mg/mL. Therefore, each µL of ZEO approximately equals to 1 mg. Essential oil analysis. GC/MS analyses were executed on Agilent 7890 A gas chromatograph equipped with column HP-5 (30 m length, 0.25 mm i.d., film thickness 0.25 μm) coupled with FID detector. Carrier gas was He with split ratio of 1: 40. Temperature was programmed at 60 °C rising to 240 °C, Injector temperature 240 °C, detector temperature 250 °C and injector volume was 0.1 μl. GC-MS was performed using Agilent 5975C coupled with Agilent 5975C mass spectrometer equipped with a column HP-5MS (see GC), using the same temperature programed and carrier gas as above and ionization source temperature of 280 °C.The constituents of the essential oils were identified by calculation of their retention indices and comparison of their mass spectra with those of the internal reference mass spectra library.
Cell lines and culture. MDA.MB.231 (highly invasive human breast cancer cell line), MCF-7 (human breast adenocarcinoma cell line), T47D (human breast cancer epithelial cell line) and L929 (normal fibroblast cell line) were purchased from National Cell Bank of Iran (Pasteur Institute, Iran). All the cells were grown in RPMI-1640 medium (Gibco) with 10% FBS (Gibco) supplemented with antibiotics (100 U/ml penicillin and 100 μg/ml streptomycin). Cells were maintained at 37 °C under humidified air containing 5% CO2 and were passaged using trypsin/EDTA (Gibco) and phosphate-buffered saline (PBS) solution. Culturing media and conditions used to grow the cells as 3D colonies was the same as monolayer cell culture.
Spheroid formation using hanging drop and liquid overlay methods. Generation of spheroids was carried out using combination of liquid overlay and hanging drop cultivation techniques. singled cell suspension of MDA-MB-231 at the concentration of 1.0 × 10 6 cells/10 ml (for 5000 cells per spheroid) in complete culture medium was deposited in 50 μl drops on the inner side of a petri dish lid then petri dish lid turned upside down and was placed on top of the plate filled with 10 ml sterilized water (to humidify the culture chamber after distribution of the drops). Cells under the force of gravity will be aggregated at the bottom of the hanging drop. Small spheroids were formed on day 3 (after 2 days) and were collected by widened end tips (100 μl pipette tip cut widely 5-6 mm from the end). After the formation of the spheroids, to increase the size of spheroids, they were transferred into 96-well plates, precoated with 50 μl of 0.5% poly-HEMA (sigma) in 95% ethanol and air dried at 37 °C for 3 days prior to use. Then homogenous sized spheroids were added to each well of a round bottom poly-HEMA-coated 96-well plate and supplied with 200 µl fresh medium. The plates were incubated under standard cell culture conditions at 37 °C, 5% CO 2 in humidified incubator.

Measurement of spheroid size and Image Analysis.
Cell spheroids with diameters ranging from 500-600 μm were obtained in about 4 days, the morphology and integrity of MDA-MB-231 cell spheroids were imaged at 5 × and 10× magnitudes using invert microscope (Ax overt 25, Zeiss, Germany). Volume and size of spheroids were analyzed with Image J software. Fluorescent Staining in monolayer and spheroids. Fluorescent staining ethidium bromide (EB)/acridine orange (AO) (Sigma-Aldrich) was performed to assess rates of cellular viability (Live/Dead). Initially cells were seeded in 6 well (monolayer) and 24 well (spheroids) cell culture plates and treated with IC50 concentration of ZEO for 24 h. then cells and spheroids washed with PBS, after which, a solution containing EB/AO was added, the stained cells were immediately visualized and imaged under an fluorescence microscope (Axioskop 2 plus, Ziess, Germany). The differentiation between viable, apoptotic, and necrotic cells is based on the difference between dye permeability into intact cell membrane. Green cells represent viable cells and are stained only with AO, green and orange cells with condensed chromatin represent early and late apoptotic cells and are stained with both AO and EB (with moderate alteration in membrane permeability), finally necrotic cells are orang and stained with EB. Ten photos were taken of randomly selected areas of the stained slides to ensure that the data obtained were representative. DNA fragmentation assay. MDA-MB-231 cells were treated with 7, 15 and 30 μg/ml of ZEO and positive control was treated with 5 μg/ml of etoposide. DNA extraction was performed by standard procedure as described before. After dissolving DNA in TE buffer, it was loaded on a 2% agarose gel and electrophoresis was performed for 2 hours and stained with ethidium bromide for DNA visualization.

Detection of apoptosis by Annexin V-FITC and PI staining in monolayer and
Reactive oxygen species (ROS) assay. ROS production was monitored by flow cytometry using 2′, 7′dichlorodihydrofluorescin diacetate (DCFH 2 -DA) (Sigma-Aldrich). This dye is readily diffuses into cells and is hydrolyzed by intracellular esterase to yield 2′, 7′ dichlorodihydrofluorescin (DCFH 2 ), which is trapped within the cells. Hydrogen peroxide or low molecular weight peroxides produced by the cells oxidizes DCFH 2 to a highly fluorescent compound 2′, 7′-dichlorofluorescein (DCF). Thus, the fluorescence intensity was proportional to the amount of hydrogen peroxide produced by the cells. Briefly, MDA-MB-231 cells (1 × 10 5 cells/well) were treated with IC50 concentration of ZEO for 12 h. Thirty minutes before the end of the experiment cells collected, centrifuged (1200 rpm, 5 min) and the pellet was washed with 1 mL of PBS, then the samples was treated with DCFH 2 -DA (20 μM) and kept in the dark. After 30 min the fluorescence was assessed by comparing two fluorescence excitation/emission 485-495 nm/525-530 nm using a flow-cytometer.

Evaluation of mitochondrial membrane potential (ΔΨm).
To determine the changes in ΔΨm levels, the MDA-MB-231 cells were treated with 0-30 μg/ml concentrations of ZEO for 12 h in the same way as called above and incubated with 400 μl 50 μM Rhodamine 123, at 37 °C for 30 min. The cells were then collected, washed twice with PBS to remove extracellular Rhodamine 123. The fluorescent intensity of the Rhodamine 123 was measured by flow cytometry with excitation and emission at 488 and 525-530 nm, respectively. The mean fluorescence intensity represented the cellular levels of intracellular MMP.
Caspase-3 activity assay. Caspase-3 activity was measured using colorimetric assay kit (BIOMOL International, USA), according to the manufacturer's instructions. Briefly, MDA-MB-231 cells (1 × 10 6 ) were treated with 0-30 μg/ml for 4 h and then each sample was harvested and washed with PBS. The cells were incubated with lysis buffer on ice and the protein concentration of each sample was determined using the Bradford method. Equivalent amounts of proteins for each sample were incubated with the appropriate caspase substrate and after 4 h, the absorbance at 405 nm was measured using a microplate reader. DNA oxidation analysis. Genomic DNA was extracted from MDA-MB-231 cells after 2 h treatment with 3, 7, 15, 30 µg/ml concentrations of ZEO along with untreated control according to standard phenol/chloroform extraction procedure. Same amounts of DNA (5 µg) was then digested with nuclease P1 (sigma N8630) and treated with alkaline phosphatase (NEB M0290S) and subjected to 8-oxo detection Elisa kit (Cayman chemical 589320) in total volume of 50 µl according to manufacturer's protocol.
Comet Assay. DNA strand breaks was measured by the alkaline comet assay, which is able to detect both single and double strand breaks in singled cells preparations 47 . Briefly, MDA-MB-231 cells (3.5 × 10 5 cells/well) were treated with IC50 concentrations of ZEO for 2 h. Subsequently Cells collected, centrifuged (1200 rpm, 5 min) and the pellet was washed with PBS. Frosted microscope slides were dipped into hot 1% normal-melting-point agarose in PBS to the frosted area. The slides were air-dried and stored at room temperature until needed. A 200 μl of cell/agarose suspension (100 μl of cell suspension containing about 10 4 cells was mixed with 100 μl of 1% low-melting-point agarose in PBS) was placed over the first agarose layer under a glass cover and incubated for 15 min in 4 °C to solidify. glass cover were removed, the slides were gently immersed in a cold lysis solution (2.5 M NaCl, 100 mM EDTA, 10 mM Tris-base, pH was adjusted to 10 with NaOH in 4 °C, 1% Triton X-100 added just before use) for 1 h at 4 °C to lyse the cells. The microscope slides were then transferred to the horizontal gel electrophoresis unit filled with fresh, chilled electrophoresis buffer (denaturing buffer) (300 mM NaOH, 1 mM EDTA, pH 13) to a level of about 0.2 cm above the slides and left for 30 min in 4 °C to allow unwinding of DNA before electrophoresis. Electrophoresis was conducted for the next 30 min at 0.6 v/cm. The slides were then drained, placed on a tray and flooded slowly with neutralization buffer (0.4 M Tris, pH 7.5), for 5 min to remove alkali. Each slide were then stained with 100 μl 1X cyber green and kept in the dark for 30 min then covered with a coverglass and were imaged in fluorescent microscope (Axioscope 2 plus, Ziess, Germany). At least 100 comet images from each slide were examined. Images were evaluated by the open comet software and individual comet images analyzed for several features including tail length, percent of DNA in tail, tail moment and olive moment.

DNA content and cell cycle phase distribution Analysis of MDA-MB-231 Cells Spheroids and
Monolayer by Flow Cytometry. Monolayer (3.5 × 10 5 ) MDA-MB-231 cells were treated with different concentration of ZEO (3, 7, 15, 30 μg/ml) for 4 and (3, 7, 15 μg/ml) for 24 h and MDA-MB-231 spheroids were treated with ZEO IC50 concentration (118 μ/ml) for 24 h. At the end of the treatment, cells were harvested, washed with 1 ml PBS, centrifuged at 1200 rpm for 5 min at 4 °C then fixed with ethanol 70% for 24 h in 4 °C. After incubation cells were centrifuged and ethanol was removed. The pellet was resuspended in PBS and incubated with 20 μg/ml RNAase (Sigma-Aldrich) and propidium iodide (20 μg/ml) (Sigma-Aldrich) in 37 °C in dark for 30 min. Finally, the distribution of different cell cycle phases was determined by flow cytometry and data analysis was carried out using the FlowJo software Version 7.6.1.
UV-Visible spectroscopy. Rat hepatocyte double stranded DNA (dsDNA) was isolated by standard phenol-chloroform method. DNA stock solution was prepared in Na 2 HPO 4 -NaH 2 PO 4 (0.1 M) buffer and stored at 20 °C. DNA concentration and quality was evaluated by NanoDrop (thermoscientific-USA) and UV-Visible spectrophotometer (Cary 100 Bio-model, Australia). UV-Visible spectra of ZEO were recorded using spectrophotometer in phosphate buffer (0.1 M with pH = 7.4) at room temperature over a wavelength range 190-600 nm. Initially the absorbance of ZEO concentrations ranging from 0 to 32 μg/ml in phosphate buffer at 298 K was measured, the solvent was taken as reference, and then similar procedure was carried out after addition of constant DNA concentration (50 μg/ml).

Fluorescence experiments.
Fluorescence experiments were carried out on a Carry eclipse (Australia) spectrophotometer. In the competitive binding studies, constant concentrations of DNA and Ethidium bromide (EB) (50 μg/ml and 2.6 μM, respectively) with increasing concentrations of ZEO (0-256 μg/ml) were used. ZEO emission spectra were recorded in the range of 530-700 nm upon excitation at 500 nm. The slit width was set as 10 nm/10 nm for ex/em, respectively. The spectroscopic experiments were performed at 310 K. CD spectral measurements. CD spectra of pure DNA and its complex with ZEO were recorded at room temperature with CD spectrophotometer (Circular Dichroism 215, Aviv, USA). Circular dichroism studies were carried out using ZEO concentration 16 μg/ml and final DNA concentration of 100 μg/ml. All spectra were recorded in far-UV range. Quartz cuvette with a path length of 1 cm was used for sampling. A spectrum of buffer solution was recorded and subtracted from the spectra of DNA and DNA-ZEO complex.
Statistical Analysis. Experimental data processing was carried out using Microsoft Excel 2013 software and results were presented as mean ± standard deviation of three or more independent experiments. The significant differences between means were determined by t-test when statistical significance was P value ≤ 0.05.