Cytotoxicity of Euphorbia peplus Extract on MCF7 Breast Cancer Cells

In this study, the effect of Euphorbia peplus aqueous extract on human breast cancer cell line MCF7 was examined. The short and long term cytotoxicity were evaluated using sulphorhodamine B and clonogenic assays respectively. Scanning and transmission electron microscopy were employed to examine Euphorbia peplus-induced ultrastructural changes in MCF7 cells. The sulphorhodamine B assay revealed that Euphorbia peplus inhibits the growth of MCF7 with an IC50 of 30.32 μg/ml. The clonogenic assay proved that Euphorbia peplus’ growth inhibitory effect is long lasting. The ultrastructural examination demonstrated that Euphorbia peplus extract induces MCF7 cell death. Scanning electron microscopy showed apoptotic blebbing. Transmission electron microscopy displayed cellular shrinkage, the formulation of apoptotic bodies, mitochondrial changes, nuclear shrinkage, chromatin condensation, autophagic vacuoles, and necrotic changes. In summary, Euphorbia peplus has displayed growth inhibitory activity against MCF7 cells and induces cell death predominantly via apoptosis and could be exploited as a breast cancer treatment after further evaluation.

Breast cancer is considered the most commonly diagnosed cancer in women and remains among the leading causes of cancer death worldwide (FERLAY et al. 2010;TORRE et al. 2015;FERLAY et al. 2015). Being characterized by genetic heterogeneity, multi-genotypic breast cancer explains why patients within clinically and morphologically similar classes show a varied response to therapy and the redundancy of the available traditional prognostic factors. Even though some breast cancer groups have been molecularly identified based on molecular gene expression profiles, further distinct molecular classification is still ongoing (PEROU et al. 2000;DOUMA et al. 2014;EUHUS & DIAZ 2015). So far, four distinct mo-lecular groups have been characterized: luminal epithelial/estrogen receptor (ER) positive, c-erb-B2 (HER2) positive, basal-like, and normal breastlike (PEROU et al. 2000). Another study divided the luminal/ER-positive group into three subtypes: luminal A, B, and C (SORLIE et al. 2003). The combination of doxorubicin and cyclophosphamide is considered to be one of the most effective chemotherapy regimens for breast cancer (MAMOUNAS et al. 2005). However, the use of these conventional chemotherapeutic drugs ignore the molecular classification of the patients and frequently result in serious adverse side effects by producing unnecessary damage to the neighboring normal and healthy cells. These current results shed the light on the need to further develop more targeted therapeutic strategies. Indeed, selective estrogen receptor modulators, such as tamoxifen and raloxifen, have been used to treat ER-positive patients, whereas trastuzumab, a HER2-targeted monoclonal antibody, is used on HER2-positive patients (PEER et al. 2007). Regrettably, the use of these drugs is associated with high incidence resistance, relapse, and some unwanted side effects (CHANG et al. 2011;NORMANNO et al. 2005;ADAMO et al. 2007). However, it has been shown that medicinal herb extracts may also be used as an adjuvant to inhibit tumor growth and to overcome these side effects (CAI et al. 2004). Moreover, combining chemotherapeutic drugs with herbal extracts could lower the chemotherapeutics doses required to treat cancer (NG et al. 2014). Herbal extracts result in a variety of effects due to being, anti-allergic, antimicrobial, anti-oxidant and anticancerous due to the presence of various components (BAK et al. 2013), and generally exert cytotoxic effects on tumor cells directly, or through the enhancement of the immune response. For this reason, the anti-cancerous activity of numerous herbal extracts is currently being investigated (CHU et al. 2009;SHOEMAKER et al. 2005).
Euphorbia peplus belongs to the genus Euphorbia, which is considered to be the largest among genera of the Euphorbiaceae family or, the spurge family. E. peplus is originally native to Europe and North Africa (ZHI-QIN et al. 2010). It is well known by the common name of Radium weed in Australia and Petty spurge in the UK. Other common names are milk weed, cancer weed, radium plant, or stinging milk weed. The family in general consists of about 7,500 plant species with a reputation for having several medicinal usages. The majority of its members ooze milky sap (latex) when their stems are cut. The milky sap that is used in traditional medicine for the treatment of nonmelanoma skin cancer and its active compounds has been determined to be diterpene esters (RAMSAY et al. 2011). The sap has been used as a treatment for solar keratosis and skin cancers in Australia for decades (ROSEN et al. 2012;BRAUN et al. 2014). In Europe, it has been used as a laxative and to treat warts, corns, waxy growths, asthma, catarrh, and skin cancers. In Ukraine, it is commonly used to treat gastric, hepatic, and uterine malignancies. Also, in Mauritius, its decoction is used to treat diarrhea and dysentery. Moreover, one of its uses in Saudi Arabia is to lower blood pressure (GREEN & BEARDMORE 1988;TCHINDA 2008). In addition, it has been shown to have antipyretic, analgesic, and antimicrobial effects (ALI et al. 2013). Many macrocyclic diterpenes with cytotoxic activity have been isolated from the sap fractionation. For instance, Ingenol 3-mebutate (angelate/PEP005), an ingenane diterpene derived from E. peplus, has emerged as a possible anticancer agent (ERSVAER et al. 2010). PEP005 has been found to be active against human melanoma xenografts in mice (GILLESPIE et al. 2004). Moreover, its effects extend to murine melanoma and lung carcinoma, human prostate, cervical and T-cell leukaemia (OGBOURNE et al. 2007). Furthermore, a previous study in murine mice revealed that PEP005 caused mitochondrial swelling and primary necrotic cell death of dysplastic keratinocytes (OGBOURNE et al. 2004). A different study showed that PEP005, in contrast to other topical agents for actinic keratosis, appears to target the residual dysplastic keratinocytes using two mechanisms; an immune-mediated response and necrosis (ROSEN et al. 2012;COREA et al. 2005). Ingenol 3-mebutate, now marketed as Picato gel, has lately been approved by the Food and Drug Administration (FDA), as a treatment of precancerous skin. Therefore, the present study examined the cytotoxic effect of E. peplus aqueous extract against MCF7 breast cancer cells by sulphorhodamine B (SRB) and clonogenic survival assays. Furthermore, E. peplus-induced ultrastructural changes were examined by both scanning (SEM) and transmission (TEM) electron microscopy.

Materials and Methods
Plant material and extraction E. peplus stem and leaves from the Asir region, Saudi Arabia (18°12¢ 59² N, 42°30¢ 19² E), were collected and authenticated by Professor Mahmoud MOUSTAFA, at the Department of Biology, College of Sciences, King Khalid University, Abha, Saudi Arabia. Ten grams of stem and leaves were washed thoroughly with distilled water and crushed directly using a grinder (Thomas Wiley Laboratory Mill, Model 4) for 15 min. The extract was filtered through 2-layered muslin cloth and the filtrate was diluted with 20 ml of distilled water and left on a rotary shaker at 100 rpm at room temperature for 2 days. The extract was stored in eppendorf tubes covered with aluminum foil and was kept at 4°C (ALAMRI & MOUSTAFA 2012).

Sulphorhodamine B assay
Cultured MCF7 cells were trypsinized, resuspended in fresh medium and counted. Then the cells were plated in 96 well tissue culture plates (5´10 3 cell/well) for 24 hrs. Three plates were used for control for each of the concentrations. After discarding the old medium, serum free medium (100 µl/well) was added to the control plates. Based on a literature search, free medium containing different concentrations of E. peplus aqueous extract (0.01, 0.1, 1, 10, 100 µg/ml) were added to the other plates for 48 hrs. After 48 hrs of incubation, the culture medium was removed and cells were fixed with 10% (v/v) trichloroacetic acid (TCA) at 4 o C for 1 h. The plates were washed three times with distilled water to remove the TCA and then stained with 70 µl/well of 0.4% sulphorhodamine B (SRB) in 1% acetic acid for 30 min at room temperature. The excess dye was removed by washing 4 times with 1% (v/v) acetic acid and the plates were allowed to dry at room temperature. The protein-bound dye was solubilized in a 10 mM Tris base solution (100 µl/well) and the optical density (OD) of the solution was determined at 570 nm using a microplate reader. The reading was repeated thrice and the mean of three readings was used for analysis. Control values were set at 100% viable and all values were expressed as a percentage of the control. The inhibitory concentration 50% (IC 50 ) of E. peplus was calculated using a AAT Bioquest IC 50 calculator (AAT Bioquest, Inc., Sunnyvale, CA). The values were plotted by using Microsoft Excel (VICHAI & KIRTIKARA 2006).

Clonogenic survival assay
MCF7 cells were grown in two 75 cm 2 cell culture flasks for 24 hrs (until~70% confluence). After that, the medium was removed. One flask was treated with the IC 50 of E. peplus (30.32 µg/ml) for 48 hrs and the other flask was used as an untreated control. Then, the cells were gently trypsinised and counted using a haemocytometer and serially diluted into 10 ml universals. Cells were seeded out at 100 cell/well into a 6-well plate: three plates for each of the control and treated cells. The 6-well plates were incubated under standard growth conditions for 14 days and stained with crystal violet (0.4% crystal violet, 50% methanol). The number of colonies was counted three times using Image J software (The National Institute of Health, Bethesda, MD) (SILVA et al. 2018). Plating efficiencies (PE) were calculated from the ratio between the number of colonies counted and the number of cells seeded. The surviving fraction (SF) was calculated using the formula: SF = PE of treated cells/PE of control × 100 (FRANKEN et al. 2006;RAFEHI et al. 2011;AL-EMAM et al. 2018a).

Scanning electron microscopy SEM
MCF7 cells were grown in complete DMEM, treated with 30.32 µg/ml of E. peplus aqueous extract and incubated for 48 hrs in a humidified CO 2 incubator. Non-treated control cells were also cultured in complete DMEM. After 48 hrs, cells were centrifuged and cell pellets were obtained. The cell pellets were immediately fixed in 2.5% (wt/vol) Na cacodylate-buffered glutaraldehyde, pH 7.4 at 4°C for 2 hrs. Then, they were post-fixed in 1% Na cacodylate-buffered osmium tetroxide, pH 7.4 for 1 h. After that, the cells were dehydrated in ascending concentrations of ethanol. Drying was done using the EMITECH-K850 critical-point drying unit. The dried cells were mounted on aluminum stubs with double sided tape and silver glue and then sputter coated with gold by BOC EDWARDS SCANCOAT. Finally, cells were observed using a Jeol SEM (JSM-6390LV, Japan), operated at 2-5 kV at the Electron Microscope Unit, College of Medicine, King Khalid University (EID et al. 2012).

Transmission electron microscopy TEM
MCF7 cells were seeded in 6-well culture plates (2´10 5 cell/well). Cultured cells were treated with the IC 50 of E. peplus aqueous extract (30.32 µg/ml) for 48 hrs. Treated cells were then collected in Hank's Balanced Salt Solution (HBSS), centrifuged at 5000 rpm and the resultant cell pellets were fixed in 2.5% (wt/vol) Na cacodylatebuffered glutaraldehyde (pH 7.2) at 4 o C for 2 hrs. The cell pellets were then washed in the same buffer and postfixed in 1% osmium tetroxide in Na cacodylate buffer. After ascending series of ethanol dehydration, the pellets were embedded in Spurr's resin. Ultrathin sections stained with uranyl acetate and lead citrate were examined by TEM (Jeol 100CXII, Japan) operated at 80 kV at the Electron Microscope Unit, College of Medicine, King Khalid University (AL-EMAM et al. 2018b).

Statistics
All experiments were done in triplicate and the data were presented as mean ± standard deviation (SD). The data were statistically analyzed using one-way ANOVA with Tukey's post hoc tests for the SRB cytotoxicity assay and the Students' t-test for the clonogenic survival assay. Analysis was performed using the statistical program IBM SPSS 22. p£0.05 was considered as statistically significant.

Results
Effect of E. peplus extract on the viability of MCF7 cells The cytotoxic effect of E. peplus aqueous extract on MCF7 cells was examined by the SRB assay, which is one of the short-term cytotoxicity assays. MCF7 cells were treated with different concentrations of E. peplus aqueous extract for 48 hrs. The results depicted in Fig. 1A demonstrate that E. peplus extract significantly reduced the viability of the cultured MCF7 by 2.79%, 4.65%, 10.30%, 41.96%, and 96.09% at the concentrations 0.01, 0.1, 1, 10 and 100 µg/ml, respectively. 100% corresponds to the viability of untreated control cells. One-way ANOVA was employed to detect differences between the groups of treated MCF7 cells and the control group. p values <0.05 were considered as statistically significant. Anova showed statistical significance and Tukey's post hoc revealed significant growth inhibition of MCF7 cells treated with 10 and 100 µg/ml concentration of E. peplus compared to nontreated control cells with p = 0.01 and p = 0.001respectively. The IC 50 was 30.32 µg/ml. Moreover, microscopic examination of the MCF7 cells treated with the IC 50 for 48 hrs revealed that the majority of the cells shran and became rounded and detached in comparison with untreated control MCF7 cells (Fig. 1B).
Effect of E. peplus extract on cell proliferation and colony survival of MCF7 cells The clonogenic survival assay was employed to assess the long-term cytotoxicity of E. peplus aqueous extract on MCF7 cells. MCF7 cells, treated with the IC 50 of E. peplus for 48 hrs, were incubated for 14 days to develop colonies. The number of colonies was counted using Image J. The plating efficiency of MCF7 cells was 80%. The surviving fraction of the colonies treated with the IC 50 of E. peplus had a significantly lower plating effeciency (12±2.7) as compared to the untreated controls (80±2.9) (p = 0.001) (Fig. 2).
Ultrastructural changes of E. peplus-treated MCF7 cells Electron microscopy is a multipurpose technique used for the examination of the surface and morphological features in tumor cells. The higher resolution of both SEM and TEM allows documenting the ultrastructural cellular changes during cell death. Moreover, it is listed among the most valid methods that can differentiate between the different modes of cell death alongside various biochemical markers (VENKATESAN et al. 2010;MILLER et al. 2002;WILLINGHAM 1999). MCF7 cells were treated with the IC 50 of E. peplus for 48 hrs and the cell pellets were processed for both SEM and TEM examination. SEM examination of E. peplus treated MCF7 cells revealed characteristic intense apoptotic membrane blebbing as shown in Fig. 3B, whereas the untreated control cells didn't show any surface zeiotic blebbing (Fig. 3A). Apoptotic blebs appear as quasi-spherical, balloon-like protrusions of the cell membrane with the cell membrane still intact.
TEM examination of untreated MCF7 cells exhibited spherical morphology with few scattered surface microvilli and normal mitochondrial mor- phology and distribution. They also had large nuclei with both eu-and heterochromatin (Fig. 4A). On the other hand, E. peplus-treated MCF7 cells revealed a loss of surface microvilli (Fig. 4B), mitochondrial distortion in the form of elongation and shrinkage and some electron dense lysosomes (Fig. 4B, C, D, E and Fig. 5B, C, D, E). A majority of treated cells showed the presence of autophagic vacuoles (autophagosomes) in their cytoplasm. Some of these vacuoles were seen containing damaged organelles and mitochondria (Fig. 4 B, C, D, E, F). Nuclear changes in the form of shrinkage, chromatin condensation, and irregular nuclear membrane were constant findings in the E. peplus-treated cells ( Fig. 4D and E). Moreover, some cells had diffuse areas of cytoplasmic degeneration (Fig. 4B, C and Fig. 5A). Also, apoptotic blebs and apoptotic bodies were frequently captured as shown in Fig. 4B, C and Fig. 5B, C, E, F, G, H. Furthermore, necrotic changes were noticed in many E. peplus-treated cells. These changes were in the form of a pyknotic nucleus, cell membrane rupture, the appearance of secondary lysosomes, and autophagic vacuoles indicating cellular digestion of organelles as shown in Fig. 6B, C, D as compared to the untreated control cells in Fig. 6A.

Discussion
The spurge family (Euphorbiaceae) is a large family of plants that have been used over the years for the treatment of various ailments including cancer. The aim of the present study was to examine the cytotoxicity of E. peplus aqueous extract against the MCF7 breast cancer cell line. The SRB assay revealed that E. peplus aqueous extract is cy- Fig. 2. A (SHIN et al. 2016), and E. supina (KO et al. 2015). The reduction of MCF7 cells' viability detected by SRB in response to E. peplus extract treatment could be attributable to the inhibition of cell proliferation, increased cell death, or both. Although SRB is one of the assays that measure the potency of a cytotoxic agent, it is considered to be a short-term cytotoxicity assay. In addition, it might underestimate the cytotoxicity of a drug or chemical agent when compared with long-term assays for cell growth or cloning efficiency. Moreover, such assays do not take into consideration the reversible damage and/or regrowth of cancer cells that are resistant to treatment (PEREZ et al. 1993). Therefore, in order to determine whether the cytotoxic effect of E. peplus against MCF7 is long lasting, we selected the clonogenic survival assay which is widely used for testing and predicting the long-term cytotoxicity of anticancer agents (YALKINOGLU et al. 1990), as it evaluates the sum of all modes of cell death, and also accounts for delayed growth arrest (MIRZAYANS et al. 2007;STRONCEK et al. 2007). Indeed our results revealed that the treatment of MCF7 cells with IC 50 of E. peplus for 48 hrs significantly reduced the number of surviving colonies compared to the untreated control cells (Fig. 2).
In agreement with this finding, previous studies have revealed that phytochemicals including apigenin, luteolin, and flavone are potent in cancer  Cytotoxic Effects of Euphorbia peplus Extract on MCF7 Cells chemoprevention. Their long-term effects on the growth of breast cancer cell lines were tested by clonogenic survival assay and they reduced colony numbers 2-3 fold (LIN et al. 2015).
In order to examine whether the cytotoxicity of E. peplus against MCF7 cells is due to the induction of cell death, we decided to study the ultrastructural changes of MCF7 cells after treatment with the IC 50 of E. peplus for 48 hrs. Interestingly, SEM examination of E. peplus-treated MCF7 cells revealed zeiotic membrane blebbing which could be an early event preceding cell death (CHARRAS 2008). Zeiotic blebbing is indeed observed during the execution phase of apoptosis which consequently leads to chromatin condensation, DNA fragmentation, and apoptotic cell death (WYLLIEE et al. 1980;EARNSHAW 1995;JACOBSON et al. 1997). Moreover previous studies, in agreement with our findings, described the same significant morphological apoptotic characteristics in MCF7 cells in response to cytotoxic herbal treatment. For instance, Elephantopus scaber Linn, a herb from the Asteraceae family, showed cytotoxic effects with apoptotic blebbing towards MCF7 cells with an IC 50 value of 15 µg/ml (HO et al. 2011). In addition, the same observation was reported by COLLINS et al. (1997). Furthermore, photodynamic therapy induced apoptosis with predominant apoptotic blebs in different cancer cell lines (HeLa, HaCaT, and MCF7) pretreated with photosensitizers (COLLINS et al. 1997). However, zeiotic membrane blebbing has been found to be closely associated with cytokinesis (FISHKIND et al. 1991), motility (BERGERT et al. 2012, cell spreading (NORMAN et al. 2010) and mitosis (MOES et al. 2011), contractolation (KHAJAH et al. 2015, and during early embryogenic migration (TRINKAUS 1973). Also, recent studies revealed that cancer stem cells which undergo apoptosis, create blebbishields from apoptotic bodies, overrule phagocytosis and undergo cellular transformation by blebbishield-mitotic cell, blebbishield-blebbishield, or blebbishield-immune cell fusion and reform tumors in mice. This exciting blebbishield emergency program linked apoptotic cancer stem cells to immune evasion, drug resistance, tumorigenesis, apoptosis evasion, and metastasis (JINESH 2017). Therefore, in order to verify the lethal effects of E. peplus on MCF7 cells, we went further to examine E. peplus-treated MCF7 cells with TEM. TEM examination revealed apoptotic changes in the form of mitochondrial elongation, shrinkage and damage, nuclear shrinkage, chromatin condensation, irregular nuclear membrane and characteristic apoptotic blebs and apoptotic bodies as shown in Figs 4 and 5. Moreover, the majority of the E. peplus-treated cells displayed an abundance of autophagic vacuoles enclosing damaged organelles and mitochondria (Fig. 4). Furthermore, some cells showed a pyknotic nucleus, cell membrane rupture, the appearance of secondary lysosomes, and autophagic vacuoles (Fig. 6), findings which are indicative of necrosis.
In agreement with our findings, several studies over the years have proved the anticancer activities of different members of the genus Euphorbia, predominantly through their pro-apoptotic activities against numerous cancer cell lines (WONGRAKPANICH & CHAROENSUKSAI 2018): for instance, E. hirta (KWAN et al. 2016), E. sogdiaa (AGHAEI et al. 2016, E. fischeriana (KUANG et al. 2016) andE. humifusa (SHIN et al. 2016) were found to be cytotoxic and induce the apoptotic cell death of breast cancer cell lines. Moreover, ingenol-3-angelate (also known as ingenol mebutate and PEP005), a diterpene isolated from E. peplus, has demonstrated anticancer activity against melanoma (OGBOURNE et al. 2004) and actinic keratoses (SILLER et al. 2009). Its mechanism of action includes primary cell death by necrosis (OGBOURNE et al. 2004), initiation of G1 and G2/M cell cycle arrest (COZZI et al. 2006), activation of protein kinase C (HAMPSON et al. 2005), and neutrophils (HAMPSON et al. 2008). It was indeed intriguing as apoptotic changes were the predominant ultrastructural changes in this study as shown in Figs 4 and 5 with a minority of cells showing both autophagic and necrotic changes (Figs 5 and 6). However, it has recently been found that 3-O-angeloyl-20-O-acetyl ingenol, a synthetic derivative of ingenol mebutate known as AAI or PEP008, inhibits cell proliferation, induces G2/M phase arrest, disrupts the mitochondrial membrane potential, and stimulates apoptosis, as well as necrosis in breast cancer and human leukemia cell line via modulation of PKCä/ERK, JAK/STAT3, and AKT signaling pathways (LIU et al. 2016). In addition, GILLESPIE et al. (2004) proved that ingenol 3-angelate can trigger necrosis alone or both necrosis and apoptosis in a cell-line-dependent manner, depending on the PKC status of the cells. Also, E. hirta aqueous extract has recently been shown to induce both apoptosis and necrosis of breast cancer cell lines (BEHERA et al. 2016). Furthermore, there are several examples in literature that documented the occurrence of both autophagic cell death and apoptosis in different cancers. For example, Paratocarpin E, a prenylated chalcone isolated from E. humifusa resulted in typical apoptosis of MCF7 cells via activating p38 and JNK and suppressing ERK signaling pathways. Paratocarpin E-treated MCF7 showed plenty of autophagic vacuoles associated with an increased ratio of LC3-II/LC3-I, activation and nuclear translocation of NF-êB, yet a reduced p62 expression. This signposts the strong pro-autophagic effects of paratocarpin E (GAO et al. 2016). Noteworthy, characteristic features of apoptosis have been noticed to occur during autophagy in the absence of essential apoptotic players such as caspases or Bax and Bak (GALLUZZI et al. 2012). Also, Cepharanthine, a natural alkaloid from Stephania cepharantha (GAO et al. 2017), and Baicalein, a bioactive compound from Scutellaria baicalensis (YAN et al. 2018) have been shown to induce apoptotic and autophagic cell death in various cancers in addition to their numerous anticancer properties. For an exhaustive list of natural autophagy inducers, interested readers are referred to a review by LIN et al. (2017). In fact, it has been confirmed that different cell death pathways including apoptosis, mitotic catastrophe, autophagy, and necrosis could be evoked by photodynamic therapy (YOO & HA 2012;MROZ et al. 2011;CASTANO et al. 2005). Moreover, several studies have shown that a shift from apoptosis to necrosis does exist. The switch in the mode of cell death is dependent on a variety of factors, comprising the nature of photosensitizers, cell genotype, and the dose of photodynamic therapy (YOO & HA 2012;MROZ et al. 2011;RUIZ-GONZALEZ et al. 2013;ACEDO et al. 2014). Furthermore, the co-existence of necrosis and apoptosis has been demonstrated in previous studies (LEDDA-COLUMBANO et al. 1991;FUKUDA et al. 1993). In addition mixed biochemical and morphological characteristics of both necrosis and apoptosis have been revealed in single cell (PENA & PILAR 2000). In fact, the concomitance of apoptotic, autophagic and necrotic changes in our ultrastuctural data could be conceivably explained by the finding that ingenol 3angelate, an E. peplus deriviative, has a dual mechanism inducing both apoptosis and necrosis (GILLESPIE et al. 2004). In addition, the abundance of autophagic changes could be viewed as a regulatory switch between the two other E. peplus-induced modes of cell death: apoptosis and necrosis. In support of this explanation, the active role of autophagy in MCF7 cell death has recently been demonstrated (GAVLAN et al. 2013). For more details on the role of autophagy in breast cancer, interested readers are referred to a review by JAIN et al. (2013). However, the more plausible explanation for the variability in terms of cell death modes observed in our current study and the aforementioned ones could be the plasticity of cell death induction and execution. In other words, cell death could be viewed as a dynamic process with a molecularly overlapping and interactive signaling network rather than a mere static pathway as reviewed by LOOS and ENNGELBRECHT (2009). Indeed, autophagy appears to have a central and upstream role in the cell death process (KARANTZA-WADSWORTH et al. 2007;KROEMER et al. 2009) and controls the onset of the other cell death modalities (YONEKAWA & THORBURN 2013). For a more detailed discussion about the dynamic regulatory interactions between autophagy and the other cell death machineries, interested readers are referred to these reviews (LIU & LEVINE 2014;GOODALL et al. 2016).
In conclusion, the data reported in this study provide sufficient proof that E. peplus aqueous extract could inhibit the growth and induce cell death of MCF7 breast cancer cells mainly via apoptosis and that it might be beneficial as an adjuvant therapy alongside conventional breast cancer treatments. The clonogenic survival assay proved that the effect of E. peplus on MCF7 cells is long lasting and abrogates the possibility of reversibility of apoptosis. However, it should be emphasized that the apoptotic, autophagic and necrotic changes witnessed by our ultrastructural examination need further investigation to understand the mechanism of cell death induction and execution of E. peplus and its derivatives in different cancer cell lines.