Inhibition of Growth and Induction of Apoptosis in Fibrosarcoma Cell Lines by Echinophora platyloba DC: In Vitro Analysis

Echinophora platyloba DC plant (Khousharizeh) is one of the indigenous medicinal plants which is used as a food seasoning and medicine in Iran. The objective of this study was to examine the in vitro cytotoxic activity and the mechanism of cell death of crude methanolic extracts prepared from Echinophora platyloba DC, on mouse fibrosarcoma cell line (WEHI-164). Cytotoxicity and viability of methanolic extract was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and dye exclusion assay. Cell death ELISA was employed to quantify the nucleosome production result from nuclear DNA fragmentation during apoptosis and determine whether the mechanism involves induction of apoptosis or necrosis. The cell death was identified as apoptosis using terminal deoxynucleotidyl transferase- (TdT-) mediated dUTP nick end labeling (TUNEL) assay. Our results demonstrated that the extract decreased cell viability, suppressed cell proliferation, and induced cell death in a time- and dose-dependent manner in WEHI-164 cells (IC50 = 196.673 ± 12.4 μg/mL) when compared with a chemotherapeutic anticancer drug, Toxol. Observation proved that apoptosis was the major mechanism of cell death. So the Echinophora platyloba DC extract was found to time- and dose-dependently inhibit the proliferation of fibrosarcoma cell possibly via an apoptosis-dependent pathway.


Introduction
Cancer is the major cause of human's death because of high incidence and mortality. e conventional modality for cancer therapy includes surgery, chemotherapy, and radiotherapy, separately or in combination but all of these have wide range of de�ciencies and side effects. ese factors highlight the essential prospects for novel therapies or therapeutic combinations to improve the survival and quality of the life of cancer individuals. An effective anticancer agent should kill cancer cells without affecting abnormal-to-normal cells. Hence, the identi�cation of new cytotoxic drug with low side effects on immune system has developed as important area in new studies of immunopharmacology. is ideal condition is feasible by inducing apoptosis in cancer cells [1]. Apoptosis (programmed cell death) is an active physiological suicide that occur normally during development and aging and as a homeostatic mechanism to maintain cell populations in tissues. Apoptosis is characterized by unique morphological and biochemical features, including cell shrinkage, membrane blebbing, chromatin condensation, and formation of apoptotic bodies [2,3]. Maintenance of organelle integrity, condensation, and DNA fragmentation, followed by removal of dying cells by macrophage-mediated phagocytosis. Dysfunction of the apoptotic program can promote tumor initiation, progression, and treatment resistance. e de�ciency of apoptosis can result in cancer, autoimmune diseases, and spreading of viral infections [4].
Traditional medicine has been used for maintaining heath, boosting immune system function, prevention, therapy, and remission of cancer [5]. Recently, extensive studies have been dedicated to the apoptosis and the role of this processes in intervention of the lethal properties of antineoplastic agents in cancer cells. Anticancer agents induce apoptosis, so that disruption of apoptotic cell death reduces treatment sensitivity. Plants have played an important role as the source of effective anticancer agents [6,7]. Extensive varieties of natural compounds possess signi�cant cytotoxic as well as chemopreventive activity, which act via apoptosis. Extracts of plants used in traditional medicine also have a similar property [8]. Echinophora is a ten-species genus of Apiaceae that contains four species native to Iran, including E. orientalis, E. sibthorpiana, E. cinerea, and E. platyloba [9]. Echinophora platyloba is widely used in western and central Iran as a food seasoning and edible vegetable. Local people add the plant to pickles and tomato pastes as an antifungal and antimicrobial preservative [10].
Taking into consideration, the potent antifungal and antibacterial activity suggested that the methanolic extract of Echinophora platyloba DC could be a potential candidate as a cytotoxic and growth inhibitory agent. e present study was undertaken to evaluate the cytotoxicity of crude methanolic extract of E. platyloba and determine the possible mechanism of cell death in malignant and nonmalignant cell lines in vitro. Cell cytotoxicity and viability was examined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and dye exclusion assay. e apoptosis was determined by cell death detection and speci�c DNA fragmentation with cell death detection (ELISA) and terminal deoxynucleotidyl transferase-(TdT-) mediated dUTP nick end labeling (TUNEL) assay.

Preparation of Plant
Extract. Echinophora platyloba DC plants were collected from the west of Iran (Shahre Kord). e Department of Botany, Institute of Medicinal Plants (IMP) of Kara�, Iran, identi�ed the plant. A voucher specimen was deposited in the herbarium of the above-mentioned entity . e aerial parts of the plant were separated, shade dried, and grinded into powder with mortar and pestle. e prepared powder was kept in tight containers protected completely from light. Extraction of methanolic extract was carried out by macerating 100 g of powdered dry plant in 500 mL of 70% methanol for 48 h at room temperature. en, the macerated plant material was extracted with 70% methanol solvent by percolator apparatus (2-liter volume) at room temperature. e plant extract was removed from percolator, �ltered through �hatman �lter paper (no. 4), and dried under reduced pressure at 37 ∘ C with rotator evaporator before being added to methanol as the solvent. e methanol extract was�ltered and concentrated using a rotary evaporator and then evaporated to dryness. Brie�y, dried extracts were dissolved in dimethyl sulphoxide (DMSO) (SIGMA, USA) to get a stock solution of 10 mg/mL. e substock solution of 0.2 mg/mL was prepared by diluting 20 L of the stock solution into 980 L serum-free culture medium, RPMI 1640 and prepared at different concentrations (the percentage of DMSO in the experiment should not exceed 0.5). e stock and substock solutions were both stored at 4 ∘ C.

Cell
Cultures. e mouse �brosarcoma cell line (�E�I-164) and mouse nonmalignant cell line (L929) were purchased from Pasteur Institute of Iran (cell Bank). e cells were grown and maintained in a humidi�ed incubator at 37 ∘ C and in 5% CO 2 atmosphere. RPMI-1640 medium (SIGMA) was supplemented with 10% heat inactivated fetal calf serum (FCS), 100 units/mL penicillin, and 100 g/mL streptomycin (all from Invitrogen Gibco) was used for cell cultures. Upon reaching appropriate con�uence, the cells were passaged. Aer being harvested from sterile T75 culture �asks (Nunc, Denmark), the cells were counted using a hemocytometer and cell viability was determined by trypan blue exclusion. Ten thousand cells from log phase cultures were seeded in 100 L of RPMI medium supplemented with 10% fetal bovine serum per well of 96-well �at-bottom culture plates (Nunc, Denmark). e cells were incubated with the different concentration of E. platyloba extract for a de�ned time (24, 36, and 48 hours) [11]. Proliferative response and cell death of the E. platyloba DC, extract-treated cells were determined using MTT assay, cell death ELISA, Trypan blue exclusion cell viability assay, and TUNEL assay, respectively.

MTT Assay. e assay detects the reduction of MTT [3-
(4,5-dimethylthiazolyl)-2,5-diphenyl-tetrazolium bromide] (Sigma) (a colorimetric technique) by mitochondrial dehydrogenase to blue formazan product, which re�ects the normal function of mitochondria and hence the measurement of cytotoxicity cell and viability. 1 × 10 4 viable cells/well were plated into the 96-well tissue culture plates (Nunc Denmark), and then incubated at 37 ∘ C overnight. e next day when cells reached >80% con�uence, the media were replaced with 200 mL of fresh complete medium containing 50, 100, 200, 300, 400, 500, 600, and 800 g/mL concentrations of crude extract, no extract was added to the negative control well and Toxol (Paclitaxel: plant-derived chemotherapeutic anticancer drug from Taxus brevifolia L.) as a control positive [12]. Aer 24, 36, or 48 h, the supernatants were removed and cell layers were washed with phosphate buffered saline (PBS, Invitrogen Gibco) and incubated with MTT (50 L, 0.5 mg/mL) in RPMI 1640 without FCS for 4 h in a humidi�ed atmosphere at 37 ∘ C according to the manufacturer's protocol. e cell cultures were centrifuged at 1000 g for 5 min and the supernatants were discarded. Subsequently, 200 L of dimethyl sulfoxide (DMSO, Sigma) and 25 L Sorenson buffer were added to dissolve the formazan crystals formed. e optical density (OD) colored solution was quanti�ed at 570 nm wavelengths by an enzyme-linked immunoabsorbent assay reader (ELISA Reader, Bio-Rad). e absorbance of untreated cells was Advances in Pharmacological Sciences 3 considered as 100%. Each extract and control was assayed in triplicate in three independent experiments. e cytotoxic effects of the extracts were estimated in terms of growth inhibition percentage and expressed as IC50 which is the concentration of compound which reduces the absorbance of treated cells by 50% with reference to the control (untreated cells). We determined that IC50 values with cytotoxicity results were more than 50% at screening concentrations. Percent growth inhibition of cells exposed to treatments was calculated as follows: % Inhibition = 100 − (Test OD/Non-treated OD) × 100) [13].

Cell Death Detection.
Cell death detection ELISA (Roche Applied Science) was used to quantify histone-complexed DNA fragments (nucleosomes) in cytoplasm of the apoptotic cells aer induction of apoptosis. Brie�y, aer incubation with the methanolic extract (at concentrations determined by MTT assay) for 24 h, cells were pelleted and lysed. Mouse monoclonal antibodies against single-strand DNA and histones (H1, H2a, H2b, H3, and H4) speci�cally detected and bound mononucleosomes and oligonucleosomes derived from cells undergoing apoptosis. Biotinylated antihistone antibodies then �xed the antibody-nucleosome complexes to the streptavidin-coated microtiter plate. e anti-DNA antibodies were conjugated with a peroxidase that reacted with the substrate ABTS [2,2V-azino-di(3-ethylbenzthiazolinsulfonate)] to form a colored product. e remaining steps were carried out according to the instructions supplied by the manufacturer. e resulting color development, which was proportional to the amount of nucleosomes captured in the antibody sandwich, was measured at 405 nm wavelength using a Benchmark microtiter plate reader (Bio-Rad). Results were expressed as the apoptotic and necrotic percentage, calculated from the ratio of absorbance of treated (apoptotic) sample to that of the untreated (control) sample [14].

Dye Exclusion
Assay. Cellular cytotoxicity induced by the E. platyloba extract treatment was measured with trypan blue exclusion assay. Brie�y, 1 × 10 4 cells were seeded into 96-well plates and treated with or without (as control) crude extract at speci�ed doses for 24, 36, and 48 h. Aer the incubation period, the cells were harvested and washed twice with PBS. e cell pellet was then resuspended with 0.5 mL PBS. en, 20 L of suspension was mixed with equal volume of 0.4% trypan blue (Sigma, USA Merck) and was count by Neubauer haemocytometer (Weber, England) by clear-�eld microscopy (Nikon, Japan). Each extract and control was assayed two times in triplicate. e cells were stained with trypan blue (Merck) and live cells were enumerated. Cell counts were expressed as mean ± standard deviation (SD).

TUNEL Assay.
To evaluate cell death by apoptosis, an in situ cell death detection kit, POD (Roche, Germany) for DNA chromatin morphologic features was used for quanti�cation. e procedures followed to the manufacture's guidelines. Brie�y, cells were cultured on glass slides and analyzed 24 hours aer treatment. Cells grown on coverslips were washed twice with PBS, air dried, and �xed for 60 min in freshly prepared 4% paraformaldehyde/PBS (Sigma-Germany), pH 7.4, at room temperature. en, the cells were washed again twice with PBS and incubated with 3% H 2 O 2 /methanol (Merck, Germany) for 10 min. Following washing with PBS, cells were permeabilized in 0.2% Triton X-100/PBS (Sigma, Germany) for 2 min at 4 ∘ C. Samples were incubated in 50 L of TUNEL reaction mixture for 2 h at 37 ∘ C in a humidi�ed chamber and in the dark, covered with para�lm. Omission of TdT provided the negative control for the assay, and preincubation of cells with 10 g/mL DNase I in 50 mM Tris-HCl, pH 7.4, 1 mM MgCl 2 , and 1 mg/mL BSA for 10 min at room temperature to induce DNA strand breaks arti�cially served as positive control. Cells were washed with PBS and incubated for 30 min in a humidi�ed chamber, at 37 ∘ C with 50 L converter-PODS (anti�uorescein antibody conjugated with peroxidase). Aer rinsing in PBS, the samples were incubated for 10 min with 100 L DAB (Sigma, Germany) substrate in the dark. At the end, the samples were mounted and analyzed under light microscope, where the apoptotic cells could be seen as condensed shrinked dark brown cells.

Statistical
Analysis. e data are expressed as mean ± standard deviation (SD) for at least three independent determinations in triplicate for each experimental point. e data were analyzed using IBM SPSS Statistics 20 soware. For all the measurements, two-way ANOVA followed by Duncan's new multiple range test ( ) was used to assess the statistically signi�cance of difference between control and E. platyloba DC, treated.

e Effects of Methanolic Extract of Echinophora platyloba on Inhibition and Proliferation of WEHI-164
Cells. e effect of E. platyloba was studied as a time-and dose-response experiment aer 24, 36, and 48 h, at concentrations of 50-800 g/mL. Proliferation of WEHI-164 cells was signi�cantly inhibited by E. platyloba in a concentration-dependent manner in 24, 36, and 48 h, as shown in Figure 1(a) with more than 80% suppression, but there was no signi�cant activity in L929 normal cell (IC50 > 800 g/mL) as shown in Figure  1 ). e concentrations producing 50% growth inhibition (IC50) of the E. platyloba extract on WEHI-164 proliferation were high potently suppressed with the IC50 value (196 673 ± 12 4 g/mL) aer incubation with the E. platyloba extract.
In 24, 36, and 48 h, Dye exclusion assay was evaluated viability of WEHI-164 cells exposed to E. platyloba extract. WEHI-164 cells were compared to elucidate the cytotoxicity of both E. platyloba extract and Toxol (chemotherapeutic agent, control positive) with more than 80% in 800 g/mL and 93% in 20 g/mL ( 1) growth suppression in 24 h ( Table 1).
Viability rate of cells exposed to E. platyloba extract at concentrations of 50 and 800 g/mL was decreased in descending form 87.3 ± 7.8% and 18.66± 2.5%, respectively ( Figure 2(a)). However, L929 cells were much less susceptible to the cytotoxic effect of E. platyloba extract (Figure 2(b)).

e Effect of the Methanolic Extract of Echinophora platyloba on Cell Death of WEHI-164 Cells.
As determined by MTT assay, E. platyloba extracts at 100, 200, and 300 g/mL in 24 h were chosen for each cell line in cell death detection ELISA. Apoptotic cell death is most likely not the only mechanism involved in downregulation of cell growth by E. platyloba. Necrosis remained at a low level ( 10%) over the whole dose range (Figure 3). e possible mechanism was via induction of apoptosis, as evidenced by the significant increase in nucleosome production at 100, 200, and 300 g/mL of E. platyloba extract aer incubation for 24 h but the ratio of apoptosis was constant approximately ± 3%. approach to the mode of cell death by E. platyloba was conducted by comparison with untreated controls showing that aer 24 h treatment of WEHI-164 cells with 200 g/mL (dose required for 50% inhibition), the number of cells decreased signi�cantly and the remaining cells reduced in size with chromatin clumping (Figure 4(b)). Untreated WEHI-164 cells with normal morphology were seen in the negative control group (Figure 4(a)).

�uanti�cation of �poptosis by T��E� �ssay.
TUNEL assay was done to test whether E. platyloba extract induced the decrease of cell viability and cytotoxicity contributes to apoptotic or necrosis death in WEHI-164 cell lines in vitro. It was found that the cells treated with extract (200 g/mL) for 24 h exhibited apoptotic body formation ( Figure 5). Typical morphological features of apoptotic cells, with condensed and fragmented nuclei in the treatment cells with E. platyloba ( Figure 5(a)), compared with homogenous nuclear chromatin in the control cells ( Figure 5(b)). It was found that the WEHI-164 cells treated with E. platyloba (200 g/mL) for 24 h exhibited apoptotic body formation ( Figure 5(c)).

Discussion
Apoptosis is recognized as programmed cell death, which follows in several pathological situations in multicultural organisms, and it is a form of common mechanism for cell replacement, tissue remodeling, and removal of damaged cell [2,3]. Dysfunction of the apoptotic program can promote tumor initiation, progression, and treatment resistance. e de�ciency of apoptosis can result in cancer, autoimmune diseases, and spreading of viral infections [4]. Traditional medicine has been used for maintaining heath, boosting immune system function, prevention, therapy, and remission of cancer [5]. Recently, extensive studies have been dedicated to the apoptosis and the role of this processes in intervention of the lethal properties of antineoplastic agents in cancer cells. Anticancer agents induce apoptosis, so that disruption of apoptotic cell death reduces treatment sensitivity. Plants have played a main role as the source of effective anticancer agents [6,7]. Extensive ranges of natural compounds possess signi�cant cytotoxic as well as chemopreventive activity, which act through apoptosis. Extracts of plants used in traditional medicine also have a similar characteristic [8].
In recent studies on medical treatment application of E. platyloba, Entezari et al. showed that the antibacterial effect on the growth of two bacteria (Staphylococcus aureus and Pseudomonas aeroginosa) of E. platyloba methanol essences and they did not observed prevention in growth of Candida albicans� Aspergillus �a�us� and Aspergillus niger [15]. e current study has demonstrated the potential effect of the extracts from E. platyloba on cell growth WEHI-164 cells. MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay is a common method of measuring the  proliferation of cells. is assay gives an indication of intact mitochondrial and also extra mitochondrial, NADHand NADPH-dependent redox enzyme systems. e effect of E. platyloba was studied as a time-and dose-response experiment aer 24, 36, and 48 h, at concentrations of 50-800 g/mL. Proliferation of WEHI-164 cells was signi�cantly inhibited by E. platyloba in a concentration-dependent manner in 24, 36, and 48 h, as shown in Figure 1(a) with more than 80% suppression, but there was no signi�cant activity in L929 normal cell as shown in Figure 1(b) ( ). e concentrations producing 50% growth inhibition (IC50) of the E. platyloba extract on WEHI-164 proliferation was high potently suppressed with the IC50 value (196.673 ± 12.4, 122.977 ± 51.7 and 58.664 ± 15.2 g/mL in 24, 36, and 48 h, resp.) aer incubation with the E. platyloba extract. In 24, 36, and 48 h, dye exclusion assay evaluated viability of WEHI-164 cells exposed to E. platyloba extract. Viability rate of cells exposed to E. platyloba extract at concentrations of 50 and 800 g/mL was decreased in descending form 87 3 ± 7 8% and 8 66 ± 2 %, respectively (Figure 2(a)). However, L929 cells were much less susceptible to the cytotoxic effect of E. platyloba extract (Figure 2(b)). Reduction of cell growth can re�ect either a decreased proliferation rate or an enhanced cell death by either necrosis or apoptosis or a combination of these mechanisms.
As determined by MTT assay, E. platyloba extract at 100, 200, and 300 g/mL in 24 h were chosen for each cell line in cell death detection ELISA. Apoptotic cell death is most likely not the only mechanism involved in downregulation of cell growth by E. platyloba. Necrosis remained at a low level ( 10%) over the whole dose range ( Figure  3). To determine whether the growth inhibitory activity of E. platyloba was related to the induction of apoptosis, the morphological character changes of WEHI-164 cells were investigated under light microscopic. In situ TUNEL