n-hexane, dichloromethane (DCM), methanol (MeOH), and dimethyl sulphoxide (DMSO) were of analytical grade and were purchased from Merck (Darmstadt, Germany). RPMI-1640 medium, fetal bovine serum (FBS), penicillin-streptomycin (100 units/mL penicillin and 100 μg/mL streptomycin), (3-[4, 5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), trypan-blue, paraformaldehyde, and ethylenediaminetetraacetate (EDTA) were obtained from Sigma Aldrich (MO, USA). Trypsin-EDTA (0.25 %) was obtained from Gibco (Paisley, UK). Cell death detection ELISA^plus kit, terminal deoxynucleotide transferase dUTP nick end labeling (TUNEL) assay kit, high pure RNA isolation kit, and protease inhibitor cocktail were purchased from Roche (Mannheim, Germany). RevertAid^TM first strand synthesis kit was obtained from Fermentas (MA, USA). SYBR Green PCR Master Mix was purchased from Applied Biosystems (CA, USA). Designed primers were purchased from Bioneer (Daejeon, Korea). Enhanced chemiluminescence (ECL) advance Western blotting detection kit was purchased from GE Healthcare (Little Chalfont, UK). Antibodies against poly ADP-ribose polymerase (PARP), β-actin, and rabbit IgG conjugated with horseradish peroxidase (HRP) were obtained from Abcam (MA, USA). Human breast cancer cell line MCF-7, mouse fibrosarcoma cell line WEHI-164, and normal human umbilical vein endothelial cells (HUVEC) were purchased from National Cell Bank of Iran (Pasteur Institute, Tehran, Iran).

The aerial parts of S. amplexicaulis were harvested from Sahand mountain, located in East Azerbaijan province, Iran. The plant was authenticated and a voucher specimen (2817) has been deposited at the Herbarium of Pharmacognosy Department, Faculty of Pharmacy, Tabriz University of Medical Sciences, Iran. The air-dried powdered aerial parts of S. amplexicaulis (1800 g) were subjected to Soxhlet apparatus and extracted sequentially with organic solvents n-hexane, dichloromethane (DCM), and methanol (MeOH). The extract solutions were then concentrated under reduced pressure using a rotary evaporator (Heildolph, Germany) at 45 °C. Afterward, 20 mg of each extract was separately dissolved in 100 µL of DMSO, diluted with RPMI-1640 medium and sterilized with 0.22 μm syringe filters (Nunc, Denmark). Finally, S. amplexicaulis extracts were stored at 4 °C for further biological experiments.

The cells were grown in RPMI-1640 medium supplemented with 10 % FBS, 100 U/mL penicillin, and 100 µg/mL streptomycin under a humidified 5 % CO₂ atmosphere at 37 °C. After 80 % confluence, the cells were detached by 0.25 % Trypsin-EDTA solution for the passage.

The cytotoxic activity of n-hexane, DCM and MeoH extracts of S. amplexicaulis on MCF-7, WEHI-164, and HUVEC cells was examined by MTT assay. Briefly, the cells in logarithmic growth phase were seeded into 96-well plates (Nunc, Denmark) at a density of 1× 10⁴ cells/well for 24 h at 37 °C with 5 % CO₂ and treated with different concentrations of extracts (10, 20, 50, 100, 150, 200, 300, 400 µg/mL) for 12, 24 and 48 h. Untreated cells and cells exposed to paclitaxel (0.5 µg/mL) served as negative and positive control, respectively. Then, 20 μL of MTT solution (5.0 mg/mL) was added to the cells and incubated for 4 h at 37 °C. Subsequently, the culture medium was removed from each well and replaced with 200 µL DMSO and 50 µL Sorenson buffer. After 30 min of incubation at 37 °C, the absorbance was measured at 570 nm and a reference wavelength of 630 nm using a microplate ELISA reader (BioTeck, Germany).

The number of viable cells in a cell suspension was determined by trypan blue exclusion assay. Briefly, 1× 10⁴ cells/well were dispensed into 96 well-plates and incubated for 24 h at 37 °C. The cells were then treated with same different concentrations of S. amplexicaulis extracts for 24 h. Untreated cells and cells exposed to paclitaxel (0.5 µg/mL) served as negative and positive control, respectively. At the end of incubation, the cells were trypsinized, washed twice with phosphate-buffered saline (PBS) and the resultant pellets were resuspended in 0.5 mL of PBS. Thereafter, equal volumes of 0.4 % trypan blue dye and cell suspensions were mixed and the number of viable and dead cells was counted using a hemocytometer (Weber, U.K.).

Apoptosis and necrosis was assessed by Cell Death Detection ELISA^plus kit according to the manufacturer's protocol. The cells were cultured at a density of 1×10⁴ cells/well into 96-well plates for 24 h at 37 °C and then treated with DCM and MeOH extracts of S. amplexicaulis at their 48-h IC₅₀ (50 % inhibitory concentration) concentrations for 24 h. Untreated cells were used as a negative control. Thereafter, the cells were trypsinized, centrifuged (1500 rpm for 5 min), the supernatants and lysates were collected and transferred into the microtiter plates coated with anti-histone antibody. After 90 min of incubation, the plates were rinsed three times with washing buffer, anti-DNA antibodies conjugated with horseradish peroxidase (HRP) were added and incubated for 2 h at room temperature (RT). Then, the plates were washed again and incubated with peroxidase substrate for 20 min at RT. The final absorbance at 405 nm was determined by a microplate ELISA reader (BioTeck, Germany).

DNA fragmentation, a prominent hallmark of apoptosis, was detected using TUNEL assay kit following the manufacturer's instructions. Briefly, cells (2×10⁵ cells/well) were seeded in 6-well chamber slides (Nunc, France) and incubated for 24 h at 37 °C. Following treatment with 48-h IC₅₀ concentrations of DCM and MeOH extracts for 24 h, the treated and untreated cells (negative control) were fixed with 4 % (w/v) paraformaldehyde solution in PBS (pH 7.4) for 1 h at RT. The slides were then washed twice with PBS and incubated with blocking solution (3 % H₂O₂ in methanol) for 10 min at the same temperature. Subsequently, the cells were washed again and permeabilized with 0.1 % Triton X-100 in 0.1 % sodium citrate for 2 min on ice. After rinsing in PBS, 50 µl of terminal deoxynucleotide transferase (TdT) reaction mixtures was added to the cells and incubated for 1 h at 37 °C. The cells were then washed three times with PBS and incubated with 50 µl Streptavidin HRP solution for 30 min at 37 °C. Finally, the cells were stained with 100 μl of DAB substrate for 10 m in a dark condition and evaluated under a light microscope (Nikon, Japan).

The cells were grown at a density of 1×10⁶ cells/well in 6 well-plates and treated with 48-h IC₅₀ concentrations of DCM and MeOH extracts for 24 h. Afterward, the treated and untreated cells (negative control) were harvested and lysed in lysis buffer (1 % Triton X-100, 1 M HEPES, 5 M NaCl and 0.5 M EDTA) supplemented with a protease inhibitor cocktail. Protein concentrations were determined using the Bradford method based on the bovine serum albumin standard. Equal amounts of protein were fractioned by 12 % sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene difluoride (PVDF) membranes (Roche, Germany). The membranes were then blocked with 4 % skim milk in Tris-buffered saline Tween-20 (TBST) for 2 h, incubated with anti-PARP (1:500 primary antibody overnight at 4 °C, washed three times with TBST, and incubated with HRP conjugated anti-rabbit IgG secondary antibody (1:1000) for 2 h at RT. After three washes with TBST, the protein bands were developed on X-ray film using ECL advance Western blotting detection kit.

Following treatment, total RNA of treated and untreated cells (negative control) were extracted using a high pure RNA isolation kit. RNA samples were reverse transcribed into cDNA by RevertAid^TM first strand synthesis kit for quantitative real-time polymerase chain reaction (qRT-PCR) according to the manufacturer's protocol. Primers for the target genes (p-53, caspase-3, caspase-9, Bax, and Bcl-2) and the reference housekeeping gene β-actin were designed using the Primer-Blast tool from the NCBI website online (http://www.ncbi.nlm.nih.gov/tools/primer- blast/index) and their sequences summarized in Table 1(Tab. 1). qRT-PCR was performed using an ABI7900HT Sequence Detection System (Applied Biosystems, USA) with SYBR Green PCR Master Mix and designed primers. Sequentially, qRT-PCR conditions were 95 °C for 5 min, 40 cycles of at 95 °C for 15 s, 57 °C for 30 s and 60 °C for 1 min. The expression levels of target genes were normalized to β-actin and the relative fold changes were calculated using the ΔΔCt method (Livak and Schmittgen, 2001[19]).

The data are expressed as the mean ± SD of three individual experiments performed in triplicate. The data were analyzed using IBM SPSS Statistics 20 software. Statistical analysis was performed by two-way ANOVA followed by Duncan's new multiple range test and P < 0.05 was considered statistically significant. The IC₅₀ values were determined from the dose-response curves using GraphPad Prisms 6.01 software.

To assess the effects of S. amplexicaulis extracts on cell growth, MTT and trypan blue assays were performed. As shown in Figure 1A-B(Fig. 1), DCM and MeOH extracts exhibited significant dose- and time-dependent cytotoxic activity against MCF-7 and WEHI-164 cancer cell lines compared to the untreated control cells (P < 0.001). However, no growth inhibition was observed following treatment with n-hexane extract. Furthermore, DCM extract inhibited the growth of cancer cells more powerfully than did the MeOH extract (P < 0.01). Considering all IC₅₀ values reported in Table 2(Tab. 2), MCF-7 cells were more sensitive to growth inhibitory effects of DCM and MeOH extracts compared with WEHI-164 cells at all tested time points. Interestingly, DCM and MeOH extracts revealed little cytotoxicity toward normal cell line HUVEC (IC₅₀ > 400 µg/mL) (Figure 1C(Fig. 1)). Microscopic cell count using a hemocytometer showed a remarkable decrease in the number of viable MCF-7 and WEHI-164 cells following exposure to DCM and MeOH extracts (Figure 2A-B(Fig. 2)). In addition, paclitaxel decreased the viability of MCF-7 and WEHI-164 cells to 59.2 % ± 0.4 and 83.5 % ± 0.7 in comparison to the control cells, respectively (data not shown).

To investigate whether DCM and MeOH extracts of S. amplexicaulis exhibit cytotoxicity due to apoptosis or necrosis, we used cell death detection ELISA kit. After 24 h of treatment with 48-h IC₅₀ concentrations of DCM extract, the apoptosis rate significantly increased from 3.8 % and 2.9 % in the control cells to 53.5 % and 40.7 % in MCF-7 and WEHI-164 cancer cells, respectively (P < 0.01) (Figure 3A-B(Fig. 3)). When the cells were treated with 48-h IC₅₀ concentrations of MeOH extract for 24 h, the percentages of apoptosis were 35.2 % and 25.5 % in MCF-7 and WEHI-164 cells, respectively (Figure 3A-B(Fig. 3)). However, DCM and MeOH extracts did not show any apoptotic or necrotic death in HUVEC normal cells (data not shown). Notably, our results implied that DCM extract induced a higher rate of apoptosis than that of MeOH extract in both cancer cell lines, particularly MCF-7 cells. Furthermore, TUNEL assay was performed to identify the presence of DNA fragmentation and confirm the induction of apoptosis in cells treated with DCM and MeOH extracts of S. amplexicaulis. As illustrated in Figure 4A-B(Fig. 4), the cells displayed dark brown stained nuclei following exposure to DCM and MeOH extracts, whereas no visible coloration was detected in the untreated control cells. We further examined the expression level of PARP cleavage in MCF-7 and WEHI-164 cells by Western blotting. Upon treatment with DCM and MeOH extract for 24 h, the expression of cleaved PARP was detected in both cancer cell lines (Figure 5(Fig. 5)). Overall, the results indicated that the cytotoxicity of DCM and MeOH extracts of S. amplexicaulis against MCF-7 and WEHI-164 cells is mainly mediated by apoptosis induction.

In order to evaluate the mRNA expression levels of apoptotic-associated genes including p-53, caspase-3, caspase-9, Bax, and Bcl-2 in MCF-7 and WEHI-164 cancer cells, qRT-PCR was carried out. As shown in Figure 6A-B(Fig. 6), treatment with 48-h IC₅₀ concentrations of DCM extract for 24 h led to significant enhancement in the mRNA expression levels of p-53, caspase-3, caspase-9, and Bax 3.2, 3.6, 4.5, and 5.6 fold in MCF-7 cells and 2.1, 2.5, 3.2, and 3.9 fold in WEHI-164 cells, respectively, when compared with the control cells (P < 0.01 and P < 0.001). Besides that, MeoH extract treatment could significantly increase the mRNA levels of p-53, caspase-3, caspase-9, and Bax 2.0, 2.3, 2.9, and 4.3-fold in MCF-7 cells and 1.6, 1.7, 2.0, and 2.2-fold in WEHI-164 cells. In contrast, the expression level of Bcl-2 mRNA significantly decreased by about 6.8 and 2.1-fold in MCF-7 cells and 1.8 and 1.4-fold in WEHI-164 cells treated with DCM and MeOH extracts, respectively (P < 0.01 and P < 0.001). These results are in line with our previous findings demonstrating a more alteration in the mRNA expression levels of the apoptotic-associated genes for MCF-7 cells treated with DCM extract compared to all other groups (P < 0.01). The results obtained herein suggest that DCM and MeOH extracts of S. amplexicaulis can induce apoptosis in MCF-7 and WEHI-164 cells through the mitochondrial pathway.