Received: 13 October 2020 | Revised: 12 March 2021 | Accepted: 21 March 2021 DOI: 10.1111/jfbc.13719

FULL ARTICLE

Secoisolariciresinol diglucoside induces caspase-­3-­mediated apoptosis in monolayer and spheroid cultures of human colon carcinoma cells

Meltem Özgöçmen1 | Dilek Bayram1 | Gülçin Yavuz Türel2 | Vehbi Atahan Toğay2 | Nilüfer Şahin Calapoğlu2

1Department of Histology and Embryology, School of Medicine, Süleyman Demirel Abstract Universtiy, Isparta, Turkey Apoptotic effects of secoisolariciresinol diglucoside (SDG) in 2D and 3D cultures of 2 Department of Medical Biology, School SW480 cells were investigated. 40–­200 M SDG was used and IC50 values were de- of Medicine, Süleyman Demirel Universtiy, μ Isparta, Turkey termined for three different time intervals as 24, 48, or 72 hr for further experiments. BrdU, TUNEL, AIF, and caspase-­3 stainings were used. SDG inhibited cell prolifera- Correspondence Meltem Özgöçmen, Department of tion almost half and half for all time intervals in 2D and 3D cultures and also, induced Histology and Embryology, Faculty of apoptosis. Apoptotic cell percentages in the control group for 24, 48, and 72 hr were Medicine, Süleyman Demirel University, Isparta, Turkey. 27.00%, 29.00%, and 28.00%, respectively, while in the SDG treatment group were Email: [email protected] 59.00%, 61.00%, and 62.00%, respectively. In the spheroid cell culture, apoptotic cell

Funding information percentages in the control group for 24, 48, and 72 hr were 6.90%, 7.20%, and 7.10%, Süleyman Demirel University, Grant/Award respectively, while in the SDG treatment group were 19.50%, 19.50%, and 20.70%, Number: 4399 -­ D2 –­ 15 respectively. Caspase-­3 and AIF antibodies were used to indicate caspase-­dependent and -­independent apoptotic pathways. Significant increases were seen in both AIF and caspase-­3 stainings when compared to the control group but caspase-­3 stain- ing results were significantly greater when compared to the AIF staining at all time intervals (p < .05). To prove this, CASP3 gene expression was evaluated by RT-­qPCR. Unlike staining results, there was no statistically significant change at 24 hr in 2D and 3D cultures. But, significant upregulation at 48 (2.32-­fold in 2D and 2.46-­fold in 3D) and 72 hr (5.04-­fold in 2D and 6.45-­fold in 3D) were seen. Practical applications Colon cancer is one of the most prevalent cancer in the developed countries and its etiology is complex. Although the underlying mechanisms are mostly unknown, the link between diet and colon cancer is known and dietary habits can promote cancer or protect against it. In recent years, flaxseed is accepted as a significant functional food ingredient and feeding with it could help in to prevent cancer. Secoisolariciresinol diglucoside is a flaxseed lignan and is metabolized to mammalian lignans by the gut. In the present study, SDG was evaluated for its apoptotic effects in colon carcinoma cell line via monolayer and spheroid cultures using immunohistochemical and gene expression techniques. Findings of this study suggest that SDG may protect against cancers and in particularly against colon cancer and further investigations has to be carried out for detailed underlying mechanisms.

J Food Biochem. 2021;00:e13719. wileyonlinelibrary.com/journal/jfbc © 2021 Wiley Periodicals LLC. | 1 of 10 https://doi.org/10.1111/jfbc.13719 2 of 10 | ÖZGÖÇMEN et al.

KEYWORDS caspase-­3, colon carcinoma, monolayer culture, secoisolariciresinol diglucoside, spheroid culture, SW480

Highlights

• SDG treatment for 100 μM at 24 hr and for 150 μM at 48 and 72 hr significantly reduced the viability of SW480 cells. • SDG-­induced apoptosis and inhibited cell proliferation at all time intervals. • Apoptotic cells increased in AIF and caspase-­3 stainings in SDG treatment groups but this increase was significantly greater in the caspase-­3 compared to the AIF. • Relative gene expression of CASP3 was significantly upregulated at 48 and 72 hr in 2D and 3D cultures in a time-­dependent manner.

1 | INTRODUCTION 2 | MATERIALS AND METHODS

Colon cancer is one of the most common cancer in the developed 2.1 | Cell line, chemicals, and reagents world. Its etiology is complex, but diet is considered to be one of the important reason of it and researchers think that lots of people SW480 (Cat. No: ACC 313, DSMZ GmbH, Germany), RPMI 1640 are at risk of colon cancer because of diet style. The link between (Cat. No: 11875085, Gibco, USA), SDG (CAS no: 148244-­82-­0, Cat diet, especially dietary fiber intakes from whole grains, and colon no: S0202, purity ≥97.0%), Streptomycin (Cat. No: S9137), Penicillin cancer is well established. (Leduc et al., 2017; Pfister et al., 2015; (Cat. No: P3032), PBS, Trypan Blue, Trypsin-­EDTA and hydrogen Saura-­Calixto, 2011). To struggle colon cancer, patients seek sup- peroxide (Sigma St. Louis, MO, USA), Ethanol, DMSO, Xylol, Mayer's plementary therapies including the use of phytoestrogen-­rich foods Hematoxylin Solution, Methyl green, Apop Tag Apoptosis detection (Mali et al., 2017). As a result of this increased awareness in people, kit (Merck KGaA, Germany), 5% heat-­inactivated fetal bovine serum the demand for functional and healthy foods is increasing, which (Biological Industries, USA), Active Caspase-­3 polyclonal antibody (Cat. pushes the food industry to increase the production of these foods No: PA1-­26426), AIF polyclonal antibody (Cat. No: PA5-­96166), BrDU (Bernacchia et al., 2014). Staining Kit (Invitrogen, Thermo Fisher Scientific Inc., USA), PureZOL In recent years, flaxseed is accepted as a significant functional RNA Isolation Reagent, iScript Reverse Transcription Supermix, and food. Studies have shown that flaxseed could help to prevent from iTaq Universal SYBR Green Supermix (Bio-­Rad Laboratories Inc., USA) diseases like cardiovascular, obesity, and cancer (Imran et al., 2015; were purchased from mentioned companies. Touré & Xueming, 2010). Secoisolariciresinol diglucoside (SDG), a lignan, is abundantly found in flaxseeds. Lignans are similar in structure to the endog- 2.2 | Cell culture enous estrogen, estradiol, and to the estrogenlanti–­estrogen, tamoxifen, and appear to act as both anti-­estrogens and weak SW480 colon carcinoma cells were cultured as monolayer and sphe- estrogens (Al-­Jumaily & Mahdi, 2013; Dietz et al., 2016). SDG roid using RPMI 1640 with 10% FBS, 5% CO2 at 37°C and, 100 U/ is metabolized to enterodiol and enterolactone by the gut ml penicillin and 100 U/ml streptomycin were added. Cells were har- and has protective effects against hormone sensitive cancers vested by detaching with 0.05% trypsin-­EDTA then RPMI 1640 was such as breast, prostate, and colon (Al-­Jumaily & Mahdi, 2013; added and centrifuged. Maximum of 15 in-­house passages were used. Fuentealba et al., 2014; Mali et al., 2017) and is known as phy- Colon spheroid culture was prepared as described in our previous pub- toestrogen because of their potential antioxidant and estro- lication (Bayram et al., 2017). Biological replicates were conducted. genic activity (Ghazanfarpour et al., 2016; Nesbitt, 1999; Patel et al., 2012). Relatively few studies have been done to demonstrate cancer 2.3 | Experimental design and cytotoxicity preventive effects of SDG in in vitro, especially in spheroid cul- 5 ture, SDG was evaluated for its apoptotic effects in colon carci- A total of 5 × 10 cells per well were seeded to 6-­well plate and noma cell line (SW480) via monolayer and spheroid cultures using cells were treated with 40 to 200 μM SDG resolved in DMSO for immunohistochemical and gene expression techniques in the pres- three different time intervals as 24, 48, and 72 hr. After incubation, ent study. cytotoxicity was determined by trypan blue and was counted with ÖZGÖÇMEN et al. | 3 of 10 a hemacytometer. The effective concentration (IC50) of SDG was 2.7 | Gene expression determined as 100 μM for 24 hr and 150 μM for 48 and 72 hr. Cells were detached using cell scraper and total RNA purification was 7 performed using PureZOL RNA Isolation Reagent from 1 × 10 cells 2.4 | BrdU staining which were treated with 100 μM SDG for 24 hr and 150 μM SDG for 48 and 72 hr. RNA concentration and purity were measured in dupli- DNA synthesis analyses of BrdU Labeling index were used. A total of 1 cate in NanoDrop (Thermo Fisher Scientific, USA). Purity was about 5 × 10 cells per well were seeded to 24-­well plate and cells were treated ∼1.9 (OD260/OD280) and RNA integrity was assessed by agarose with 100 or 150 μM SDG according to time intervals on glass cover gel electrophoresis as described here (Çelik et al., 2020). Reverse slips for monolayer cultures. For spheroid culture, spheroids were re- transcription was completed using iScript Reverse Transcription. For moved, fixed, and washed with tap water. Then incubated with ethanol cDNA, 1 µg of RNA was used and synthesized cDNA was diluted series and xylene, embedded in paraffin, and sectioned by microtome 1/20 for each reaction. Triplicate real-­time qPCR was performed on and attached to a slide coated with polylysine. Sections were depar- CFX96 Real-­Time PCR Detection System (Bio-­Rad Laboratories Inc., affinized with xylene and were rehydrated in a series of graded alcohol. USA) with iTaq Universal SYBR Green Supermix. Amplification pa- Remaining procedure was same for both monolayer and spheroid cul- rameters were, 1 cycle: Polymerase Activation & DNA Denaturation tures and described in our previous publication (Bayram et al., 2017). at 95°C for 30 s and 40 cycles: Denaturation at 95°C for 5 s and Annealing/Plate Read at 60°C for 30 s.

2.5 | Apoptosis analysis 2.8 | Statistical analysis TUNEL assay was used for the detection of apoptosis. A total of 1 × 5 10 cells per well were seeded to 24-­well plate and treated with 100 p < .05 were considered significant. One-­way ANOVA and Dunnett's or 150 μM SDG. Then, the medium was removed and cells were fixed two-­tailed post hoc t test were used for evaluation in SPSS 18 soft- with paraformaldehyde and incubated in 2:1 ethanol–­acetic acid at ware. Data were presented as mean + standard error/standard −20°C for 5 min. For spheroid cultures, the procedure was the same deviation. qPCR data were evaluated using the ΔΔCt method via as for cell proliferation analysis. Remaining procedure was same for REST2009 software and ACTB used as housekeeping. both monolayer and spheroid cultures described in our previous publication (Bayram et al., 2018). 3 | RESULTS

2.5.1 | AIF and caspase-­3 staining 3.1 | Cytotoxicity

5 A total of 1 × 10 cells were seeded to 24-­well plate per well and SDG was found to reduce the viability of SW480 cells depending on were treated with 100 or 150 μM SDG. Following, medium was re- the concentration. Doses higher than 100 μM for 24 hr and 150 μM moved and washed with PBS. Cells were incubated in 70% ethanol for 48 and 72 hr inhibited 50% or more of the cells. Accordingly, and for immunohistochemistry, were incubated in PBS, washed with IC50 values were determined as 100 for 24 hr, and 150 μM for 48 distilled water and inhibited in 3% H2O2 in methanol. After 10 min, and 72 hr and remaining procedure was processed accordingly. cells were incubated for 30 min in 4 N-HCl.­ The remaining procedure Cytotoxicity was repeated with selected doses and similar results is the same as for apoptosis analysis. are obtained. The results were seen in Figure 1 (p < .05). Non-­specific binding was prevented using Ultra-­V Block. Primary antibodies, AIF (at 1:100 dilation) or caspase-­3 (at 1:20 dilation), were used for 1 hr. Following, the secondary antibody was used and 3.2 | BrdU staining then, sections were washed with streptavidin peroxidase for 20 min. Afterward, DAB was used as chromogen, sections were counter- As shown in Figure 2a, treatment with SDG significantly affected the stained with hematoxylin and were closed with ultramount sealer cell proliferation rate compared to the untreated control in all time and evaluated with light microscope. intervals of both monolayer and spheroid cultures as expected (p < .05). SDG reduced the proliferation by more than half. In monolayer cell culture, the proliferative cell percentages in the control group 2.6 | Hematoxylin and eosin staining for for 24, 48, and 72 hr were 59.00%, 60.00%, and 62.00%, respec- spheroid culture tively, while in the SDG treatment group were 27.00%, 28.00%, and 26.00%, respectively. In spheroid cell culture, the proliferative cell Samples were sectioned by microtome, were deparaffinized in xy- percentages in the control group for 24, 48, and 72 hr were 20.20% lene, and were stained with H&E. 19.50%, and 18.80%, respectively, while in the SDG treatment group 4 of 10 | ÖZGÖÇMEN et al.

FIGURE 1 Effects of SDG on cytotoxicity. Results were presented as Mean Cell Count ± SEM of three independent experiments for all time intervals and p < .05 was considered significant. *Statistically significant when compared to the internal control group were 6.90%, 7.20%, and 7.60%, respectively. Microscopic photo- 7.20%, and 7.10%, respectively, while in the SDG treatment group graphs can be seen in Figure 2b for monolayer and in Figure 2c for were 9.50%, 9.70%, and 9.90%, respectively. Microscopic photo- spheroid cultures. graphs can be seen in Figure 4b for monolayer and in Figure 4c for spheroid cultures.

3.3 | Apoptosis analysis 3.3.2 | Caspase-­3 staining Results presented that apoptotic cells were significantly enhanced by the treatment of SDG at all time intervals in both cultures when com- Caspase-­3-dependent­ apoptotic cells were increased in SDG treat- pared with the untreated control (p < .05, Figure 3a). In monolayer ment groups when compared with the untreated control in caspase-­3 cell culture, the apoptotic cell percentages in the control group for 24, staining at all time intervals in 2D and 3D cultures. (p < .05, Figure 5). 48, and 72 hr were 27.00%, 29.00%, and 28.00%, respectively, while Greater increase occurred in caspase-­3 staining compared to AIF, in the SDG treatment group were 59.00%, 61.00%, and 62.00%, re- suggesting that the main apoptotic pathway is caspase-­3-­dependent. spectively. In spheroid cell culture, the apoptotic cell percentages in In monolayer cell culture, the caspase-­3-­dependent apoptotic cell the control group for 24, 48, and 72 hr were 6.90%, 7.20%, and 7.10%, percentages in the control group for 24, 48, and 72 hr were 23.00%, respectively, while in the SDG treatment group were 19.50%, 19.50%, 25.00%, and 24.00%, respectively, while in the SDG treatment group and 20.70%, respectively. Microscopic photographs can be seen in were 59.00%, 60.00%, and 62.00%, respectively. In spheroid cell Figure 3b for monolayer and in Figure 3c for spheroid cultures. culture, the caspase-­3-­dependent apoptotic cell percentages in the control group for 24, 48, and 72 hr were 7.10%, 7.20%, and 7.10%, re- spectively, while in the SDG treatment group were 18.50%, 19.50%, 3.3.1 | AIF staining and 20.20%, respectively. Microscopic photographs can be seen in Figure 5b for monolayer and in Figure 5c for spheroid cultures. Caspase-­3-­independent apoptotic cells were increased slightly but significantly in SDG treatment groups when compared with the untreated control in AIF staining at all time intervals in 2D and 3D 3.4 | Hematoxylin and eosin staining for cultures. (p < .05, Figure 4) But, when compared with caspase-­3 spheroid culture staining results, apoptotic cell increase was greater in the caspase-­3 (p < .05). In monolayer cell culture, the caspase-­3-­independent ap- Spheroid structure was normal in the control groups unlike SDG optotic cell percentages in the control group for 24, 48, and 72 hr treatment. SDG treatment degenerated spheroid structures as were 23.00%, 25.00%, and 24.00%, respectively, while in the SDG shown in Figure 6. Quite smooth, round, and non-­dispersed sphe- treatment group were 30.00%, 32.00%, and 31.00%, respectively. roids were found in the control group (Figure 6a1), while the sphe- In spheroid cell culture, the caspase-­3-­independent apoptotic cell roids with impaired limits and rarely smooth and more dispersed percentages in the control group for 24, 48, and 72 hr were 7.10%, spheroids were found in the SDG treatment groups (Figure 6a2-­4). ÖZGÖÇMEN et al. | 5 of 10

FIGURE 2 Results of BrdU staining. Graphical demonstration (a). Results were presented as Mean Percentage ± SEM of three independent experiments for all time intervals and p < .05 was considered significant. *Statistically significant when compared to the SDG treatment group. Microscope images (×40) of monolayer (b1-­4) and spheroid (c1-­4) cultures. One picture as a representative for internal controls were used for all three time intervals. The abundance of brown stained nuclei with BrdU shows proliferation. Positive BrdU staining of control group (b1; c1) Proliferation decreased in SDG treated groups (b2-­b4; c2-­c4) when compared with control groups

3.5 | Gene expression assay the colon cancer is one of them for over three decades (Jenab & Thompson, 1996). These lignans significantly reduced the prolifera- There was no statistically significant change at 24 hr analyses of 2D tion in colon cancer cell lines (Sung et al., 1998). However, it is still and 3D cultures. CASP3 fold changes were almost the same as in the unclear how this mechanism exactly works. control in 24 hr groups. However, gene expression was significantly Bommareddy et al. reported that enterodiol and enterolactone upregulated at 48 (2.32-­fold in 2D and 2.46-­fold in 3D) and 72 hr (mammalian submetabolites of SDG) with different concentrations (5.04-­fold in 2D and 6.45-­fold in 3D) groups in a time-­dependent caused a significant increase in apoptosis and decrease in prolifer- manner. Result can be seen in Figure 7. ation in human colon adenocarcinoma Caco-­2 cells (Bommareddy et al., 2010). Likewise, Qu et al. studied enterolactone and enterodiol, alone or in combination in the SW480 cells and reported dose- ­and 4 | DISCUSSION time-­dependent decrease in cell numbers. Although cell proliferation was not affected at all doses, all treatments induced cell cycle arrest Human and animal studies have shown that SDG and its sub- at the S-­phase and enterodiol alone or combined treatments induced metabolites, which appeared to be very beneficial when used apoptosis (Qu et al., 2005). Same researchers also investigated SDG in appropriate doses, provide protection against cancer (Dietz in SW480 cells. Although researchers used almost 1/3 of our doses, et al., 2016; Ghazanfarpour et al., 2016; Mali et al., 2017; Mason treatment with SDG at 0–­40 μmol/L resulted in a dose-­ and time-­ & Thompson, 2014; Qu et al., 2005; Saggar et al., 2010) and dependent decrease in cell numbers and SDG demonstrated similar 6 of 10 | ÖZGÖÇMEN et al.

FIGURE 3 Results of TUNEL staining. Graphical demonstration (a). Results were presented as Mean Percentage ± SEM of three independent experiments for all time intervals and p < .05 was considered significant. *Statistically significant when compared to the internal control group. Microscope images (×40) of monolayer (b1-­4) and spheroid (c1-­4) cultures. One picture as a representative for internal controls were used for all three time intervals. The abundance of brown stained nuclei with TUNEL shows apoptosis. Apoptosis increased in SDG treated groups (b2-­b4; c2-­c4) when compared with control groups (b1; c1)

effects on cell growth, cytotoxicity, and cell cycle arrest when com- found a slight increase in AIF expression. It is also possible that the pared with its metabolite enterolactone but high-­performance liq- pathways affected by SDG at different times and doses may change, uid chromatography analysis, indicating that SDG in the media was but we think that SDG can activate both caspase-­3-­dependent and much more stable than enterolactone. However, authors claimed -­independent pathways in a limited way. Cregan, Dawson, and Slack that the inhibition of cell growth by SDG did not mediate by cytotox- mentioned that as a response to apoptotic stimulation, mitochondria icity, but by a cytostatic mechanism associated with cyclin A expres- can also release caspase-­3-­independent cell death effectors such as sion. (Ayella et al., 2010). Similar to our result, these studies showed AIF and Endonuclease G in tumor cells (Cregan et al., 2004). that SDG or its metabolites are effective against colon cancer. In our Similar to these results, Danbara et al reported that entero- study, casp3 staining and relative casp3 gene expression levels in- lactone suppressed human colon cancer (colo 201) cell growth, creased in time-­dependent manner in both monolayer and spheroid increased apoptosis and caspase-­3 protein, and decreased PCNA cultures. We are aware of the difficulty of interpreting our 24 hr re- protein. Antiapoptotic Bax and Bcl-­xl unchanged. In their in vivo sults. We hope to be able to explain the difference in 24h in CASP3 treatment, enterolactone inhibited tumor growth in colo 201 cell gene and protein expression via our ongoing further experiments. To transplanted athymic mice (Danbara et al., 2005). Shin et al. in- clarify this results, we believe that pro-­caspase-­3 should be investi- vestigated enterodiol's apoptotic effects in CT26 colorectal cell gated. Although the exact mechanism is not yet clear, SDG probably line. Enterodiol treatment resulted in an apoptosis rate of up to used caspase-­3-­dependent apoptosis pathway. However, we also 40%, decreased the expression levels of anti-­apoptotic proteins, ÖZGÖÇMEN et al. | 7 of 10

FIGURE 4 Results of AIF staining. Graphical demonstration (a). Results were presented as Mean Percentage ± SEM of three independent experiments for all time intervals and p < .05 was considered significant. *Statistically significant when compared to the internal control group. Microscope images (×40) of monolayer (b1-­4) and spheroid (c1-­4) cultures. One picture as a representative for internal controls were used for all three time intervals. The abundance of brown stained nuclei with AIF shows caspase-­independent apoptosis. Apoptosis slightly increased in SDG treated groups (b2-­b4; c2-­c4) when compared with control groups (b1; c1) and suppressed the migration of cells in a dose-­dependent manner but enhanced the expression of caspase-­3 (Ezzat et al., 2018). In but showed no cytotoxicity (Shin et al., 2019). Despite the fact that another study, SDG was investigated both in vivo and in vitro using different cancer cell lines, Buckner et al. suggested that flax- via enterolactone. In the triple-­negative breast cancer model in seed oil can specifically inhibit cancer cell growth and induce apop- C57BL/6 mice, 100 mg/kg SDG supplementation significantly re- tosis through caspase-­3-­dependent pathway in a dose-­dependent duced tumor volume and expression of Phospho-­NF-κ­ B p65 gene. manner while non-­malignant cell lines showed small increases in Additionally, ENL treatment inhibited viability, survival, and NF-κ­ B cell growth (Buckner et al., 2019). Likewise, in a combine treatment activity and gene expression in E0771, MDA-­MB-­231 and MCF-­7 of SDG and Bortezomib, Li and Yang reported increased caspase-­3 cells (Bowers et al., 2019). activity and the expression of caspase-­3 mRNA in A549 cell line Shah and Patel used 500 mg/kg SDG-­rich extract to demon- (Li & Yang, 2012). Ezzat et al. investigated six different flaxseed strate the anticancer effects in diabetic colon cancer-­induced rats cultivars in vivo and in vitro. One of the cultivars, which contains and suggested improvement in condition (Shah & Patel, 2016). the highest concentration of SDG, showed the most significant Salim et al. studied Egyptian flaxseed oil with two different doses cytotoxicity and increase in caspase-­3-dependent­ apoptosis in in rats with colon cancer and found significant decrease in distribu- two different cell lines. As in in vitro, dietary intake of flaxseed tion and total numbers of aberrant crypt foci, putative preneoplas- in vivo resulted in reducing the tumor volume, the expression of tic lesions, and the numbers and sizes of colon tumors. Flaxseed estrogen, insulin growth factor, progesterone, VEGF and MMP-­2, oil, reduced the abnormalities in the cells that secrete mucus and 8 of 10 | ÖZGÖÇMEN et al.

FIGURE 5 Results of Caspase 3 staining. Graphical demonstration (a). Results were presented as Mean Percentage ± SEM of three independent experiments for all time intervals and p < .05 was considered significant. *Statistically significant when compared to the internal control group. Microscope images (×40) of monolayer (b1-­4) and spheroid (c1-­4) cultures. One picture as a representative for internal controls were used for all three time intervals. The abundance of brown stained nuclei with Caspase 3 shows caspase-­dependent apoptosis. Apoptosis increased in SDG treated groups (b2-­b4; c2-­c4) when compared with control groups (b1; c1)

FIGURE 6 Microscope images of H-­E staining (×40). Spheroid structure of control group was normal (a1) unlike SDG treated groups (a2, a3, a4) kept them at near normal levels and as a result in a dose-­dependent mechanism has not yet been fully clarified, our results and some other manner suppressed DMH-­induced colon carcinogenesis in rats similar studies have suggested that these effect of SDG appears to (Salim et al., 2011). be via caspase-­3-­dependent apoptosis. As a result, SDG is a potential As a result, it is understood that SDG may protect against can- agent for protection and treatment against the cancer and further in- cers and in particularly against colon cancer. Although the underlying vestigations has to be carried out. ÖZGÖÇMEN et al. | 9 of 10

FIGURE 7 Effects of SDG treatment on relative gene expressions of CASP3. Results were presented as Mean Fold Change ± SEM of 3 replicates of 3 independent experiments for all time intervals and p < .05 was considered significant. *Statistically significant when compared to the internal control group

(MMA) cell lines. IOSR Journal of Pharmacy, 3(1), 18–­24. https://doi. ACKNOWLEDGMENTS org/10.9790/3013-31201824­ This research was supported by Süleyman Demirel University Ayella, A., Lim, S., Jiang, Y., Iwamoto, T., Lin, D., Tomich, J., & Wang, W. Scientific Research Support Coordination Unit with grant [4399 -­ D2 (2010). Cytostatic inhibition of cancer cell growth by lignan secoiso- – ­ 15]. lariciresinol diglucoside. Nutrition Research, 30(11), 762–­769. https:// doi.org/10.1016/j.nutres.2010.10.002 Bayram, D., Armagan, İ., Özgöcmen, M., Senol, N., & Calapoglu, M. CONFLICT OF INTEREST (2018). Determination of apoptotic effect of juglone on human All authors declare that they have no conflict of interest. bladder cancer TCC-­SUP and RT-­4 cells: An in vitro study. Journal of Environmental Pathology, Toxicology and Oncology, 37(2), 173–­181. https://doi.org/10.1615/JEnvi​ronPa​tholT​oxico​lOncol.20180​25226 AUTHOR CONTRIBUTIONS Bayram, D., Çetin, E., Kara, M., Özgöçmen, M., & Candan, I. (2017). The Meltem Özgöçmen: Conceptualization; Data curation; Formal apoptotic effects of silibinin on MDA-­MB-­231 and MCF-­7 human analysis; Investigation; Methodology; Validation; Visualization; breast carcinoma cells. Human & Experimental Toxicology, 36(6), 573–­ Writing-­original draft. Dilek Bayram: Conceptualization; Formal 586. https://doi.org/10.1177/0960327116​ 658105​ analysis; Funding acquisition; Investigation; Methodology; Project Bernacchia, R., Preti, R., & Vinci, G. (2014). Chemical composition and health benefits of flaxseed. Austin Journal of Nutrition and Food administration; Resources; Supervision; Validation; Writing-­ Sciences, 2(8), 1045. review & editing. Gülçin Yavuz Türel: Investigation; Validation; Bommareddy, A., Zhang, X. Y., Kaushik, R. S., & Dwivedi, C. (2010). Writing-­original draft. Vehbi Atahan Toğay: Formal analysis; Effects of components present in flaxseed on human colon adeno- Investigation; Visualization; Writing-­original draft. Nilüfer Şahin carcinoma Caco-­2 cells: Possible mechanisms of flaxseed on colon cancer development in animals. Drug Discoveries and Therapeutics, Calapoğlu: Funding acquisition; Resources; Supervision; Writing-­ 4(3), 184–­189. review & editing. Bowers, L. W., Lineberger, C. G., Ford, N. A., Rossi, E. L., Punjala, A., Camp, K. K., Kimler, B. K., Fabian, C. J., & Hursting, S. D. (2019). The flaxseed DATA AVAILABILITY STATEMENT lignan secoisolariciresinol diglucoside decreases local inflammation, suppresses NF B signaling, and inhibits mammary tumor growth. Research data are not shared. κ Breast Cancer Research and Treatment, 173(3), 545–­557. https://doi. org/10.1007/s10549-​ 018-­ 5021-­ 6­ ORCID Buckner, A. L., Buckner, C. A., Montaut, S., & Lafrenie, R. M. (2019). Meltem Özgöçmen https://orcid.org/0000-0003-3190-4486 Treatment with flaxseed oil induces apoptosis in cultured malignant cells. Heliyon, 5(8), e02251. https://doi.org/10.1016/j.heliy​on.2019. e02251 REFERENCES Çelik, D. A., Gurbuz, N., Toğay, V. A., & Özçelik, N. (2020). Ochratoxin A Al-­Jumaily, E. F., & Mahdi, A. A. (2013). In vitro cytotoxic study for causes cell cycle arrest in G1 and G1/S phases through p53 in HK-­2 partial pure lignan extract from flax seed (Linum usitatissirnurn) on cells. Toxicon, 180, 11–17.­ Rhabdomyosarrcoma (RD) and murine mammary adenocarcinoma 10 of 10 | ÖZGÖÇMEN et al.

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