Κυριακή 18 Οκτωβρίου 2020

The anti-cancer effect of flaxseed lignan derivatives on different acute myeloid leukemia cancer cells

 

Highlights

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ENL has more prominent anti-leukemic effects than other flaxseeds lignans.

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ENL promotes selective anti-proliferative and pro-apoptotic effects on AML cells.

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The intrinsic apoptotic pathway is activated upon Enterolactone exposure.

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This study offers new insights into the health benefits of flaxseeds.

Abstract

Flaxseeds have been known for their anti-cancerous effects due to the high abundance of lignans released upon ingestion. The most abundant lignan, secoisolariciresinol diglucoside (SDG), is ingested during the dietary intake of flax, and is then metabolized in the gut into two mammalian lignan derivatives, Enterodiol (END) and Enterolactone (ENL). These lignans were previously reported to possess anti-tumor effects against breast, colon, and lung cancer.

This study aims to investigate the potential anti-cancerous effect of the flaxseed lignans SDG, END and ENL on acute myeloid leukemia cells (AML) in vitro and to decipher the underlying molecular mechanism. AML cell lines, (KG-1 and Monomac-1) and a normal lymphoblastic cell line were cultured and treated with the purified lignans. ENL was found to be the most promising lignan, as it exhibits a significant selective dose- and time-dependent cytotoxic effect in both AML cell lines, contrary to normal cells. The cytotoxic effects observed were attributed to apoptosis induction, as revealed by an increase in Annexin V staining of AML cells with increasing ENL concentrations. The increase in the percentage of cells in the pre-G phase, in addition to cell death ELISA analysis, validated cellular and DNA fragmentation respectively. Analysis of protein expression using western blots confirmed the activation of the intrinsic apoptotic pathway upon ENL treatment. This was also accompanied by an increase in ROS production intracellularly. In conclusion, this study demonstrates that ENL has promising anti-cancer effects in AML cell lines in vitro, by promoting DNA fragmentation and the intrinsic apoptotic pathway, highlighting the protective health benefits of flax seeds in leukemia.

Abbreviations

SDG
Secoisolariciresinol Diglucoside
END
Enterodiol
ENL
Enterolactone

Keywords

Flaxseeds lignans
Enterolactone
Acute myeloid leukemia
Anti-cancer
Apoptosis

1. Introduction

Flaxseed, also known as linseed, is one of the oldest crops grown worldwide and it is derived from the flax plant which is a part of the Linaceae family [1,2]. According to theplantlist.org (2020), flax belongs to the species Linum usitatissumum L., meaning “very useful” [3,4]. In fact, flaxseeds have many different functions whether utilized industrially or in food. Flax is the term mainly used for the dietary product [3]. One of the major traditional interest in flaxseed was its important use in the prevention and treatment of many diseases, including cancer. To investigate the organic components of flaxseeds, scientists have characterized a water‐soluble extract, and reported an abundance of proteins, water‐soluble carbohydrates, phenolic compounds, essentially secoisolariciresinol diglucoside (SDG), ferulic acid and p–coumaric acid [5]. Flaxseeds have the highest content of lignans of all plant foods, up to 800 times more than other plant foods used for human consumption [3,6].

Lignans are polyphenols that can act as both antioxidants and phytoestrogens. Their content in flaxseed is principally composed of SDG, pinoresinol, lariciresinol and matairesinol, respectively from highest to lowest concentration [7]. During intake of flax, the most abundant compound, SDG, is ingested, then metabolized and converted into two mammalian lignans, Enterodiol (END) and Enterolactone (ENL) by the gut microbiota [8,9]. In vivo, the lignans are acted upon in the upper part of the bowel, by the gastrointestinal microflora to release Secoisolariciresinol (SECO), the non-sugar moiety of SDG. Further hydroxylation and demethylation by the microflora lead to the production of the mammalian lignan END, which is then oxidized to produce ENL [7,[10], [11], [12]]. Once produced, END and ENL can either be efficiently absorbed into the bloodstream or conjugated by enterocytes into inactive compounds excreted in the gut [13].

These lignans constitute potential candidates for cancer therapy as previous studies have reported their anti-cancerous effects in vivo and in vitro. In the case of SDG, experiments have shown its anti-metastatic effect on melanoma cells in mice [14,15], as well as its anti-proliferative effect on human breast cancer cells in vitro [16]. The research flow of END is much slower as compared to ENL. The latter has been tested and studied extensively, with much fewer studies performed on END; however recently, Shin et al. investigated the pro-apoptotic effect of END on colorectal cancer cells and its possible metastasis inhibition [17]. On the other hand, END has been reported to exhibit antitumor effects in vivo on murine melanoma [18], and in vitro on different types of cancers namely breast cancer [[18], [19], [20]] and lung cancer [21].

Even though flaxseeds lignans have shown so far a significant potential in the prevention and treatment of various types of cancers, no studies have addressed their potential effects on leukemia. Acute myeloid leukemia (AML), one of the most commonly diagnosed types of acute leukemia in adults [22], results from clonal expansion of myeloid progenitors in the bone marrow or peripheral blood. Previous studies have indicated that the accumulation of genetic alterations with age is a factor for this disorder which validates the severity of the disease with age [23,24]. The main purpose of this study is to determine the effects of flaxseed derivatives on two AML cell lines, namely KG-1 and Monomac-1 cells, and to elucidate the mechanism involved in their activity.

2. Materials and methods

2.1. AML cell culture

Acute Myeloid Leukemia cell lines KG-1 and Monomac-1 cell lines were obtained from the American Type Culture Collection (ATCC) and Leibniz Institute (DSMZ), respectively. KG-1 (ATCC® CCL-246) is a leukemia cell line derived from the bone marrow of a male individual with erythroleukemia that developed into AML in 1978 [25]. Monomac-1 (DSMZ ACC 252) is a leukemia cell line derived from a male individual with acute monocytic leukemia at relapse in 1985 following myeloid metaplasia [26,27]. Both cell lines were kindly provided from the lab of Dr. Ralph Abi Habib (Lebanese American University) [28]. They were cultured in RPMI-1640 medium (Rosewell Park Memorial Institute, SIGMA-Aldrich, St. Louis, MO, USA), supplemented with 10 % FBS (Fetal Bovine Serum, Gibco, Dublin, Ireland) and 100 U/mL Penicillin and 100 μg/mL Streptomycin (Pen-Strep, Lonza, Basel, Switzerland) in a 5% CO2 humidified incubator at 37 °C. In order to check cell viability, the cells were visualized using the ZOE Fluorescent Cell Imager (Abcam, Cambridge, UK), and cell counting was done using Trypan Blue exclusion method before each experiment [29].

2.2. Healthy human B-Lymphoblasts culture

Human B-Lymphoblasts healthy (BL-H) cell line (GM03299) was obtained from the Coriell cell repository, extracted initially from an 8-year old healthy female. Cells were cultured under the same conditions as AML cell lines. BL-H cells were split every 3 days at a ratio of 1:2. Before any experiment, 10 μL of the cells were mixed with 10 μL of trypan blue and counted using a hemocytometer to check the cell viability.

2.3. Flax seed derivatives preparation

A concentrated stock of each of Enterolactone ENL (MW of 298.33 g/mol), Enterodiol END (MW of 302.36 g/mol), and Secoisolariciresinol diglucoside SDG (MW of 686.7 g/mol) (Sigma-Aldrich) was prepared with DMSO. Upon use, each sample of the concentrated stock with media (RPMI) to reach a concentration of 1 mM that will be used for dilutions on cells. The concentration of DMSO never exceeded 0.5 % (v/v).

2.4. Cell proliferation assay

Cells were plated in 96-well plates (1 × 105 cells/well), seeded overnight, treated with increasing concentrations of SDG, END, or ENL and incubated for 24 h, 48 h, or 72 h. Control cells were treated with RPMI media. BL-H cells were treated with the highest concentration of lignans used (100 μM) for 48 h to check whether the lignans exhibit cytotoxic effects on healthy cells. WST-1 cell viability reagent (Roche, Basel, Switzerland) was added after the respective incubation times, based on the manufacturer’s guidelines. Cell proliferation was assessed by spectrophotometry absorbance measurement at 450 nm using a Multiskan FC Microplate Photometer to identify metabolically active cells. The percentage of proliferation was then calculated relative to control untreated cells and displayed graphically [30].

2.5. Cell death ELISA for apoptosis detection

AML cells were plated and incubated overnight in a 12 well plate (1 × 105 cells/well), then treated for 24 h and 48 h with two increasing concentrations of ENL (40 μM–100 μM). Control cells were treated with RPMI media, whereas positive control cells were treated with 100 μM of etoposide (Abcam, Cambridge, UK). The experiment was done using the Cell Death ELISA kit (Roche, Basel, Switzerland), as previously described. Briefly, cells were extracted and lysed with incubation buffer before the isolation of the fragmented cytosolic DNA which was present in the supernatant of the samples after centrifugation. The extracted DNA was incubated in wells that were pre-coated with anti-histone monoclonal antibody before adding anti-DNA antibodies linked to an enzyme. The colorimetric substrate was then added and the changes in activity were measured spectrophotometrically at an optical density of 405 nm using the Multiskan FC Microplate Photometer. DNA fragmentation enrichment factor (absorbance of treated cells/ absorbance of non-treated cells) was calculated based on the manufacturer’s instructions [31].

2.6. Analysis of cell cycle using PI staining

AML cells were seeded and treated as described above. Cells were fixed overnight using ice-cold absolute ethanol, stained with PI (Abcam, Cambridge, UK) and the DNA matter was evaluated using the Guava EasyCyte™ System flow cytometer as previously described [32]. The distribution of cells in each cell cycle phase was determined by assessing the DNA content: sub-G0/G1 phase cells (Pre-G or dead cells) have <2n, G0/G1 phase cells have 2n, S phase cells have between 2n and 4n, and G2/M phase cells have 4n [33].

2.7. Detection of apoptosis using Annexin V staining by fluorescence microscopy

KG-1 cells were treated with ENL as described above and then stained with Annexin-V provided by the Annexin-V Fluorescein Isothiocyanate (FITC) Apoptosis Detection Kit (Abcam, Cambridge, UK). The cells were visualized under the ZOE Fluorescent Cell Imager using bright-field conditions as previously described [34].

2.8. Quantification of apoptosis by Annexin V/PI staining

KG-1 cells were seeded in a 6-well plate (2 × 105 cells/well), treated with ENL for 48 and incubated overnight and then stained with Annexin V and PI (Annexin V–FITC Apoptosis Detection Kit, Abcam, Cambridge, UK). The analysis was performed using the Guava EasyCyte™ System flow cytometer. Binding of Annexin V indicates the flipping of the exposed charged head groups of phosphatidylserine to the outer leaflet of the cell membrane, which is a hallmark of apoptosis. The cell membrane integrity dismisses PI in viable and early apoptotic cells, but not in late apoptotic and necrotic cells. Therefore, dual parameter FACS (Fluorescence-activated cell sorting) study allows, through dual staining, the differentiation between 4 distinct populations of cells in a sample: Ann-/PI- representing normal healthy cells, Ann+/PI- representing early apoptotic cells, Ann+/PI + representing late apoptotic cells, Ann-/PI + representing necrotic cells. These populations are represented on a four-quadrant graph with Annexin V binding on the x-axis and PI binding on the y-axis [35].

2.9. Analysis of protein expression through western immunoblot

KG-1 cells were plated and incubated overnight in 6-well plates (5 × 105 cells/mL). After treatment for 48 h with 2 increasing concentrations of ENL (40 μM, 100 μM, and control cells were treated with RPMI media) proteins were extracted using the Q-proteome Mammalian Protein Kit (Qiagen, Hilden, Germany) followed by quantification using the DC (Detergent Compatible) protein assay (Bio-Rad). Proteins were separated by SDS-PAGE, transferred to PVDF (Polyvinylidene fluoride) membranes followed by blocking with 5% skimmed milk as previously described [36]. Membranes were then incubated overnight in 2% milk with primary antibodies anti-β-actin (Santa Cruz, Biotechnology, Dallas, TX, USA), anti-Cytochrome-c (Ab 27129) (Elabscience, Houston, TX, USA), anti-cleaved PARP-1 (Ab 32064) (Abcam, Cambridge, UK), Bax (Ab 27044) (Elabscience, Houston, TX, USA), Bcl-2 (Ab 22004) (Elabscience, Houston, TX, USA), Caspase-9 (Ab 27061) (Elabscience, Houston, TX, USA), Caspase-8 (Ab 27060) (Elabscience, Houston, TX, USA), Caspase 3 (Ab 30004) (Elabscience, Houston, TX, USA), and p53 (Ab 1431) (Abcam, Cambridge, UK), all at a concentration of 1:1000. After incubation with the secondary antibody (1:2500) (Bio-Rad, Hercules, CA, USA), image development was done using the Clarity Western ECL Substrate (Abcam, Cambridge, UK) on the ChemiDoc machine (Bio-Rad, Hercules, CA, USA). Protein expression was quantified and calculated through blot bands analyses using the ImageJ computer program [37].

2.10. Cytochrome c release analysis through Cytosolic and Mitochondrial fractionation

KG-1 cells were plated and incubated overnight in 6-well plates (5 × 105 cells/mL). After treatment for 48 h with 2 increasing concentrations of ENL (40 μM, 100 μM, and control cells were treated with RPMI media), proteins were extracted using a protocol adapted from Abcam subcellular fractionation protocol. The cells were centrifuged and lysed using the fractionation buffer prepared [38]. Through a series of centrifugation steps, the cytosolic and mitochondrial fractions were separated, extracted, and quantified. Western immunoblotting was then carried out, as described in the previous section (2.9.), in order to detect the release of cytochrome c (Ab 110325) (Abcam, Cambridge, UK) from the mitochondria to the cytosol of the cells.

2.11. Reactive oxygen species detection

The DCFDA Cellular ROS Detection Assay kit (Abcam, Cambridge, UK) was used to detect the levels of Reactive Oxygen Species (ROS) in the cells as previously described [39]. Briefly, KG-1 cells were incubated with the cell-permeant 20,70-dichlorodihydrofluorescein diacetate (H2DCFDA), and then treated with increasing concentrations of ENL. Positive control cells were incubated with the ROS inducer Tert-Butyl Hydrogen Peroxide (TBHP) at a concentration of 75 μM. Fluorescent 20,70-dichlorofluorescein (DCF) was quantified by fluorescent spectroscopy using the Varioskan LUX multimode microplate reader (Thermo Fisher Scientific, Waltham, MA, USA).

2.12. Statistical analysis

All the experiments were done in triplicates and repeated three independent times. Statistical analyses were performed using GraphPad Prism 8 (San Diego, CA, USA). The values obtained were reported as the mean ± Standard Deviation SD and the p-values were calculated by t-tests or two-way ANOVA, depending on the experiment. Significant differences were reported, with * indicating a p-value: 0.01 < p < 0.05, ** indicating a p-value: 0.001 < p < 0.01, *** indicating a p-value: p < 0.001.

3. Results

3.1. Cytotoxic effect of Enterolactone as compared to SDG and Enterodiol

WST-1 cell proliferation reagent was used on the AML cell lines, KG-1 and Monomac-1 treated with SDG, END, and ENL, in order to assess the cytotoxic effect of these flaxseed lignans on the cells. Results show that SDG and END only possess a minimal anti-proliferative effect on KG-1 cells whereas ENL showed a prominent dose-dependent anti-proliferative effect on both KG-1 cells and Monomac-1 cells and a time-dependent anti-proliferative effect mainly on KG-1 cells. In fact, when treated with SDG or END, KG-1 cells did not show a significant decrease in proliferation, after 24 h of treatment (Fig. 1a), and on the contrary, showed an increase in proliferation after 48 h of treatment (Fig. 1b). Whereas in the case of Monomac-1 cells, there was an increase at both 24 h (Fig. 1c) and 48 h (Fig. 1d) with increasing concentrations of SDG or END. However, both KG-1 and Monomac-1 cells showed a gradual decrease in proliferation with the increase in ENL concentration and an increase in treatment duration, validating that its effect is dose-dependent in both cell lines and time-dependent in KG-1 cells (Fig. 2, Supplementary Table). After 24 h, 48 h, and 72 h, the percent proliferation of the cells treated with 100 μM of ENL significantly decreased, reaching 55 % (p-value<0.0001), 46 % (p-value<0.0001), and 29 % (p-value<0.0001) respectively in the KG-1 cell line (Fig. 2a), and 55 % (p-value<0.0001), 46 % (p-value<0.0001), and 40 % (p-value<0.0001) respectively in the Monomac-1 cell line (Fig. 2b). This cytotoxic effect of ENL was more prominent on the KG-1 cell line with the IC50 being around 60 μM at 48 h as compared to the Monomac-1 cell line with an IC50 around 90 μM at 48 h. In order to test the selective inhibitory effect of all 3 lignans on AML, healthy human B-lymphoblasts proliferation with all lignans after 48 h was tested with the results showing a slight non-significant change in cell proliferation at the highest concentration of the 3 lignans: SDG (100 μM) = 88.6 % (p-value = 0.2895), END (100 μM) = 114 % (p-value = 0.1669), ENL (100 μM) = 92.5 % (p-value = 0.6003) (Fig. 3). These results reveal a clear much more prominent effect of ENL on KG-1 and Monomac-1 cells indicating ENL’s selective inhibition of AML cell proliferation.

Fig. 1
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Fig. 1. Cytotoxic effects of SDG, END, and ENL on KG-1 and Monomac-1 cells after 24 h (a and c) and 48 h (b and d). Results show a minimal decrease/ increase in KG-1 and Monomac-1 proliferation with SDG and END treatment.

Fig. 2
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Fig. 2. The anti-proliferative effects of ENL on KG-1 cells (a) and Monomac-1 (b) cells after 24 h, 48 h, and 72 h. A large decrease in proliferation was shown with ENL treatment, especially after 48 h. Results also show a dose and time-dependent decrease in AML cell proliferation with an IC50 of 60 μM and 90 μM for KG-1 and Monomac-1 respectively. Significant differences are reported in the Supplementary file as Supplementary Table.

Fig. 3
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Fig. 3. Effect of SDG, END, and ENL on healthy human B-lymphoblasts after 48 h. No significant decrease in BL-H proliferation was detected upon treatment with SDG, END, and ENL.

Moreover, since SDG and END did not promote any prominent effects on cell proliferation, ENL was used to pursue further experimentation, using mainly 2 concentrations (40 μM and 100 μM) since the IC50 was shown to be between these 2 concentrations in both cell lines.

3.2. DNA fragmentation through cell death ELISA

In order to elucidate the mechanism of cell death induced by ENL, Cell death ELISA was performed. This assay is based on the fact that DNA fragmentation is a direct indication of apoptosis and can be assessed by quantifying the presence of cytosolic nucleosomes (fragments of DNA wrapped around a histone core). After collecting the cytosolic portions of KG-1 and Monomac-1 cells treated with increasing concentrations of ENL, the Sandwich ELISA quantifies the DNA using anti-histone antibodies coated on the well of the plate and then with anti-DNA antibodies tagged with the enzyme. The increase in DNA fragmentation is shown through the increase in the color intensity of the spectrophotometric indicator ABTS; the results obtained show almost a 2-fold increase in DNA fragmentation between KG-1 and Monomac-1 treated with 100 μM of ENL after both 24 h (p-value = 0.0318) and 48 h (p-value = 0.0247) (Fig. 4). These results validate the presence of a dose dependent increase in apoptosis in both KG-1 and Monomac-1 cells upon treatment with ENL. These results are consistent with the proliferation and cytotoxic effects shown previously.

Fig. 4
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Fig. 4. DNA fragmentation enrichment factor in KG-1 and Monomac-1 cells after 24 and 48 h of ENL treatment. Results showed almost a 2-fold increase to 2.2 and 1.7 respectively after 24 h and 48 h of ENL treatment in both KG-1 and Monomac-1 cell lines. Significant differences are reported, with * indicating a p-value: 0.01 < p < 0.05, ** indicating a p-value: 0.001 < p < 0.01, and *** indicating a p-value: p < 0.001.

3.3. Apoptosis analysis through Annexin V staining

In order to confirm the apoptotic effect of ENL on AML, Annexin V staining was analyzed qualitatively using the ZOE fluorescent microscope (Figs. 5); etoposide (100 μM) was used as a positive control. The results visually showed increase in Annexin binding to KG-1 cells treated for 24 h with 100 μM of ENL compared to control cells treated with RPMI media. The increase was visually more prominent after 48 h of ENL treatment. In order to verify the increase visualized, these results were later on quantified using the ImageJ program, and they revealed a significant increase in Annexin V binding with increasing concentration of ENL at 48 h (p-value = 0.0421 and p-value = 0.0180 for 40 μM and 100 μM respectively).The dose-dependent and time-dependent increase in Annexin V staining indicates the increased abundance of phosphatidylserine on the outer membrane leaflet of the AML cells, indicating a stronger activation of the apoptotic pathway with increased ENL concentration and treatment duration.

Fig. 5
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Fig. 5. Annexin V staining of KG-1 cells treated with ENL for 24 h (a) and 48 h (b), visualized microscopically and quantified. Results show a slight increase in Annexin V staining in KG-1 cells after 24 h of treatment but a more prominent significant increase after 48 h. Significant differences are reported, with * indicating a p-value: 0.01 < p < 0.05, ** indicating a p-value: 0.001 < p < 0.01, and *** indicating a p-value: p < 0.001.

3.4. Annexin V / propidium iodide staining analysis

For quantitative analysis of apoptosis, dual Annexin V and Propidium Iodide (PI) staining was performed using flow cytometry (Fig. 6). Annexin V is used to stain the phosphatidylserine on the outer membrane leaflet of the cell membrane, which is a major indicator of apoptosis. PI only stains DNA in cells with breached cell membranes, which is an indicator of necrosis or advanced apoptosis (late apoptosis). After 48 h of treatment with increasing concentrations of ENL, KG-1 cells showed a significant increase in proportion of cells in early and apoptosis (from 10.8 % in the control to 22.8 % in cells treated with 100 μM of ENL (p-value = 0.0035)) without any significant increase in the proportion of necrotic cells. The analysis of KG-1 cells treated with etoposide indicates the validity of the experiment in testing increase in apoptosis. This indicates that ENL possesses pro-apoptotic effects on KG-1 cells.

Fig. 6
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Fig. 6. Annexin V/PI staining of KG-1 cells treated with ENL ((a) control, (b) 40 μM, (c) 100 μM, (d) Etoposide (100 μM)) for 48 h; (e) a bar graph representation of the changes in percentage of cells in each quadrant. The results revealed a significant increase in apoptosis (whether early or late) in cells treated with the highest concentration of ENL (100 u M). Significant differences are reported, with * indicating a p-value: 0.01 < p < 0.05, ** indicating a p-value: 0.001 < p < 0.01, and *** indicating a p-value: p < 0.001.

3.5. Cell cycle analysis through propidium iodide staining

In order to examine the effect of ENL on the cell cycle progression of KG-1 AML cells, Propidium Iodide staining followed by flow cytometry was used. Upon treatment with increasing concentrations of ENL for 48 h, KG-1 cells showed a major shift in cell cycle phases distribution. In fact, a decrease in the number of cells in the S phase (from 26.7%–15.7 % (p-value = 0.0780)) and G2-M phase (from 17.6 % to 5.2 % (p-value = 0.0452)) is coupled to a significant increase in the proportion of cells in the pre-G0 stage (from 7.6%–35.8% (p-value = 0.0001)) which is a stage of very low DNA content (<2n), typical of extremely damaged cells and fragmented cell portions (Fig. 7). This indicates that ENL could be activating a pathway leading to cell death by fragmentation, possibly an apoptotic pathway.

Fig. 7
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Fig. 7. PI staining of KG-1 cells treated with ENL ((a) control, (b) 40 μM, (c) 100 μM) for 48 h. KG-1 cells showed a shift distribution in the cell cycle phases, revealing an increase in fragmentation in the pre-G phase. Significant differences are reported, with * indicating a p-value: 0.01 < p < 0.05, ** indicating a p-value: 0.001 < p < 0.01, and *** indicating a p-value: p < 0.001.

3.6. Apoptotic proteins expression through western immunoblot

To explore the molecular events leading to apoptosis, western immunoblotting was done to examine the change in protein expression in KG-1 cells as a result of 48 h treatment with the different concentrations on ENL (0 μM, 40 μM, and 100 μM) (Fig. 8). The pathways hypothesized were the intrinsic versus extrinsic apoptotic pathway, based on the change in protein expression. Primarily, actin was used as a loading control as it is a product of a constitutively expressed housekeeping gene. A significant increase in pro-apoptotic proteins was shown to occur upon treatment with 100 μM of ENL, with an increase in cleaved PARP (1.9-fold increase (p-value = 0.0027)), cytochrome c (2.7-fold increase (p-value = 0.0006)), the cleaved forms of caspase-9 (1.5-fold increase (p-value = 0.0415) and caspase-3 (2-fold increase (p-value = 0.0011)). A very minimal non-significant increase was detected for expression of p53 (p-value = 0.0566) and the cleaved form of caspase-8 (p-value = 0.4571). The significant increase in the Bax to Bcl-2 ratio (2.2 fold-increase (p-value = 0.0083)) also indicates the induction of apoptosis in KG-1 cells upon treatment with ENL.

Fig. 8
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Fig. 8. Expression of proteins extracted from KG-1 cells treated with ENL (0 u M, 40 μM, 100 μM) for 48 h. Increase in pro-apoptotic proteins was shown, with a significant increase in the Bax to Bcl-2 ratio, cleaved PARP, cytochrome c, cleaved caspase-9 and cleaved caspase-3. Significant differences are reported, with * indicating a p-value: 0.01 < p < 0.05, ** indicating a p-value: 0.001 < p < 0.01, and *** indicating a p-value: p < 0.001.

3.7. Cytochrome c Release from the Mitochondria to the Cytosol of the cell

The extraction and subcellular fractionation revealed, through western immunoblotting (Fig. 9a), the release of cytochrome c from the mitochondria into the cytosol as shown through a significant 1.8-fold increase in the cytosolic cytochrome c expression (p-value = 0.0250) versus a significant decrease in the mitochondrial cytochrome c expression (p-value = 0.0007) leading to the derivation of a significant increasing cytosolic/mitochondrial cytochrome c ratio (p-value = 0.0219) (Fig. 9b).

Fig. 9
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Fig. 9. (a) Western Immunoblotting of cytochrome c in the cytosolic versus mitochondrial fraction of the cell. (b) Graphical representation of change in the protein expression of cytochrome c release from the mitochondria to the cytosol of the cell with increasing concentration of ENL treatment of KG-1 cells for 48 h. The results indicate an increase in cytosolic cytochrome c versus a decrease in mitochondrial cytochrome c resulting in an increasing ratio of cytosolic/mitochondrial cytochrome c release. Significant differences are reported, with * indicating a p-value: 0.01 < p < 0.05, ** indicating a p-value: 0.001 < p < 0.01, and *** indicating a p-value: p < 0.001.

3.8. Reactive oxygen species analysis upon ENL treatment

In order to further describe the effects of ENL on AML cells, Reactive Oxygen Species (ROS) levels were measured in KG-1 cells using the DCFDA assay. KG-1 cells exhibited a weak yet significant increase in intracellular ROS levels upon treatment with ENL. In fact, a significant, yet minimal increase (1.3 fold-increase (p-value<0.0001)) in ROS levels was detected upon treatment with 100 μM of ENL. This indicates that ENL leads to an increase in ROS levels. The apoptotic effects of ENL may be partly attributed to the increase in ROS levels (Fig. 10).

Fig. 10
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Fig. 10. ROS levels change upon ENL treatment at different concentrations. Up to 1.3-fold increase in ROS production in KG-1 cells is detected upon treatment with the highest concentration of ENL (100μM). Significant differences are reported, with * indicating a p-value: 0.01 < p < 0.05, ** indicating a p-value: 0.001 < p < 0.01, and *** indicating a p-value: p < 0.001.

10.1016/j.biopha.2020.110884

4. Discussion

With flaxseeds/ linseeds used in ancient traditions as an antitumor product [40], this seed has shown promising therapeutic anti-cancerous effect, whether in vivo [16,41] or in vitro [42]. Based on these previous studies, flaxseed derivatives, namely SDG, END, and ENL, were analyzed to check their effects on AML cell lines in vitro. The primary results in this study exhibited that out of the three flax derivatives, ENL was the major compound that showed a dose-dependent and time-dependent anti-proliferative effect on the AML cells used, KG-1 and Monomac-1, with distinct IC50 concentrations of 60 μM and 100 μM respectively at 48 h of ENL treatment. In fact, many previous studies have shown that ENL is the most potent among the lignans derivatives of SDG, especially with anti-cancer properties on breast cancer [43]. Moreover, ENL has a higher bioavailability in the bloodstream upon its formation in the gut, knowing that the production, metabolism, absorption and excretion of these lignans are highly variable among individuals, due to the unique individual microflora and hepatoportal system [13,44]. Also, the effects of ENL observed on AML cells lines were similar to the effects observed on prostate cells (same range of effect for the concentration up to 100 μM) and to those observed in breast cancer cells like MDA-MB-231, knowing that lignans are known to be very active against breast cancer cells [45].

The effect of flaxseeds lignans was also investigated on healthy human B-lymphoblasts, and the results validated their selectivity in targeting cancerous cells rather than normal cells. This is in line with previous studies where ENL was shown to inhibit the proliferation of a prostate cancer cell line LNCaP as well as a modified prostate cell line WPE-1 with minimal effects on the normal prostate cells [46].

Throughout this study, since similar cytotoxic effects were observed on both AML cell lines, the rest of the experiments were carried out using KG-1 cells, in order to identify the mechanism of ENL cytotoxicity at 24 and/or 48 h even though the best and most effective therapeutic outcome was after 72 h of treatment, since the interest was to elucidate the early molecular mechanisms triggered by ENL leading to cell death. The results obtained show that apoptosis was the main mechanism involved in this effect. This was confirmed through an increase in cellular fragmentation, the turnover of the phosphatidylserine moiety towards the outer side of the cell membrane explaining the increase in Annexin V staining, in addition to an increase in DNA fragmentation which was determined through Cell Death ELISA and Propidium Iodide analysis after 48 h of treatment. These apoptotic trademarks are a result of a cascade of proteins that lead to the activation of apoptosis. A major role is played by the bcl-2 family proteins formed by a group of apoptosis-promoting proteins (bax, bak, bcl-xS) or apoptosis-inhibiting proteins (bcl-2, mcl-1, bcl-xL) [47]. These proteins regulate the permeability of the mitochondrial outer membrane leading to the irreversible release of intermembrane space proteins, and subsequent caspase activation and apoptosis [48]. In this study, cells treated with ENL exhibit a slight upregulation of Bax and a downregulation of Bcl-2, thus resulting in an increase in the Bax/Bcl-2 ratio. Additionally, the overexpression of the downstream molecule Cytochrome c confirmed the initiation of apoptotic mechanisms upon increased ENL exposure as compared to the control. Throughout this process, cytochrome c is primarily released from the mitochondria into the cytosol [49], which was verified through the increase in cytosolic/mitochondrial ratio of cytochrome c, followed by the activation of the caspase pathway [50].

In order to reveal if the apoptotic pathway is intrinsic or extrinsic, the activation of Caspase 9 and Caspase 8 was investigated. Upon cytochrome c release from the mitochondria, Caspase 9 is expected to be cleaved and activated. This caspase is the initiator of the intrinsic pathway, making it critical to the apoptotic machinery in many tissues. In fact, the results show an increase in the activation of Caspase 9 upon treatment with ENL, which is revealed through a significant increase in the cleaved form, therefore substantiating that the apoptotic pathway is intrinsic [51,52]. As opposed to the intrinsic pathway, which is activated by caspase 9, the extrinsic pathway is initiated by Caspase 8. Through this extrinsic apoptotic signaling pathway, pro caspase 8 becomes cleaved and activated through the recruitment of the receptor-specific adaptor protein Fas-associated death domain (FADD). This activation will either directly cleave and activate the downstream caspases or cleave the BH3 Bcl2 interacting protein; thus leading to the release of cytochrome c from the mitochondria and furthermore the activation of Caspase 9. This also leads to the activation of downstream caspases (including caspase 8) [[53], [54], [55]]. The western blot results showed a non-significant alteration in the cleaved form of caspase 8, at the highest concentration of ENL treatment, confirming that the apoptotic pathway triggered by ENL is mostly intrinsic rather than extrinsic. Following the activation of caspase 9, the signaling caspase typically leads to the apoptotic activation of caspase 3. Aside from the typical apoptosis hallmarks, caspase 3 plays a crucial role in apoptotic chromatin condensation and DNA fragmentation [56]. The results obtained upon ENL treatment showed an increase in the cleaved form of caspase 3, further confirming that the intrinsic apoptotic pathway is responsible for the pro-apoptotic effect of ENL in AML cell lines. Throughout this pathway, PARP is then catalyzed by caspase 3. PARP activation and subsequent cleavage have active and complex roles in apoptosis [57]. The results obtained confirmed the significant increase in cleaved PARP, further validating apoptosis induction in the cells upon ENL treatment.

Moreover, p53 plays a role in apoptosis, due to its role as a tumor suppressor gene. Its activation could occur in both the extrinsic and intrinsic pathways [58,59]. However, since the intrinsic mitochondrial pathway was shown to be more evident in this case, p53 dependent apoptosis would be expected to occur through the intrinsic pathway rather than the extrinsic death receptor pathway. This is expressed through the slight increase in p53, even though the increase was not significant. Previous studies have also reported p-53 independent apoptotic pathways which are consistent with our results [60,61]. Overall, the data obtained in this study validate the anti-cancerous effect of ENL on AML cells mainly through the intrinsic apoptotic mechanism.

Previous studies have shown the antioxidant properties of ENL in lipid and aqueous in vitro models, mainly at low concentrations (100 μM), by linoleic acid peroxidation assay and deoxyribose assay, where a decrease in ROS levels was detected [62,63]. Usually, oxidative stress caused by increased ROS levels leads to damage in DNA, proteins and cellular membranes, so antioxidant molecules tend to protect cells from this damage. However, in this study, using the DCFDA assay, a cellular-based model, we report a slight increase in ROS levels in KG-1 cells upon treatment with ENL. In fact, pinoresinol, another lignan in the flaxseed, was shown to possess antioxidant properties (radical scavenging) using an aqueous assay (Trolox Equivalent Antioxidant Capacity (TEAC) assay), but DCFDA assay performed on nematodes (C. elegans) showed no difference in ROS levels [64]. A possible explanation for the results of the ROS assay in our study can be the fact that ENL does not have any intracellular antioxidant effects on AML cells. The slight increase in ROS upon treatment of KG-1 cells with ENL can however be attributed to the process of apoptosis initiated in the cells after ENL treatment. Apoptosis itself can generate ROS resulting in part from the release of cytochrome c from the mitochondria and the disruption of the electron transport chain in the mitochondria [65]. The role of the generated ROS in apoptosis in not well established yet but some studies describe their role in oxidation of phosphatidylserine intracellularly before their translocation to the outer leaflet of the cell membrane [66].

5. Conclusions

The overall experimental approach of this study allowed the determination and validation of the selective anti-cancerous effect exhibited by the flaxseed lignan, ENL, on AML cells in vitro. ENL is a gut metabolite of SDG, the main lignan in flaxseeds. The results showed that this cytotoxicity occurs through the apoptotic pathway, specifically the intrinsic pathway. Furthermore, necroptosis induction is a possible pathway that has recently brought attention in research, thus testing the expression of key proteins like MLKL are needed [67], to confirm whether necroptosis is also activated by flax lignans in AML cells. Even though the results reported in our study clearly demonstrate the health benefits and protective effects of the flax seed lignan ENL in AML, further investigations are needed to confirm its effects in vivo.

Authors contributions

Data curation, project administration: ST, TH, JD and MHH. Formal analysis: ST and TH. Writing, original draft: ST. Conceptualization, Supervision, Funding Acquisition, Editing and finalizing manuscript: SR. All authors have contributed to the work, have read and approved the final version of the manuscript.

Funding sources

This study was financially funded by intramural funds from the Department of Natural Sciences (School of Arts and Sciences, Lebanese American University).

Declaration of Competing Interest

The authors report no declarations of interest.

Appendix A. Supplementary data

The following are Supplementary data to this article:Download : Download Word document (23KB)

References

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© 2020 Published by Elsevier Masson SAS.
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