Herbal Treatment with flaxseed oil induces apoptosis in cultured malignant cells

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Treatment with flaxseed oil induces apoptosis in cultured malignant cells
Alison L. Buckner,a Carly A. Buckner,a,b Sabine Montaut,a,c and Robert M. Lafreniea,b,c,d,∗
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Abstract
Flaxseed oil is widely recognized for its exceptional nutritional value, high concentration of fiber-based lignans and large amounts of ω-fatty acids. It is one of a generic group of functional foods that is often taken by cancer patients as a potential treatment. We have examined the anti-cancer effects of flaxseed oil by studying its direct effects on cancer cell growth in vitro. Treatment of a variety of cancer cell lines with flaxseed oil decreased their growth in a dose-dependent manner while non-malignant cell lines showed small increases in cell growth. Cells treated with a mixture of fatty acids, including α-linolenic acid, docosahexaenoic acid, and eicosapentaenoic acid and lignans including enterodiol and enterolactone was also able to decrease the growth of cancer cells. Treatment of B16-BL6 murine melanoma and MCF-7 breast cancer cells with flaxseed oil induced apoptosis as determined by changes in cell morphology, annexin V staining, DNA fragmentation and/or caspase activation. In addition, treatment with flaxseed oil also disrupted mitochondrial function in B16-BL6 and MCF-7 cells. These results indicate that flaxseed oil can specifically inhibit cancer cell growth and induce apoptosis in some cancer cells and suggests it has further potential in anti-cancer therapy.
Keywords: Cancer research, Cell biology, Cell culture, Cell death, Food science, Natural product, Nutrition, Flaxseed oil, Cancer cells, Cell death, Omega fatty acids
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1. Introduction
Flax (Linum usitatissimum), in the form of seeds or seed-derived oil, is a “functional food” recognized for its nutritional quality, high concentration of fiber-based lignans, and large amounts of ω−fatty acids [1, 2]. Flax was used to treat various health complaints in ancient Greece and Rome and was described in ancient Ayurvedic and Egyptian sources [3]. The recent popularity of flax has been bolstered by a number of studies describing health-promoting benefits such as reducing cardiovascular disease, decreasing cancer risk, inhibiting inflammatory activity, promoting gastrointestinal regularity, and alleviating menopausal symptoms [4, 5, 6]. Studies conducted in aged hens showed a diet rich in whole flaxseed decreased the risk and severity of ovarian cancer by decreasing proinflammatory prostaglandin and estrogen signaling pathways [7, 8]. The growth of estrogen-dependent MCF-7 breast cancer cells in xenotransplanted mice was also inhibited by feeding the mice flaxseed and involved decreasing hormone and growth factor signaling [9, 10]. A meta-analysis of the impact of flax on patients with breast cancer has indicated that regular consumption of flax decreased breast cancer risk, improved symptoms and survival, and was associated with improved mental health among breast cancer patients [11].
Flaxseed is one of the richest plant sources of α-linolenic acid, an ω-3 polyunsaturated fatty acid (PUFA) (49–60%), and contains a modest level of linoleic acid, an ω-6 PUFA (12–17%) [3]. It is suggested that a healthy diet should consist of a 1:4 ratio of ω-3 to ω-6 fatty acids. However, the average western diet which consists of a 1:10 to 1:30 ratio of ω-3 to ω-6 fatty acids has been linked to high levels of chronic disease and cancer [12, 13, 14]. Increasing the ω-3/ω-6 PUFA ratio has been shown to decrease cancer risk [15, 16] while increasing ω-6 PUFA has been shown to increase cancer risk [17, 18] in some human epidemiological studies, although not in all patient cohorts [6]. In vitro experiments and animal models suggest that diets rich in ω-3 PUFA can be protective against several cancers, such as colon or breast cancers [19, 20, 21, 22] while treatment with ω-6 fatty acids can increase cancer cell proliferation [15]. For example, mice fed diets enriched in α-linolenic acid, which increases plasma levels of α-linolenic acid and its metabolites eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), decreases the growth of transplanted prostate, colon, and breast cancer cells [23, 24, 25, 26]. In vitro studies have shown that treatment of cancer cells with ω-3 fatty acids such as α-linolenic acid, DHA and/or EPA can inhibit their growth and promote apoptosis. For example, treatment of cells with α-linolenic acid can inhibit the growth and promote apoptosis of cervical, pancreatic, colon and breast cancer cells [25, 26, 27, 28, 29, 30]. In addition, treatment of colon cancer cells [31] or MCF-7 breast cancer cells [32] with α-linolenic acid, EPA or DHA was able to induce apoptosis through a mitochondrial-mediated pathway. Other experiments have shown that α-linolenic acid, DHA, and EPA can affect cell survival by altering the expression of oxidative response signaling [33], MAP kinase and NF-kB survival pathways [27], or miR-21 expression [34].
Flaxseed is also a rich source of plant lignans, such as secoisolariciresinol diglucoside (SDG), which have been shown to block cell proliferation and reduce tumor growth in experimental models possibly by modulating estrogen receptor- or growth factor-dependent signaling [9, 35]. For example, treatment of breast cancer cells with flaxseed enriched in lignans, including SDG, was able to inhibit cell growth likely by modifying estrogen signaling and downregulating the expression of ERα and ERβ [10, 19]. However, it is thought that the combination of SDG and ω-3 fatty acids is important to mediate the anti-inflammatory and anti-cancer activities [9, 16, 36].
Our experiments investigated the effects of treatment of cultured cells with flaxseed oil in order to investigate the mechanisms underlying changes in cell growth. The results indicate that treatment with flaxseed oil preferentially inhibits the growth of malignant cell cultures and were able to induce apoptosis in treated cancer cells.


 

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2. Materials and methods
2.1. Tissue culture
B16-BL6 (murine melanoma) [37], MCF-7, MDA-MB-231, MDA-MB-468 (breast cancer), HeLa (cervical cancer), HEK293 (embryonic kidney cells) (obtained from the American Type Culture collection, ATCC, Manassas, VA), HSG (human epithelial cells [38]), and HBL-100 (breast epithelial cells [39]) (obtained from KM Yamada, NIH, Bethesda, MD) were maintained in Dulbecco's Modified Essential Medium (DMEM, Hyclone Logan UT) supplemented with 10% fetal bovine serum (Hyclone), 100 μg/ml streptomycin, and 100 U/ml penicillin (Invitrogen, Burlington, ON). The U937 and THP-1 (monocytic leukemia) (ATCC) cells were cultured in RPMI1640 medium supplemented with 10% fetal bovine serum and 100 μg/ml streptomycin, and 100 U/ml penicillin. The cells were cultured at 37 °C in 5% CO2. For experiments, cell cultures were treated with media containing different concentrations of flaxseed oil or sunflower oil.
2.2. Flaxseed oils and characterization
Flaxseed oils were obtained by extraction of flaxseeds or from commercial suppliers including Life Brand (Shoppers Drug Mart, Toronto, ON), Weber Naturals (WN Pharmaceuticals, Coquitlam BC), Swiss Natural (Valeant Pharmaceuticals, Laval, QB), and Polar Foods Inc. (Fisher Branch, MB). The Life Brand of flaxseed oil was used throughout the experiments. The sunflower oil was obtained from a commercial source. For analysis, the fatty acids were extracted and methylated according to Phippen et al. [40]. Oils were treated in 1 ml 0.5 M KOH in methanol at 60 °C for 1 h, 1 ml 1 M H2SO4 for a further 15 min, and then extracted into hexane. LC-MS analysis was performed on an Agilent G1311A/G1213A LC system and Agilent 6120 MS using a 2.1 × 250 mm Grace Smart C18, 60A, 5 μm column (Grace Discovery Sciences). The mobile phase was applied at 0.5 ml/min starting with 55% phase A (0.1% formic acid in water)/45% phase B (0.1% formic acid in acetonitrile) for 10 min and then ramped to 5% phase A/95% phase B for a further 20 min. The electrospray interface for the MS operated at 350 °C, capillary voltage was 4000V positive, 3500V negative, nitrogen gas was used at a flow rate of 10 l/min, and full scans were collected between m/z 100–1000. The amount of methylated fatty acids in the oils was determined from a standard curve of pure standards (FAME, Sigma-Aldrich Chemical Co., Oakville, ON) run under the same conditions.
Individual fatty acids including α-linolenic acid, docosahexaenoic acid, eicosapentaenoic acid, linoleic acid, oleic acid, and palmitic acid and the lignans enterodiol and enterolactone were purchased from Sigma-Aldrich Chemical Co. The fatty acids were solubilized in DMSO, combined in an equimolar mixture, with each component suspended in culture media at 10−5 M.
2.3. Trypan blue cell survival assay
The cell cultures (3 × 105/ml) were plated on 60 mm tissue culture plates on day 0. The cells were treated with 0.3% (v/v, low concentration) or 0.9% (v/v, high concentration) flaxseed oils on day 1 and were maintained without changing the media for the duration of the experiment. Replicate plates were harvested each day for 4–6 days the cell pellets suspended in PBS, pH 7.4 containing 0.015% trypan blue, and clear (live) cells were counted on a hemocytometer. Cell counts were performed in octuplet, the numbers for each experiment averaged, and the mean ± standard deviation for fold increase reported for 3 independent experiments.
2.4. Methyl tetrazolium blue viability assay
The viability of the cells was measured using the methyl tetrazolium (MTT) blue assay. The cells were plated on 96-well plates at 2 × 103 cells/well on day 0 and treated with the 0.9% (v/v) of flaxseed or sunflower oil on day 1 which was maintained for the duration of the experiment. Each day, 5 μl/well of a 5 mg/ml MTT solution was added to a replicate plate and after a 3 h incubation the media was removed, the cells solubilized in 100 μl DMSO, and the absorbance measured at 540 nm. Each experiment was performed using 6 wells/condition and the average determined. The percent inhibition of growth compared to cells treated only with media was determined and the mean for 3 independent experiments was reported.
2.5. Flow cytometry
Cell proliferation and apoptosis were measured by flow cytometry following staining with propidium iodide or annexin V. B16-BL6 and MCF-7 cells were treated with media or media containing 0.3% (v/v) or 0.9% (v/v) flaxseed or sunflower oil for 24–72 h and harvested with trypsin. For cell cycle analysis, the cells were fixed by incubation in cold 70% ethanol and then washed and incubated in 1 ml PBS, pH 7.4, containing 5 μl propidium iodide for 30 min and analyzed on a FC500 Beckman flow cytometer. For annexin V staining, the freshly harvested B16-BL6 cells were incubated in PBS, pH 7.4, containing 1% FBS for 45 min, washed, and then stained for 1 h in 1 ml PBS, pH 7.4, containing 5 μl of annexin V-FITC (Roche Diagnostics, Laval QB). The cells were then washed and analyzed on a FC500 Beckman flow cytometer. The % cells shown for each condition were calculated as the mean percent positive cells from 3 independent experiments.
2.6. Fluorescence microscopy – vital staining
Cell morphology was examined in cells stained with acridine orange and ethidium bromide. B16-BL6 and MCF-7 cells were plated overnight on glass coverslips and then treated with media or media containing 0.3% (v/v) or 0.9% (v/v) flaxseed or sunflower oil or 10−6 M camptothecin (Sigma-Aldrich Chemical Co.) as a positive control, for 24–48 h. Cells were also treated with an equimolar mixture of flaxseed oil components (fatty acids including α-linolenic acid, docosahexaenoic acid, eicosapentaenoic acid, linoleic acid, oleic acid, and palmitic acid and the lignans enterodiol and enterolactone) in culture media each at a concentration of 10−5 M. Cells were incubated in 10 μg/ml acridine orange and 10 μg/ml ethidium bromide for 15–30 min and visualized on a LSM5 Zeiss fluorescence microscope for apoptotic morphology. Alternately, live cell monolayers were incubated in 100 nM MitoTracker Red CMXRos (Molecular Probes, Fisher Scientific, Whitby, ON) or 100 nM Lysotracker Red DND-99 (Molecular Probes) for 15–30 min and visualized on a LSM5 Zeiss fluorescence microscope to determine the effects on organelle function.
2.7. Fluorescence microscopy – TUNEL
The TUNEL assay was used to detect DNA fragmentation [41]. The cells were plated on glass coverslip and then treated with media or media containing 0.3% (v/v) and 0.9% (v/v) flaxseed oil or sunflower oil. As a positive control for apoptosis, B16-BL6 cells were treated with 10−6 M camptothecin for 24–48 h and MCF-7 cells were treated with Ultraviolet radiation for 30 min and incubated for 24 h. The cells were fixed in 1 ml of 10% formaldehyde solution (Sigma-Aldrich Chemical Co.) for 5 min, permeabilized in 1 ml 1% Triton X-100 in PBS for 5 min and incubated with 50 μl of the TUNEL reagent (Roche Diagnostics) for 60 min at 37 °C. The coverslips were mounted on glass slides and visualized using an LSM5 Zeiss fluorescence microscope. The percent positively stained cells is shown for at least 5 independent microscope fields (>100 total cells counted/condition).
2.8. Immunoblot analysis
B16-BL6 or MCF-7 cells were treated with media, 0.3% (v/v) or 0.9% (v/v) flaxseed oil, and/or sunflower oil, or 10−6 M camptothecin for 48 h. The cells were harvested and lysed in RIPA buffer (1% Triton X-100, 0.5% SDS, 0.5% sodium deoxycholate, 150 mM sodium fluoride, 1 mM sodium orthovanadate) containing protease inhibitors (Roche Diagnostics). Total cell lysates were subjected to electrophoresis on 10% PAGE gels containing SDS and electrophoretically transferred to nitrocellulose membranes (Schleicher and Schuell, Xymotech Biosystems, Toronto, ON). The membranes were blocked by incubation in 5% BSA in Tris-buffered saline, pH 7.5 and 0.1% Tween-20 (TBST) and then incubated with antibodies against caspase-3 (#9668, Cell Signaling Technology, Danvers, MA), caspase-9 (sc-8355, Santa Cruz Biotech., Santa Cruz, CA) or PARP (sc-7150, Santa Cruz Biotech.) in 0.5% BSA in TBST. The filters were washed and incubated with appropriate anti-IgG-horseradish peroxidase conjugates (Santa Cruz Biotech.) and the HRP detected by incubation in Supersignal Reagent (Pierce Chemical Co., Rockford, IL) and exposed to Hyperfilm-ECL X-ray film (Amersham-Pharmacia, Oakville, ON). Densitometry was performed using AlfaEaseFC software (Protein Simple, San Jose, CA) and fold changes in band intensity were compared to untreated cells and reported for 3 independent experiments.
2.9. Caspase activity assays
The activity of caspase-2, 3, 6, 8, and 9 were assayed using the Apotarget Caspase Protease Assay kit (Invitrogen Corporation, Thermo-Fisher, Whitby, ON). B16-BL6 cells (5 × 106 cells/assay) were treated with media, 0.9% (v/v) flaxseed oil or sunflower oil for 24 h, harvested using trypsin, and lysed in 50 μl of chilled Cell Lysis Buffer for 10 min. The lysates were centrifuged at 16,000 × g for 1 min and the supernatant diluted to a protein concentration of 2 mg/ml. A mixture of 50 μl of cell lysate, 50 μl of 2× reaction buffer and 5 μl of 4 mM colorimetric substrate (200 μM final concentration) was incubated at 37 °C for 1.5 h in a 96-well plate and absorbance read at 405 nm. The fold increase in caspase activity was determined by subtracting the background (no lysate control) and then dividing the flaxseed- or sunflower oil-treated samples by the media-treated controls. The results are the mean of 3 independent experiments.
2.10. Statistical analysis
Data were expressed as the mean ± SD of at least three independent analyses. The mean values were subjected to a one-way ANOVA followed by a Tukey post hoc analysis to test significant differences (p < 0.05) between groups. Comparison between treatment and control samples for the caspase assays were performed using a Students t-test.
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3. Results
3.1. Flaxseed oil inhibits cancer cell growth
The effect of flaxseed oil on cell growth was determined for a variety of malignant and non-malignant cells lines. The cells were treated by adding different concentrations of flaxseed oil directly to the media and then the number of cells counted each day for 4–6 days. Treatment with flaxseed oil inhibited the proliferation of malignant cell lines in a dose-dependent manner. Treatment with 0.3% (v/v) flaxseed oil (low dose) for 4 days decreased the number of B16-BL6 cells by ∼50% and treatment with 0.9% (v/v) flaxseed oil (high dose) completely inhibited cell growth. Other malignant cells showed similar inhibition: 4 days after treatment with 0.3% (v/v) flaxseed oil, growth was reduced by 25% for HeLa, 50% for MDA-MB-231 and MDA-MB-468 cells, and 75% for MCF-7 cells while treatment with 0.9% (high dose) flaxseed oil reduced growth by 60% for HeLa cells, 75% for MDA-MB-231 and MDA-MB-468 cells, and 90% for MCF-7 cells (Fig. 1A). In contrast, treatment with flaxseed oil increased the number of the non-malignant human cell lines including HSG epithelial cells, HBL100 breast cells, and HEK293 embryonic kidney cells. The MTT viability assay also showed that the growth of malignant cells was inhibited by 40–60% after 4 days of treatment with flaxseed oil while non-malignant cells were not affected (Fig. 1B). In order to control for changes in the overall lipid content of the treatment media, sunflower oil was used as a control. Treating the cells with similar amounts of sunflower oil had no effect on the growth of any of the cells tested.

Treatment with flaxseed oil induces apoptosis in cultured malignant cells
 

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There is a recommendation of flax seed oil and cottage cheese being eaten together for this purpose, by Dr. Budwig:

The Flaxseed Oil And Cottage Cheese Mix – How Does it Work?

The flaxseed oil and cottage cheese mix is known for having a tremendously powerful impact on those who have cancer. What’s the science behind the combination?

First, watch this video and then keep reading:

About 3 minutes

How Does The Flaxseed Oil and Cottage Cheese Combination Work?

View: https://www.youtube.com/watch?v=ibpzNio862k


Dr. Budwig’s Research
According to Dr. Budwig’s research, solar electrons are exceptionally high in essential fatty acids. Because Flaxseed oil contains essential fatty acids, they have a negative electrical charge. In contrast, cottage cheese is loaded with sulphurated amino acids, and thus have a positive charge. This contrast provides the basis for an electrical circuit on a biological setting. Flaxseed oil is the electron donor; it stores energy and releases it on demand. The more energy stored, the healthier a person is.

The Flaxseed oil and Cottage Cheese Combination
Flaxseed on its own does not readily enter the cells of the body because it is not water soluble, but when mixed with cottage cheese, it becomes water soluble. So, this combination provides energy and oxygenation that the cells require. Experiments from as far back as the 19th century (before Dr. Budwig was even born), showed that animals that were fed a high-fat diet without protein died. Likewise, when they were fed a high protein diet without fat, they also died. However, when they were fed flax oil and protein together, they quickly recovered health and vitality. Hence, to supply the cells with energy and oxygen, we need the protein and healthy natural fats combination that the flaxseed oil and cottage cheese mix provide.

Read testimonials and success stories from our patients here.

Interestingly, Dr. Budwig also encouraged her patients to sunbathe and spend time outdoors. When a person consumes the flaxseed oil and cottage cheese and also sunbathes, the solar electrons within that person resonate with the photons/electrons from the sun increasing energy levels even more, which significantly contributes to the healing process. Dr. Budwig said the worse your health is, the more you need to spend time outdoors, even on cloudy days.

Do you or a loved one have cancer? If so, we can help. Contact us for a free consultation. We also invite you to head to our Instagram Profile. We have some very active followers who regularly share updates on how they are doing as the follow the Budwig Diet.

CLICK HERE to learn how to make the Flaxseed Oil and Cottage Cheese Mix. And for more information about the budwig mix, natural cancer treatments or Dr. Budwig herself, download our FREE guide.

 
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