Methyl donor S-adenosylmethionine (SAM) supplementation attenuates breast cancer growth, invasion, and metastasis in vivo; therapeutic and chemopreventive applications

DNA hypomethylation coordinately targets various signaling pathways involved in tumor growth and metastasis. At present, there are no approved therapeutic modalities that target hypomethylation. In this regard, we examined the therapeutic plausibility of using universal methyl group donor S-adenosylmethionine (SAM) to block breast cancer development, growth, and metastasis through a series of studies in vitro using two different human breast cancer cell lines (MDA-MB-231 and Hs578T) and in vivo using an MDA-MB-231 xenograft model of breast cancer. We found that SAM treatment caused a significant dose-dependent decrease in cell proliferation, invasion, migration, anchorage-independent growth and increased apoptosis in vitro. These results were recapitulated in vivo where oral administration of SAM reduced tumor volume and metastasis in green fluorescent protein (GFP)-tagged MDA-MB-231 xenograft model. Gene expression analyses validated the ability of SAM to decrease the expression of several key genes implicated in cancer progression and metastasis in both cell lines and breast tumor xenografts. SAM was found to be bioavailable in the serum of experimental animals as determined by enzyme-linked immunosorbent assay and no notable adverse side effects were seen including any change in animal behavior. The results of this study provide compelling evidence to evaluate the therapeutic potential of methylating agents like SAM in patients with breast cancer to reduce cancer-associated morbidity and mortality.


SUPPLEMENTARY MATERIALS Cell proliferation and viability assay
For proliferation assay, MDA-MB-231 and Hs578T cells plated in each well of 6-well plates were treated with 100 and 200 μM SAM or vehicle every second day for six days ( Figure 1A). The cells were trypsinized and counted at different time points starting from day 1 (no treatment) until the end of each treatment period (on day 7 from the initial plating) using a Coulter counter (Model ZF; Coulter Electronics, Hertfordshire, UK). For viability assay, cells were trypsinized, stained with 0.4% trypan blue (Sigma) and the viable cells were counted under a light microscope.

Cell migration/ wound healing assay
For in vitro wound healing analysis, MDA-MB-231 and Hs578T cells were treated with 100 and 200 μM SAM or vehicle following the treatment strategy mentioned in Figure 1A in the presence of regular cell culture media supplemented with 10% FBS in 10 cm Petri dishes. Afterward, the cells were trypsinized, and 500,000 cells were plated in each well of 6-well plates to form a monolayer and then wounded manually with a sterile 200 μL pipette tip in the center of each well forming a crosslike section of the wound. Cells were then washed twice with serum-free culture medium to get rid of the detached cells and debris. From this point, the cells were grown in the presence of culture media supplemented with 2% FBS and migrating cells were photographed at different time points (0, 6, 24, 48 hours after initial wounding) with an inverted bright field microscope under the 4X objective. Analysis and quantification of the cell-free area were carried out using the Image Pro-Plus software (Media Cybernetics, Inc, Rockville, MD, USA). The measurements obtained from the software were calculated as percentage wound healing using the equation: % wound healing = [1 − (wound area at T x h/wound area at T 0 )], where T x is the respective time point, and T 0 is the initial time immediately after the scratch.

Boyden chamber matrigel invasion assay
The changes in the invasive capacity of control and SAM-treated samples of MDA-MB-231 and Hs578T breast cancer lines was tested using a two-compartment Boyden chamber invasion assay (Costar Transwell, Corning Corporation, Sigma-Aldrich, Oakville, Ontario, Canada). The 8-μm-pore polycarbonate filters provided by the manufacturer were first coated with basement membrane Matrigel (50 μg/filter). Briefly, 2.5 × 10 5 viable cells from different treatment groups were resuspended in 100 μL of serum-free culture media and added to the upper chambers of the Matrigel Boyden wells. 800 μL of conditioned media was added to the lower chamber as the chemoattractant. After an incubation period of 18 hours at 37°C with 5% CO 2 , the invasion assay was stopped by moving the cells out of the incubator. The upper chamber was then washed with PBS to remove the non-invading cells from the top of the membrane. Then invading cells at the bottom of the membrane were fixed using 2% paraformaldehyde and 0.5% glutaraldehyde (Sigma-Aldrich) in 0.1 M phosphate buffer saline (PBS) with a pH of 7.4, at room temperature for 30 minutes. The membranes were then stained with 1.5% toluidine blue, washed with PBS, mounted onto glass slides, and the number of invading cells from randomly selected fields were counted under a light microscope and averaged.

Colony formation assay
To determine the effect of SAM on anchorageindependent growth, a measure of cellular transformation in vitro, soft agar colony formation assay was performed as previously described [1]. Briefly, 5 × 10 3 MDA-MB-231 and HS578T cells from control SAM-treated groups were counted and seeded in triplicates onto 6-well Petri dishes (BD Falcon TM ) in the presence of 4 ml of complete culture medium containing 1.5% agar (Bioshop ® ; Catalog# AGR001) solution. The culture medium was replenished every second day and the colonies formed after 2 weeks were counted under a light microscope.

Western blot
Cell lysates from control and SAM-treated cells were prepared using radioimmunoprecipitation assay buffer (RIPA) containing a cocktail of protease and phosphatase inhibitors. Equal amounts of proteins were loaded and resolved on a 15% sodium dodecyl sulfate-polyacrylamide gel and then transferred to polyvinylidene difluoride (PVDF) membrane using standard protocols. After transfer, non-specific binding was blocked by using 5% milk in www.impactjournals.com/oncotarget/

Oncotarget, Supplementary Materials 2017
Tris-buffered saline (TBS). Mouse monoclonal Bcl-2 antibody (Santa Cruz Biotechnology, Cat# sc-7382) was used to detect the anti-apoptotic Bcl-2 protein, and antimouse β-tubulin (BD Pharmingen cat# 556321) was used a loading control. The anti-mouse secondary antibodies used in this study were purchased from Bio-Rad, and the proteins were visualized by using an enhanced chemiluminescence detection kit (Amersham, GE Healthcare Life Sciences).

Novel object recognition test
For the novel object recognition test to assess whether SAM-treatment has any adverse effect on memory, animals were allowed to explore two identical copies of the first object in an open field enclosure for 10 minutes. By this time, the first object is now familiar to the mouse. Then there was a 60-minute break before the assessment of short-term memory began. During this period, the animals explored a familiar object (first object used during training) for eight minutes and a novel object (different shape) for 5 minutes and the time spent in exploration of the objects were recorded. Discrimination index (DI), a ratio of the time spent with the novel object in comparison with the familiar object, was determined and used for comparing between control and SAM-treated animals [2].

Open field test
The open field test to assess any potential increase in the anxiety levels upon SAM-treatment was conducted by placing mice from control and treatment group in an open field arena measuring 45 × 45 × 60 cm. The mice were tested individually for 5 minutes, their movement activity during this period was recorded using a camera and later analyzed by ANY-maze software. During the analysis stage, the open field arena was partitioned into nine squares having similar areas using the ANY-maze software. The square in the center was regarded as the central zone, and the surrounded areas were called the peripheral zone. Different parameters like the frequency and time spent in the center, total distance traveled within the central zone as well as the whole open field box along with their locomotion speed were calculated by ANYmaze, and the results were shown in bar graphs.