Main Research Findings

1) Treatment with S-4 in ovariectomized female rats resulted in reduced body fat and the prevention of bone loss.

2) S-4 is able to negatively regulate the PI3K/Akt/mTOR signaling pathway resulting in a display of potent anti-cancer activity.

 

Selected Data

1) In the study conducted by Kearbey et. Al, the research team obtained 120 female Sprague-Dawley rats from Harlan Biosciences. The animals were given ad libitum access to food and water and were randomly housed three animals to a cage while being kept on a strict 12 hour dark, 12 hour light cycle. At 23 weeks of age the animals underwent an ovariectomy (OVX) or a sham-operation prior to random assignment into one of 12 treatment groups. The treatment groups were defined as follows: 1) OVX + S-4 (0.1 mg/day); 2) OVX + S-4 (0.3 mg/day); 3) OVX + S-4 (0.5 mg/day); 4) OVX + S-4 (0.75 mg/day); 5) OVX + S-4 (1.0 mg/day); 6) OVX + S-4 (3.0 mg/day); 7) OVX + DHT (1 mg/day); 8) OVX + S-4 (0.5 mg/day) + bicalutamide (1.0 mg/day); 9) OVX + vehicle; 10) intact + S-4 (1 mg/day); 11) intact + DHT (1 mg/day); 12) intact + vehicle. It is important to note that the researchers administered DHT treatment to both intact and OVX animals to act as a positive control group and highlight effects of a pure steroidal androgen on skeletal muscle and bone tissue. Other negative control groups were defined as intact and OVX animals receiving treatment with a vehicle, as well as a group that co-administered the antiandrogen, bicalutamide, and S-4. The purpose of the co-administration group was to delineate the AT-mediated effects in comparison to AR-independent effects of the SARM. Daily dosing solutions were prepared by dissolving the proper treatment in DMSO and diluting in PEG 300; the doses were subcutaneously injected daily, for 120 days [1].

The animals were weight and DEXA scans were performed on day 0 and day 120 in order to measure changes in total bone mineral density (BMD), percent fat mass (FM), body weight (BW), bone mineral content (BMC), bone mineral area (BMA), and lean mass (LM). Immediately after the day 120 DEXA scans, groups 1-11 were euthanized while the lumbar vertebra, femur, and tibia were extracted. Group 12 was euthanized on day 210 and bone parameters were excised and evaluated by scanning through a 3-inch deep room temperature water bath in order to simulate soft tissue. Additionally, the proximal femur, distal femur, proximal tibia, L2-L4 vertebrae, and L5-L6 vertebra were analyzed for BMD through the use of DEXA scans.

The final assessment required the research team to extract the right femurs from Group 5: OVX + 1.0 mg/day S-4, Group 6: OVX + 3.0 mg/day S-4, Group 7: OVX + 1.0 mg/day DHT, Group 9: OVX control, Group 10: intact + 1 mg/day S-4, and Group 12: intact control, in order for pQCT analysis and biomechanical testing to occur. The femur was initially analyzed at the mid-shaft and distal regions. Scout scan views were used to determine the length of the femur while the mid-shift region was chosen at 50% of the length of the femur and the distal region was chosen at 20% of the length of the femur. Additionally, a 0.5 mm perpendicular slice of the long axis of the femur was used for analytical purposes.Total and cortical BMC, BA, and BMD, as well was cortical thickness, periosteal perimeter, and endosteal perimeter were all determined at the mid-shaft. On the other hand, total and trabecular BMC, BA, and BMD were determined at the distal region of the femur [1].

Following pQCT analysis, the whole femur was used in a three-point bending test. The femur was placed on a three-point bending fixture with the anterior side of the bone facing down into an Instron Mechanical Testing Machine. The length was set accordingly while the upper loading device was aligned to the center of the shaft in order for a load to be applied at a constant displacement until the femur broke. The testing machine measured maximum load, stiffness, and energy absorbed. The research team calculated the axial area moment of inertia from the data collected from the pQCT analysis. Finally, statistical analyses were performed by Fisher’s Protected Least Significant Difference test for multiple comparisons [1].

2) The research team of Yavuz et. Al examined the potential of S-4 to elicit anti-cancer effects in hepatocellular carcinoma (HCC) cell lines. The first step of the study was to obtain HCC cells lines from the SK-HEP and HEP-3B stocks. Both cell lines were cultured in Eagle’s minimal essential medium (EMEM), supplementation with 10% heat-activated FBS, and 1% Penicillin/Streptomycin. The medium was replaced every other day while cells were passaged at 80% confluent. In the following assays performed, the research team utilized 0.4% Trypan Blue solution via Thoma chamber to could the sells, while the samples were seeded in 3 replicates for reproducibility purposes.

25 mg of S4 was purchased from Sigma Aldrich and was solubilized in 2.26 ml DMSO in order to produce a 25 mM stock solution which was then aliquoted and stored until further use. Stock S4 was further diluted in a medium in order to collect tested concentrations ranging from 0.0001 mM to 0.4 nM. After S4 treatment cellular viability was investigated through cell proliferation assay. In order to determine cytotoxicity of the SARM, cells were seeding in a 96-well plate in 100 uL of culture medium. The cell plates were incubated for 24 hours then the culture was replaced with varying concentrations of S-4 containing medium. The culture medium was used as a negative control while the medium containing 10% DMSO was used as the positive control. After all cells were properly prepared, an MTT assay was performed while the half-minimal inhibitory concentration values for 24- and 48-hour treatments were calculated [2].

In addition to a cell proliferation and MTT assay, the researchers performed a colony formation assay (CFA) in order to determine the ability of the cells to form colonies following treatment with S-4. The cells were seeded in a 96-well plate in a 100 uL of medium. The cells were incubated for 24 hours before the culture medium was replaced with a medium containing S-4. The S-4-free culture medium was used for a negative control while a 10% DMSO-containing medium was used as the positive control. The medium was replaced every other day and the cells were cultured until the negative control group reached 80% confluency. This was followed by a wound healing assay performed to assess the migration capacity of the cells upon drug administration. Cells were seeded in a 24-well plate in 1 mL of medium. The cells were incubated for 24 hours then the medium was replaced with S-4-containing medium. After an additional 24 hour incubation period, wounds were created on the cells by scratching them with a pipette tip; the scratches were photographed and analyzed following creation of the wound [2].

These tests were followed by a soft agar assay, used in order to determine the tumorigenicity of the cells after treatment with S-4. Culture medium or medium containing S-4 were used for control and treatment groups; the cells were cultured for three weeks and the upper layer was replaced twice a week. At the end of the assy, spheroids were photographed and analyzed using ImageJ software. The next assessment was an Annexin-V assay conducted by seeding cells in a 12-well plate in 1 mL of a medium. The medium was changed to S-4-containing medium after the cell cultures were incubated for 24 hours. Cells were incubated for an additional24 hours followed by harvesting and washing with 1 mL of cold PBS; washed cells were suspended in 100 uL of Annexin-binding buffer followed by n5 uL Alexa Fluor488 dye and 1 uL 100 ug/mL of PI solution. Cells were incubated again for 15 minutes prior to analysis using flow cytometry.

A final EdU assay was conducted in order to detect the rate of cell proliferation. THe cells were seeded in a 96-well plate and incubated for 24 hours. All mediums were then replaced with S-4-containing medium or a culture medium in order to form control and treatment groups. After 24 hours of incubation the cells were incubated again for 2 hours with EdU at a final concentration of 10 uM. Cell cultures were then fixed in 150 uL of 100% methanol for 20 minutes followed by washing with 100 uL PBS containing 3% BSA. 100 uL of saponin-based permeabilization reagent was added to the culture and the cells were incubated for 20 minutes, followed by washing with PBS containing 3% BSA. After these procedures were followed the cells were labeled with Hoechst dye containing 100 uL of PBS followed by fluorescent microscopy in order to efficiently count EdU and Hoechst-positive cells [2].

Finally, a real-time qRT-PCR was conducted using the PI3K/Akt/mTOR gene panels. Cells were seeded to a 6-well plate sustained in 2 mL of medium which was replaced the next day with a medium that did or did contain a 24-hour IC50 S-4 concentration for each cell line. Cells were harvested after 24 hours of incubation followed by the isolation of RNA. Isolated RNA was converted to cDNA so qRT-PCR could be performed using human mTOR signaling RT2 Profiler PCR Array. The researchers thought it was important to know that all experiments were performed at least three times to verify conclusions. Data normality was checked using the Shapiro-Wilk test while the one-way ANOVA and Tukey’s post-hoc analysis was used to assess multi-variable data [2].

 

Discussion

1) Results of the study conducted by the research team of Kearby et. Al determined that ovariectomy leads to decreased BMD in comparison to the intact control group. Treatment with 0.1 mg/kg doses of S-4 led to partial or full prevention of BMD loss in OVX animals. When S-4 was administered with the anti-androgen, bicalutamide, the effects elicited by the SARM were prevented. This indicated that the androgen receptor is crucial to regulation of bone response to the drug. Additionally, body weight and body composition was observed through DEXA scans. OVX groups exhibited greater body weight than the intact groups, suggestions that animal growth is influenced by estrogen-deprivation. Treatment with 3 mg/day of S-4 led to further increases in body weight in OVX animals, however S-4 administration in intact animals resulted in a decrease in body weight in comparison to OVX and intact controls [1].

Percent fat mass was also measured through DEXA scans. The OVX control exhibited higher levels of FM than intact control groups. Additionally, S-4 treatment resulted in dose-dependent decrease in FM to a similar level to the intact controls. Vertebral bone loss was also measured by DEXA analysis. L5YL6 vertebrae were extracted to observe vertebral BMD. S-4 treatment led to a dose-dependent reduction in bone loss. The 3 mg/day dose prevented bone loss completely while the 0.5 and 1 mg/day doses partially prevented bone loss. The elicited effects were inhibited when the anti-androgen bicalutamide was administered in conjunction with S-4 [1].

Figure 1: Changes in BMD in response to different treatments

The femurs of the rats were analyzed by pQCT at the mid-shaft for cortical thickness, periosteal circumference, cortical content, and cortical density, and at the distal femur for trabecular BMD. Following OVX, S-4 treatment led to an increase in cortical thickness above the levels reported in the intact control groups. Additionally, S-4 was able to completely inhibit the loss of cortical content at the femoral midshaft after OVX was completed. The 3 mg/day dose of S-4 led to an increase in cortical content, however, the change was deemed insignificant, however, S-4 treatment in OVX rats prevented a decrease in periosteal circumference. While cortical thickness was increased in intact animals administered S-4, periosteal circumference was decreased.

pQCt was also used to measure cortical bone mineral density (CD) of the femoral mid-shaft; S-4 treatment prevented the OVX-induced loss of CD. In comparison to OVX and intact controls, CD was increased in intact animals receiving 1 mg/day of S-4. OVX also led to significant trabecular bone loss in the distal femur. S-4 partially prevented this loss; in intact animals S-4 treatment was shown to increase trabecular BMD to a higher level than the intact controls. This indicates that S-4 promotes anabolic activity in the trabecular bone regions. Furthermore, a three-point bending analysis was used to measure biomechanics strength. S-4 treatment in OVX animals was found to prevent the loss of femoral biomechanical strength [1].

Figure 2: pQCT analysis of the femur. A) cortical thickness, B) periosteal circumference, C) cortical content, D) cortical bone density, E) trabecular density

Figure 3: Maximum load determined by three-point bending evaluation.

2) MTT assay was the first evaluation performed examining the cytotoxicity of S-4 on HCC cells. When S-4 was administered to HEP3-B cells over a 24 hour time period, cell viability was decreased at all testing concentrations. For concentrations of 0.05 and 0.1 mM viability of HEP-3B cells were found to be 52 and 29%, respectively. When treated with S-4 for 48 hours, HEP3-B cell viability decreased to 58% with a concentration of 0.05 mM and further to 40% with a concentration of 0.1 mM. When treated with 0.2 mM of S-4 only 9% of cells survived while 0% survived following treatment with 0.4 mM of S-4 [2].

Figure 4: MTT assay assessed S4 cytotoxicity

Similar to the HEP-3B cells, SK-HEP-1 cells were incubated in an S-4-containing medium for either 24 or 48 hours. This process resulted in the significant diminishment in cell viability for S-4 concentration from 0.05 mM to 0.4 mM. The viability of SK-HEP-1 cells was decreased to 87% when 0.05 mM of S-4 was administered. 60% viability was seen with concentrations of 0.1 mM S-4, however, treatment with 0.2 mM S-4 resulted in only 29% viability. Complete toxicity of S-4 on SK-HEP-1 cells was observed after 24 hours of treatment with 0.4 mM S-4. Similar results were reported when the SK-HEP-1 cells were treated with S-4 for 48 hours; the 0.05 mM S4 treatment group experienced a decrease in cell viability to 64%. The 0.1 mM S4 treated reduced viability of SK-HEP-1 cells to 6%. The cells were not viable following administration of S-4 in concentrations of 0.2 and 0.4 mM during the 48 hour incubation period [2].

Additional functional tests were conducted treating HCC cells with S-4. MTT data provided the information needed to determine the S-4 concentration that inhibited half of the cell population (IC50). The IC50 values for 24 hour treatment with S-4 was found to be 0.065 and 0.14 mM for HEP-3B and SK-HEP-1 cells, respectively. The IC50 values for 48 hour treatment with S-4 was found to be 0.070 and 0.058 mM for HEP-3B and SK-HEP-1, respectively.

The effects of S-4 treatment on colony-forming ability of HEP-3B cells were evaluated through the use of 0.025, 0.05, and 0.1 mM of the SARM. HEP-3B colony-forming capacity inhibited the effects of S-5 treatment; the 0.025 mM group experienced a 1.5-fold decrease in HEP-3B colonies. Furthermore, both the 0.05 and 0.01 mM concentrations of S4 resulted in a 6- and 13-fold respective decrease in HEP-3B colony number. Soft agar assay was the next evaluation performed in order to determine the tumorigenicity of SK-HEP-1 cells. The end of the assay found that the control cells formed 78 spheroids measuring 108 um in ferret diameter while S-4 treatment formed 13 spheroids with 1 um feret diameter [2].

A wound healing assay was conducted next in order to measure migration capacity. The assay emphasized the anti-migratory impact of S-4 treatment that occurred 6 hours after 10% of the scratch control cells were closed. Additional data revealed that following an 18 hour incubation period, S-4 treatment led to 7% wound closure. This was compared to the control group that experienced a 1.5-fold decrease in migration capacity in the cells treated with S-4. EdU stainings were completed next in order to evaluate the rate of apoptosis and proliferation. Apoptosis in SK-HEP-1 cells experienced a 1.5-fold increase following treatment with S-4, while a 2.5-fold decrease in proliferation rate was observed. Overall, the researchers were able to conclude that S-4 has a strong anti-carcinogenic impact on HEP-3B cells through the stimulation of apoptosis and decreasing migration capacity and colony-forming ability. Additionally, anti-carcinogenic activity on SK-HEP-1 cells was elicited through the promotion of apoptosis and suppression of migration and proliferation [2].

 

Disclaimer

**LAB USE ONLY**
*This information is for educational purposes only and does not constitute medical advice. THE PRODUCTS DESCRIBED HEREIN ARE FOR RESEARCH USE ONLY. All clinical research must be conducted with oversight from the appropriate Institutional Review Board (IRB). All preclinical research must be conducted with oversight from the appropriate Institutional Animal Care and Use Committee (IACUC) following the guidelines of the Animal Welfare Act (AWA).

 

Citations

[1] Kearbey JD, Gao W, Narayanan R, Fisher SJ, Wu D, Miller DD, Dalton JT. Selective Androgen Receptor Modulator (SARM) treatment prevents bone loss and reduces body fat in ovariectomized rats. Pharm Res. 2007 Feb;24(2):328-35. doi: 10.1007/s11095-006-9152-9. Epub 2006 Oct 25. PMID: 17063395; PMCID: PMC2039878.

[2] Yavuz M, Takanlou LS, Avcı ÇB, Demircan T. A selective androgen receptor modulator, S4, displays robust anti-cancer activity on hepatocellular cancer cells by negatively regulating PI3K/AKT/mTOR signalling pathway. Gene. 2023 Jun 15;869:147390. doi: 10.1016/j.gene.2023.147390. Epub 2023 Mar 27. PMID: 36990257.

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