Sesamol and its derivative investigated as antiandrogen - A potential prevention to prostate cancer in rats

Androgen signaling is essential for the development of prostate cancer (PCa) initiated from prostatic basal cells with collocation of androgen receptor gene mutations. Phytoestrogens, the naturally occurring compounds are AR antagonist. These compounds downregulate prostate-specific antigen (PSA) expression and cell proliferation. Thus, this gives a track to research these compounds as a possible treatment for PCa. In this work, STITCH and molecular docking predict the conformation of ligands inside the suitable target binding site. Therefore, a study was planned to know the interactions among SM and its derivatives with AR. It was further, evaluated for in vitro evaluation on LNCaP, PC-3, and DU-145 using MTT studies. The two lead compounds shortlisted from MTT studies were further analyzed for androgen-regulated genes by using RT-PCR, western blot studies and an animal model of prostate cancer. We found that SM and its derivative (3’-MA) may prevent the development of PCa by androgen pathway.


Introduction
Prostate cancer (PCa) is the second leading malignancy amongst men worldwide. Androgen signaling is the driving force for the progression of PCa. [1] [2][3] AR regulates multiple cellular events, proliferation, apoptosis, migration, invasion, and differentiation. [4] [5] According to the latest finding by Yongfeng he (2018), androgen signaling is essential for the development of PCa initiated from prostatic basal cells [6] Collocation of androgen receptor gene mutations in PCa. [7] Thus, AR is the primary drug target for PCa treatment. The androgens are key players for the growth of PCa in the initial stages, which leads to a treatment choice as androgen deprivation therapy (ADT). The AR is essential in all stages of PCa progression, it may be a relapse of the tumor, which can be even androgen-independent in nature.
[8] The oral dosage form of antiandrogens are freely available and based on chemical structure, it can be classed as steroidal and non-steroidal. Cyproterone acetate being steroidal and bicalutamide, flutamide, and nilutamide are non-steroidal. [9] Although ADT is a highly effective treatment option, it is associated with considerable side effects. It includes hot flashes, insulin resistance, cardiovascular (CV) diseases, abridged body mass and muscle strength, others relating to bones osteoporosis and major concerns maybe depression and sexual dysfunction. [10] In this context, there is a need for investigating natural antiandrogens to alleviate adverse effects arising from ADT and subsequently enhance the patient's quality of life. The examples of natural antiandrogens are Mahanine, 2′-hydroxy genistein-7-O-glucoside, 3, 3′-Diindolylmethane, N-butyl benzene sulfonamide etc. [11] Phytoestrogens, the naturally occurring compounds are AR antagonist. These compounds downregulate the activation of PSA and cell proliferation. Thus, this gives a track to research these compounds as a possible treatment for PCa. [12] [13] 4 Sesamolin is also a precursor to sesamol and SM is formed in frying procedures. [14] Sesame seed is a rich source of dietary lignans. [15] Lignan (sesamol, sesamin, and sesamolin) profile was identified in sesame seeds. [16][17] SM is the main reason for the oxidative stability of sesame seed oil. [18] Pianjing P, et al. investigated the phytoestrogenic potential of Sesame Lignans and their metabolites on human breast cancer cells. [19] In the present study, we claimed and tested the SM and its derivatives to be used as a prophylactic treatment for prostate cancer.

STITCH
Understanding the interactions between SM and androgen-regulated genes is essential to predict the molecular mechanism for the androgen signaling pathway. The online database tool "STITCH ('search tool for interactions of chemicals') was used to understand the interaction of Sesamol and associated genes as androgen receptor gene (AR), FKBP5 (FKBP Prolyl Isomerase 5), Prostate-specific antigen (PSA/KLK3), Transmembrane Serine Protease 2 (TMPRSS2) was used (http://stitch.embl.de) [17]. The input and predicted functional partners are shown in table 2 and table 3 respectively.

Ligand preparation
The lowest energy 3D structures with corrected chiralities were produced by ligand optimization using the tool LigPrep tool. OPLS 2005 force field used and the process performed at neutral pH.

Protein preparation and grid generation
Before the docking study, the biological unit of protein subjected to the protein preparation where it added with missing side chains and missing residues by the Prime tool. Further refining of the protein done by heavy atom and water molecule removal in restrained 5 optimization. After the protein preparation, the receptor grid was generated using the OPLS 2005. The cubic box with specific dimensions generated around the centroid of the active site.

Ligand Docking
The standard precision (SP) and extra precision (XP) flexible glide docking were used to screen the analogs, by using the Glide tool. For ligand atoms, Van der Waals factor and partial charge cut off were selected to be 0.80 and 0.15 respectively and ligands that are optimized used for this purpose.

Quantitative Polymerase Chain Reaction (q-PCR) analysis
RNA expression studies of these lead compounds were further evaluated by q-PCR for the androgen signaling pathway. Androgen signaling genes like AR, FKBP5, PSA, and Primers used in the study were as follows:

In-vivo Model
Testosterone undecanoate (TU) (Cernos Depot 1Gm Injection) was purchased from Sun Pharmaceutical. Sesamol and 3′,4′-(Methylenedioxy) acetophenone were purchased from Sigma-Aldrich. All the analytical grade chemicals were procured from Merck Limited, India and Hi-media. Plastic labware and syringes were procured from tarson and BD biosciences respectively. Rat PSA Prostate Specific Antigen (PSA) ELISA Kit (Catalog number: E-EL-R0796) is purchased from Elabscience, Houston, USA.

Animals grouping and experimental design:
The experimental design along with the grouping of animals are described in table 1.

Histopathological examination
The prostate tissues were fixed and embedded in paraffin wax followed by sectioning. The thin sections were stained using hematoxylin and eosin. The stained sections were mounted on a glass slide and are observed under an inverted microscope and images were taken.

Determination of antioxidant parameters
Antioxidant assays of ventral prostate homogenate {Catalase, Lipid Peroxidation, Nitrite, GSH} were screened. [19] 2.4.6 Prostate homogenate preparation: Ventral prostate tissue was homogenized in ice- color turns yellow and is measured at 450 nm ± 2 nm optical density (OD). The concentration of rat PSA in the samples was calculated from the standard calibration curve of PSA.

Statistical Analysis
The data were analyzed statistically and expressed as the mean ± SEM(n=6/3). Groups were compared using ANOVA followed by Tukey's test for multiple comparisons. The level of significance set at p < 0.05.

Stitch analysis
The network shows the correlation (Figure 2.I) of sesamol and its interactions with androgen receptor-regulated genes. Sesamol is lignan in nature; may shows phytoestrogen activity.
Sesamol interacts with the Androgen receptor and regulates the androgen-regulated genes.
The interaction of sesamol with AR genes, PSA (KLK3), TMPRSS2 indicates the possibility of the androgen-mediated pathway.

Schrodinger based Molecular docking for SM, and its derivatives
For the rational structure-based drug design, Sesamol and its eight different derivatives were investigated their binding affinity against AR. The human androgen receptor was downloaded from the Protein Data Bank using PDBID -1E3G and 2AM9 respectively and docked using the Maestro Molecular Modelling platform by Schrodinger, LLC. The Dock score is represented in table 4 with a comparison of androgen receptors type.

Cytotoxicity Assay:
The compounds SM and 3' MA showed the least IC 50 (mM) as 3.94 and 4.43 (Figure 2.II) respectively on the other hand rest compounds were found to have very high IC 50 values (Table 5).

0
The IC 50 (mM) in the androgen negative cell line was found to be more than 10 mM in PC-3 and DU-145 for the nine compounds. The values obtained indicated the drugs may be acting through the androgen signaling pathway so the androgen-independent cell lines were unaffected. The Vero cell lines, normal cells were also treated with these compounds and found to be safe in this assay; IC 50 was more than 15 mM in each case.

qPCR Studies:
The experiment showed positive results as the expression was reduced by the SM and 3' MA in all the genes at a higher dose. PSA notably was reduced by both in dose-dependent manners ( Figure 2.III and Figure 2.IV). Thus, this confirmed the activity as anti-androgenic at the RNA level, and further protein expressions were confirmed by western blotting.

Western blot analysis
The protein estimations of PSA in both cases were found to be dose-dependent inhibition in figure 2.V.A and 2.V.B.

Prostate Weight
Animals treated with TU and MNU (disease control group) showed a significant increase in prostate weight of 78.68% compared to the normal control group.
In comparison with the disease control group, SM treatment (50 and 100 mg/kg/day, concomitantly administered with TU and MNU) significantly decreased the prostate weight by 25.14 % and 32.93%. Similarly, in the case of 3' MA, at the dose of 50 and 100 mg/kg, there is a significant decrease in prostate weight as 31.43% and 57.44% which is comparable to the standard drug, finasteride (% reduction is 60.65) (Figure 3.I) 1 1 Effect of SM and 3'MA treatment in two different doses (50 and 100 mg/kg, orally) onantioxidant markers as nitrite, lipid peroxidation, catalase, and GSH level were evaluated and quantified as mentioned in Table 6.

Effect of SM and 3'MA treatment on antioxidant status of ventral prostate
Catalase and glutathione (GSH) level enhanced significantly in SM and 3'MA treatment group in a dose-dependent manner. While Nitrite and MDA level decreased in SM and 3'MA treated groups signifies the antioxidant nature. This antioxidant potential may help in reducing prostate cancer disease progression.

Prostate Specific Antigen Estimation
The measurement of prostate-specific antigen in serum is an early detection marker in PCa. A significant decrease in PSA level (Figure 3.II) signifies, the influence of androgen signaling mediated pathway in SM and 3'MA treated groups.

Histopathology -Prostate
The histopathological evaluation indicated marked changes in the prostatic histoarchitecture in SM and 3'MA treated groups and indicated protection in PCa (Figure 3.III).

Conclusion
Results from the studies reveal that SM and 3'MA could be hopeful molecules for further assessment for PCa treatment option for preventive therapy. In the present investigation, sesamol and its derivative (3'-MA) prevented the development of PCa by interfering androgen-regulated pathway.