Targeting Epithelial to Mesenchymal Transition in Breast Cancer with Bioactive Compounds derived from Solanum surattense: A GC-MS and Molecular Docking approach

 

Nagesh Kishan Panchal, Jerine Peter S, Chandrayee Sil, Pratiksha Chhetri, Evan Prince Sabina*

Department of Biomedical Sciences, School of Biosciences and Technology, Vellore Institute of Technology Vellore, India.

 

ABSTRACT:

The epithelial to mesenchymal transition (EMT) has been associated with the invasion and migration of tumor cells, thus playing a fundamental role in cancer progression and metastasis. Breast cancer is a heterogeneous disease, which has been regarded as one of the leading causes of death amongst women globally. The initiation of EMT in breast cancer embraces the involvement of multiple signaling pathways. The involvement of a few factors present in EMT has left a prominent impact on shaping breast cancer development. Solanum surattense, belonging to the family Solanaceae, possesses numerous therapeutic properties due to the presence of several substances. In this study, we focused on major key players genes in promoting EMT, which increases the severity of cancer. Using GC/MS analysis, the active compounds from Solanum surattense were predicted. Molecular docking studies for this extracted active compounds were performed against the genes responsible for EMT using the PatchDock server. The docked complexes were then visualized using PyMOL. Results obtained from the molecular docking showed the inhibitory effect against the EMT pathway, thus inhibiting breast cancer progression.

 

 

1. INTRODUCTION:

Breast cancer is the common cause of cancerous death amongst women globally. It is a heterogeneous disease that is characterized by a high grade of cell plasticity arising from the involvement of a diverse range of factors. However, when we talk about breast cancer, we refer to diverse tumors with different histological forms, molecular variations, presentation, and distinct clinical outcomes¹. According to the most recent classification, breast cancer can be subdivided into 5 categories luminal A, luminal B, HER-2, basal-like, and claudin-low^2–5. Recent progress in understanding the importance of various molecular mechanism underlying breast cancer have clarified the role of various genes, kinases, and transcriptional factors in causing the epithelial to mesenchymal transition (EMT)⁶.

 

The EMT is a complex process where epithelial cells gain a mesenchymal phenotype and motility through a cascade of biological events. EMT is divided into three major types; Type 1: which relates to embryogenesis, gastrulation, and neural crest formation; Type 2: involves tissue regeneration and wound healing; Type3: is associated with malignancy, invasion, and metastasis^7–10. According to a survey, 90% of deaths in breast cancer are due to invasion, and metastasis, which are two major features of EMT¹¹. The EMT and stem cell-like features have been linked with each other and are also associated with gaining therapeutic resistance^12,13. Cancer stem cells have self-renewal and tumor-initiating capabilities, which is one of the important factors involved in the metastasis of breast cancer. Many transcriptional factors play a vital role during the de-differentiation of cancer cells. They are responsible for inducing EMT through transcriptional control of E-cadherin along with SNAIL1/2, ZEB1/2, TWIST1/2, FOXC1/2, TCF3, and various other factors. Amongst them, SNAIL and TWIST are able alone, if activated, to induce an EMT^14,15. Moreover, these genes are overexpressed in EMT, as many studies have been carried out. Plants have been a vital source of medicine for millions of years, 25% of the world's medicines used are today are majorly based on plants. Solanum surattense is one of the potent plant effective against several diseases^16,17. This plant is commonly known as Kantakari as it’s rich in several phenolic, flavonoids, alkaloids, sterols, saponins, glycosides, tannins, amino acids, and carbohydrates. Therefore in this study, we explored the inhibitory response of bioactive compounds derived from Solanum surattense against the EMT causing genes such as TWIST1/2, ZEB1, FOXC2, SNAIL1, and MMP3 using the molecular docking approach.

 

2. MATERIALS AND METHODS:

2.1 Identification and collection of the plant:

The plant Solanum surattense was obtained from the Salt Lake region, Kolkata, and authenticated by Plant Anatomy Research Center Chennai (Reg no: PARC/2020/4282). After authentication, the plant was subjected to air-drying under shade.  Once the drying process was completed, the leaves were ground into a fine powder for further analysis.

 

2.2 Identification of plant  active compounds:

After the fine grounding of Solanum surattense leaves, using mortar and pestle, they were subjected to GC-MS analysis respectively.

 

2.3 Protein 3D Structures:

3D structures of Twist1 (PDB id: 2MJV), Foxc2 (PDB id: 6AKO), Snail1 (PDB id: 3W5K), MMP3 (PDB id: 4G9L), Zeb2 (PDB id: 2DA7) for analysis was retrieved from RCSB PDB database (https://www.rcsb.org/) (Figure 1).    

 

 

2.4 Molecular Modeling of 3D protein structure:

As the 3D structure of TWIST2 was not available, we modeled it using the I-TASSER server (https://zhanglab.ccmb.med.umich.edu/I-TASSER/) as shown in (Figure 1). A quality check for the modeled structure was performed using the Rampage server.

 

 

Figure 1: Protein 3D structures of A) TWIST1, FOXC2, SNAIL1, MMP3, ZEB2; B) Modeled–TWIST2

 

Figure 2: Bioactive compounds derived from Solanum surattense leaves using GC-MS

 

2.5 ADME analysis:

All the active compounds were subjected to Lipinski’s rule of 5 and the ADME check-in SwissADME server. Compounds following Lipinski’s rule of 5 and ADME check were further taken for molecular docking analysis.

 

2.6 Molecular Docking Analysis:

The ligand and receptor were subjected to a Patch-Dock server, which is an online molecular docking server. The results obtained from the server consist of the score, area and atomic contact energy (ACE) of the docked compounds with the highest score were noted. Molecular docking of the complexes obtained from the Patch-Dock server was visualized using PyMol.

 

3. RESULTS:

3.1. Bioactive compounds isolated from Solanum surattense:

Gas chromatography-mass spectroscopy examination of the powdered leaves of Solanum surattense was performed. After GC-MS profiling 39 bioactive compounds were reported from leaves (Figure 2). Active compounds obtained after GCMS analysis were subjected for ADME check to the SwissADME server. Compounds following the ADME check were further considered for molecular docking studies.

 

3.2 Molecular docking analysis:

3.2.1 Molecular docking of ZEB2:

Molecular docking analysis of ZEB2 was performed with active compounds derived from Solanum surattense leaves. Results were obtained after the molecular docking study are shown in (Figure 3) (Table 1).

 

3.2.2 Molecular docking of TWIST1:

Molecular docking analysis of TWIST1 was performed with active compounds derived from Solanum surattense leaves. Results were obtained after the molecular docking study are shown in (Figure 4) (Table 2).

 

2.2.1.         Molecular docking of MMP3:

Molecular docking analysis of MMP3 was performed with active compounds derived from Solanum surattense leaves. Results were obtained after the molecular docking study are shown in (Figure 5) (Table 3)

 

 

Figure 3: Molecular Docking of ZEB2 A) Aspidospermidin-17-ol; B) Tetradecanoic Acid; C) 1,3-Bis-T-butylperoxy-Phthalan;
D) 11-Eicosenoic Acid, Trimethylsilyl Ester; E) 4-Dimethyl(Phenyl)Silyloxypenta; F) 3,6-Methano-8h-1,5,7- Trioxacyclopenta[Ij]Cyclopro;
G) Decanoic Acid, 10-Fluoro-, Trimethylsilyl Ester; H) 2-Methyl-3-Decanol; I) 4-Heptanol, 2-Methyl;
J) Bicyclo[3.2.1]Oct-3-En-2-One, 3,8-Dihydroxy; K) Di-N-Propylmalonic Acid; L) D,L-Xylitol, 1-O-Undec-10-Enoyl;
M) Heptanedioic Acid, Bis(Trimethylsilyl) Ester; N) N-(5-Chloro-2-Hydroxyphenyl)Dodecanamide;
O) 4-Fluoro-1-Methyl-5-Carboxylic Acid, Ethyl(Ester); P) Cyclopropanetetradecanoic Acid, 2-Octyl-,Methyl;
Q) Cyclopentadecanone, Oxime; R) 4- (Trimethylsilylmethyl)Benzoylcyclopentane.

Table 1: Docking Results of ZEB2

 

Figure 4: Molecular Docking of TWIST1 A) Aspidospermidin-17-ol; B) 1,3-Bis-T-butylperoxy-Phthalan; C) Androstane-11,17-Dione, 3-[(Trimethylsilyl)Oxy]; D) 4-Dimethyl(Phenyl)Silyloxypenta; E) 1- Heptyn-4-Ol; F) D,L- Xylitol; G) Emylcamate; H) n-(5-chloro-2-hydroxyphenyl)dodecanamide -; I) 4-Fluoro-1-Methyl-5-Carboxylic Acid, Ethyl(Ester); J) Cyclopentadecanone,oxime; K) 4- Heptanol;

 

Table 2: Docking Results of TWIST1

 

Figure 5: Molecular Docking of MMP3 A) Aspidospermidin-17-ol; B) Tetradecanoic Acid; C) 1,3-Bis-T-butylperoxy-Phthalan; D) Androstane-11,17-Dione,3-[(Trimethylsilyl)Oxy]-, 17; E) 4-Dimethyl(Phenyl)Silyloxypenta; F) 3,6-Methano-8h-1,5,7- Trioxacyclopenta[Ij]Cyclopro; G) 9-Octadecenoic Acid, (2-Phenyl-1,3-Dioxolan-4-Yl)Methyl; H) 2-Methyl-3-Decanol; I) Bicyclo[3.2.1]Oct-3-En-2-One, 3,8-Dihydroxy; J) Di-N-Propylmalonic Acid; K) D,L-Xylitol, 1-O-Undec-10-Enoyl; L) Emylcamate; M) ) N-(5-Chloro-2-Hydroxyphenyl)Dodecanamide; N) 4-Fluoro-1-Methyl-5-Carboxylic Acid, Ethyl(Ester); O) 4- (Trimethylsilylmethyl)Benzoylcyclopentane ; P) 4- Heptanol;

 

 

Figure 6: Molecular Docking Of SNAIL1 A) Aspidospermidin-17-Ol; B) 1,3-Bis-T-Butylperoxy-Phthalan; C) Androstane-11,17-Dione,3-[(Trimethylsilyl)Oxy]-, 17;D) 11-Eicosenoic Acid, Trimethylsilyl Ester; E) 4-Dimethyl(Phenyl)Silyloxypenta; F) 3,6-Methano-8h-1,5,7- Trioxacyclopenta[Ij]Cyclopro; G) 9-Octadecenoic Acid, (2-Phenyl-1,3-Dioxolan-4-Yl)Methyl; H) Decanoicacid,10-Fluoro-,Trimethylsilylester; I) Bicyclo[3.2.1]Oct-3-En-2-One, 3,8-Dihydroxy; J) 1-Heptyn-4-Ol; K) Di-N-Propylmalonic Acid; L) D,L-Xylitol, 1-O-Undec-10-Enoyl; M)  N-(5-Chloro-2-Hydroxyphenyl)Dodecanamide; N) Cyclopentadecanone,Oxime; O) 4- (Trimethylsilylmethyl)Benzoylcyclopentane; P) 4- Heptanol;

3.2.4. Molecular docking of SNAIL1

Molecular docking analysis of SNAIL1 was performed with active compounds derived from Solanum surattense leaves. Results were obtained after the molecular docking study are shown in (Figure 6) (Table 4).

 

Table 3: Docking Results of MMP3

 

Table 4: Docking Results of SNAIL1

 

Figure 7: Molecular Docking of FOXC2 and TWIST2 respectively :: a) A) Tetradecanoic Acid ; B) 1,3-Bis-T-Butylperoxy-Phthalan; C) Androstane-11,17-Dione,3-[(Trimethylsilyl)Oxy]-, 17;D) 11-Eicosenoic Acid, Trimethylsilyl Ester; E) 4-Dimethyl(Phenyl)Silyloxypenta; F) 3,6-Methano-8h-1,5,7- Trioxacyclopenta[Ij]Cyclopro; G) 9-Octadecenoic Acid, (2-Phenyl-1,3-Dioxolan-4-Yl)Methyl; H) Decanoicacid,10-Fluoro-,Trimethylsilylester; I) Bicyclo[3.2.1]Oct-3-En-2-One, 3,8-Dihydroxy; J) 1-Heptyn-4-Ol; K) Di-N-Propylmalonic Acid; L) D,L-Xylitol, 1-O-Undec-10-Enoyl; M) Emylcamate; N) N-(5-Chloro-2-Hydroxyphenyl)Dodecanamide; O) Cyclopentadecanone, Oxime; P) 4- (Trimethylsilylmethyl)Benzoylcyclopentane ; Q) 4- Heptanol :: b) A) 1,3-Bis-T-Butylperoxy-Phthalan; B) 3,6-Methano-8h-1,5,7- Trioxacyclopenta[Ij]Cyclopro; C) 4-Heptanol, 2-Methyl ; D) D,L-Xylitol, 1-O-Undec-10-Enoyl; E) Emylcamate; F) Heptanedioic Acid, Bis(Trimethylsilyl) Ester

Table 5: Docking Results of FOXC2 and TWIST2

 

3.2.5 Molecular docking of FOXC2 and TWIST2:

Molecular docking analysis of FOXC2 and TWIST2 was performed with active compounds derived from Solanum surattense leaves. Results were obtained after molecular docking study are shown in (Figure 7) (Table 5)

 

4. DISCUSSION:

There is growing evidence for plant-derived active compounds as potential inhibitors in several stages of cancers. EMT results in the invasion of cells into the bloodstream^11,12. Following the aforementioned literature related to Solanum surattense leaves extracts has shown promising effects against several diseases^16,18. The Solanum surattense and Solanum nigrum belong to the same genus. Solanum nigrum has demonstrated antitumor effects in various types of cancer, which include breast cancer, cervical cancer colorectal, and several others^18,19. Therefore here we evaluate the anticancer activity of Solanum surattense using GC-MS and molecular docking^20–22. In the present study, the active compounds derived from Solanum surattense were examined against the EMT causing genes, which ultimately leads to the spread of breast cancer. Here, we focused on the major key player of EMT, causing genes like TWIST1/2, ZEB1, FOXC2, SNAIL1, and MMP3. Molecular docking of active compounds such as  Tetradecanoic Acid, 1,3-Bis-T-Butylperoxy-Phthalan, Androstane-11,17-Dione,3-[(Trimethylsilyl)Oxy]-, 17, 11-Eicosenoic Acid, Trimethylsilyl Ester, 4-Dimethyl(Phenyl)Silyloxypenta, 3,6-Methano-8h-1,5,7- Trioxacyclopenta[Ij]Cyclopro, 9-Octadecenoic Acid, (2-Phenyl-1,3-Dioxolan-4-Yl)Methyl, Decanoicacid,10-Fluoro-, Trimethylsilylester, Bicyclo[3.2.1]Oct-3-En-2-One, 3,8-Dihydroxy, 1-Heptyn-4-Ol, Di-N-Propylmalonic Acid,  D, L-Xylitol, 1-O-Undec-10-Enoyl, Emylcamate, N-(5-Chloro-2-Hydroxyphenyl)Dodecanamide, Cyclopentadecanone, Oxime,4-(Trimethylsilylmethyl) Benzoylcyclopentane, 4- Heptanol with the TWIST1/2, ZEB1, FOXC2, SNAIL1, and MMP3 has shown interactions respectively as shown in above figures and tables. The result obtained from molecular docking studies clearly shows that the aforementioned active compounds show interaction with EMT makers and can act as potent inhibitors. Further, in future in-vitro and in-vivo studies might help to evaluate the effective concentration of the compounds by administrating it to an animal model. Therefore the active compounds extracted from Solanum nigrum can serve as one of the better medicinal options in treating EMT in breast cancer.

 

5. CONCLUSION:

Breast cancer is a common type of cancer amongst the female population globally. EMT in breast cancer makes it difficult for the treatment. Although there are many therapeutic options for treating, these results are not satisfactory. The compounds namely 1,3-Bis-t-butyl peroxy-phthalan, 3,6-Methano-8h-1,5,7- trioxacyclopenta, D,L-Xylitol, 1-o-undec-10-enoyl showed high affinity and best interaction with all the receptors TWIST1, TWIST2, FOX C2, SNAIL1, MMP3, and ZEB2 with many hydrogen bonds, which will have a beneficial effect against the EMT pathway and thus can inhibit breast cancer. Therefore from this study, we conclude that these mentioned active compounds and be used as therapeutic options against EMT. Molecular dynamic simulations studies along with in-vitro and in-vivo studies need to be performed for further development and understanding.

 

6. ACKNOWLEDGMENT:

We would like to thank VIT for giving us the opportunity to work and giving us all the necessary resources to successfully carry out this work.

 

7. DISCLOSURE:

Authors declare that they do not have any conflicts of interest.

 

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Accepted on 23.11.2021           © RJPT All right reserved

Research J. Pharm. and Tech 2022; 15(12):5637-5644.