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	�:	Journal of Molecular Biomarkers & Diagnosis
Short name: J Mol Biomark Diagn
Volume 11: 6, 2020	
The Effects of Thymoquinone on Inhibiting the Expression of SENP1 Gene in the MCF-7 Cell Line
Neda Shaghaghi1, Farzaneh Sabouni2*, Reza Mohammadzadeh1*, Maryam Kheyrollah2
�Cell and Molecular Biology Department, Faculty of basic Science, University of Maragheh, Iran
2National Institute of Genetic Engineering and Biotechnology, Tehran, Iran
Abstract
SUMOylation, as post-translational modifications, plays essential roles in various biological functions including cell growth and migration, stress response, and tumorigenesis. In SUMOylation, SENP1 catalyzes the SUMO protein maturation to combine with target proteins. Breast cancer is a common malignancy in women and also SENP1 progression is high in this cancer. Thymoquinone is a biologically active substance and a secondary metabolite found in the black seed, and recent researches indicate antioxidant, anti-inflammatory, anti-cancer and other important biological activities. The effects and mechanisms of Thymoquinone on SENP1 are not well studied. For this purpose, the first MCF-7 tumor cell line and normal MCF-10A cell line were cultured in normal conditions and then treated with specific doses of thymoquinone. The lethal activity was evaluated by MTT assay and exhibited that the toxicity of thymoquinone on MCF-7 cell line was higher than healthy cell and the intensity of its effect was different from MCF-10A cell. Then SENP1 gene expressions were measured. Gene expression changes in tumor cells were then compared with normal cells and it was found that thymoquinone was able to reduce SENP1 gene expression in the tumor cells.  Then docking of Thymoquinone with SENP1 protein was performed. The amount of binding energy between Thymoquinone and protein SENP1 is -54 and with dimer of Thymoquinone is -80.  Based on what researchers have concluded in this study, it is possible that  the mentioned gene and SENP1 Protease can be considered as a candidate for breast cancer treatment and drug target.
Keywords: SUMOylation � Breast Cancer � Black Seed � Cysteine Protease � SUMO
*Address for Correspondence: Sabouni F, National Institute of Genetic Engineering and Biotechnology, Tehran, Iran, E-mail: sabouni@nigeb.ac.ir
Mohammadzadeh R, Cell and Molecular Biology Department, Faculty of basic Science, University of Maragheh, Iran, E-mail: rmohamadzadeh82@gmail.com
Copyright: � 2020 Shaghaghi N, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Received: 21 July, 2020; Accepted: 16 October, 2020; Published: 23 October, 2020
Introduction
Post Translation Modification (PTM) is widely observed in the regulation of protein activity. Proteins can be altered by small chemical groups, sugars, lipids and even by covalent binding to another polypeptide. The most well-known example is the ubiquitin polypeptide modifier. The role of ubiquitin is to target the protein to the proteasome and its proteolytic digestion. In addition to ubiquitin, there are several other ubiquitin-like proteins (UbLs) that can bind to the protein substrate and alter its function. SUMOylation, as post-translational modification, plays key role in various biological functions including cell growth and migration, stress response, and tumorigenesis. The imbalance of SUMOylation and deSUMOylation were associated with the occurrence and progression of various diseases. In Compared with ubiquitin, although SUMOylization does not promote protein digestion, it modifies proteins against some functional parameters, depending on the protein type. Some of these parameters includes intracellular protein translocation, gene expression, chromatin structure, message transfer, genome stability, DNA binding or alteration of transcription factor activity and some other activities. It is likely that the effects of ubiquitination and SUMOylation are largely due to the binding of proteins and the position of their interacting domains. Ubiquinitization and SUMOylation changes are both reversible [1]. SUMO (Small Ubiquitin-like Modifier) is a small protein with 97 amino acids that is structurally similar to ubiquitin and is also known as Smt3p, Pmt2p, PIC-1, GMP1, Ubl1 and Sentrin [2]. Sumo, like ubiquitin, has been shown to bind covalently to some of the lysine subunits in protein targets [3]. The SUMO family is a PTM protected form, in all eukaryotes. There is only one SUMO gene in germinated yeast, while there are at least 8 types of SUMO in plants. The SUMO protein in mammalian cells has four types: SUMO-1, SUMO-2, SUMO-3 and SUMO-4. SUMO-2 has 95% of homology to SUMO-3 .SUMO-2 and SUMO-3 differ by only three residues at the N-terminus and have approximately 45% homology to SUMO-1. SUMO-4 has the lowest SUMO family feature. SUMO-4 is probably unbound in physiological conditions [4]. Similar to ubiquitination, SUMOyaltion involves series of enzymatic processes. The mature SUMO is activated by conjugation to the E1 enzyme (SAE1/SAE2), transferred to the E2 enzyme (Ubc9) and ligated to the specific lysine residue of the target proteins by an E3 enzyme [5]. Members of the Ulp / SENP family are the Ulp1 and Ulp2 proteins of Saccharomyces cerevisiae initially identified by Li and Hochstrasser [6]. SENPs are enzymes of the maturation and decongestion of SUMO. In addition to protected catalytic domains, SENP proteases have specific N-terminal domains, SENPs have an enzymatic dual function for immature SUMO maturation and SUMO deconjugation. In deconjugation reaction, SENP separates an isopeptide bond that transfers SUMO fragments into the �-amino group in lysine [4].The human protease (Sentinel-specific protease 1) has 643 amino acids and 73 kDa weighing . Its gene is located on the 12th human chromosome. It is a super family of cysteine proteases. Cysteine is at position 602, histidine at position 533 and aspartic acid at position 550. Cysteine is at the alpha-helix N terminus of the protein nucleus with two other amino acids, aspartate and histidine are at the end of beta [7]. SENP1 is found in both nucleus and the cytosol depending on the cell type, which is seen to be transmitted from the nucleus to the cytosol via the NES located at the C terminus. SENP1 is predominantly located in the nucleus in mammals. SENP1 Catalyzes the maturation of the SUMO Protein (Small Ibbokitin Reducer), which Hydrolyzes the SUMO peptide bond in the G-Gly- Conserved Sequence | -Ala-Thr-Tyr becomes terminal at C [8].
Thymoquinone (TQ) is a 164.2 g/mol phytochemical substance extracted from Nigella sativa and its different effects has been shown, such as analgesic, anticancer  and antioxidation in the heart [9]. TQ is used to treat a variety of cancers such as brain, breast, colon, liver, lung, and prostate cancers [10]. Thymoquinone is an anti-neoplasic agent reported to have the therapeutic potential in the treatment of breast cancer. The anti tumour activity of thymoquinone has been reported in cells, derived from ovarian, breast and colon cancers. The apoptotic activity of thymoquinone has been reported to induce the total Bax / Bcl-2 ratio in MCF7, HCT-116 and HL-60 cancer cells. These findings were based on flow cytometry, western blot analysis and multi-color fluorescence in situ hybridization (mFISH). Therefore, further investigation is needed to determine its effects on human genome expression using cDNA microarray technology [11].
Docking of the small molecular constituents at the receptor binding site and the affinity estimation of this complex are an important part of the structure-based drug design process. For a complete understanding of the structural principles that determine the strength of a protein / ligand complex, a fast and accurate binding protocolH the ability of visualize binding and interaction geometries are required [12]. There are three basic tasks any docking procedure must accomplish: (1) characterization of the binding site; (2) positioning of the ligand into the binding site (orientation); and (3) evaluating the interaction strength for a specific ligand-receptor complex (�scoring�) [13]. 
Materials and Methods
Thymoquinone solution preparation
The 1 mM stock solution of thymoquinone (Sigma-Aldrich, Saint-Quentin-Fallavier, France) was prepared with DMSO (Sigma-Aldrich).
Cell culture and treatment
We received the breast cancer cell line MCF7. We also used the normal breast cell line, MCF10 as a non-cancer control. All cell lines were cultured in Dulbecco�s Modified Eagle�s Medium (DMEM, ThermoFisher Scientific PN: 11965118) supplemented with 10% Fetal Bovine Serum (Atlanta Biologicals PN: S11150), grown in a monolayer at 37 �C and 5% CO2. Cells were passaged one to two times per week, or until cells reached approximately 80% confluence. The MCF7 cells were seeded at 10000 cells/well in 96-well plates. They were cultured at 0.5% CO2 in a humidified incubator at 37 �C.
MTT assay
Determination of the thymoquinone dose and MTT implementation test: Cells were first treated with thymoquinone: 1) 200 �l of solution (DMEM + FBS +cell) was added to each plate. 2) 24-hour incubation. 3) The solution was removed from the plates. 4) Add TQ in each plate with the concentrations of 20/5/0 �M. 5)24-hour incubation. After performing the MTT test the absorbance was measured in 570 nm.
RT PCR (RT-PCR)
All cell lines were seeded at 500000 cells/well in a 6-well dish and grown at 37 �C and 5% CO2 for approximately 24 hours. RNA was isolated from the MCF7 and MCF10 cells using the RNX Plus Mini kit (Qiagen, Valencia, CA, USA) according to the manufacturer�s instructions. The quantity of RNA was measured using a spectrophotometer (NanoDrop 7*000c; Thermo Scientific). Samples with the RNA concentration (A260/A280 e"1.8 ng/�l) and purity (A230/A260 e"2.0 ng/�l) were selected. cDNA was generated using the cDNA Synthesis Kit (iNTRON) using 20ng of RNA, d(T)23 VN primers and following the recommended protocol (Table 1).
Bioinformatics
Setup and execution of docking runs: The human SENP1 protein structure was downloaded from the PDB site and also TQ structure was downloaded from the pubCHEM site. The MVD software was used for performing the docking process. MVD need receptor and ligand representations with a file format called pdb which is a modified protein data bank format containing atomic charges, atom type definitions and, for ligands, topological information (rotatable bonds). These file preparations are carried out by the plugin using scripts from the MVD Tools package. Ligands for subsequent docking runs can either be prepared one by one through MVD selections. After binding site definition and the preparation of receptor and ligand, docking runs can be directly launched from MVD. In this study, the specific molecular docking software (MVD) was used to investigate the molecular interaction between drugs and protease enzyme. This software provides a three-dimensional (3D) view of the interaction between compounds and the protease enzyme virus and the amino acids participating in the interaction. In the present study, all docking conditions including the interaction frequency, the study area of interaction, the protease enzyme and the rate of docking were considered to minimize error. During this molecular docking study, the number of interaction run is 10, the diameter of interaction area 30 (angstrom) with the ability to investigate the hydrogen- electrostatic and Van der Waals interactions in the total active site of the enzyme and the results were compared.
Results
MTT Assay Result
Thymoquinone has been reported to suppress cell proliferation in many types of cancers, but its effect on healthy cells and target SENP1 have not been clearly studied. To determine the anti-tumor activity of Thymoquinone on MCF7 cells and compare with MCF10, the researcher carried out growth curve assay and cell viability assay using two cell lines. According to the repeated MTT assay, the effect of thymoquinone at various concentrations on MCF-7 cell line showed that the IC50 value of thymoquinone is 20 �M (p <0.05) (Table 2). The following table shows the effect of thymoquinone on different concentrations on MCF- 10A cell line, indicating that the toxicity of thymoquinone is less than cancer cells (p <0.05) (Table 3) (Figure 1-3).
Results of RT PCR
In order to analyze the expression of SENP1 gene and �-actin gene, gel electrophoresis was examined by image J software and the following results were obtained .The area under each peak indicates the amount of gene expression (Figure 4). According to PCR data, gene expression decreased with increasing dose of thymoquinone in MCF7 cells (Figure 5). Also, the expression level of SENP1 gene was significantly increased in tumor cell lines compared to the normal cells. The aforementioned results have been obtained in previous studies. 
Docking Result
Docking of Thymoquinone with SENP1 protein was done by Molegro Virtual Docker software and the result showed that Thymoquinone interacts with the SENP1 protein and the best binding energy of the various conformations with protein was -54 and with Dimer of Thymoquinone is -80 (Figure 6). In docking of the Thymoquinone with the SENP1 protein, two types of hydrogen bond and ester bonds are formed.
The results showed that the site of interaction of the compounds was in the conserved region of the enzymatic flap. Regarding the binding energy among these compounds the binding of 5 important amino acids due to their presence in the highly conserved region has an active site and a key role in enzymatic catalysis. These amino acids are Gly531, Leu530, His529, Phe 496, Thr 499. The results of these compounds are summarized in Tables 4-6.
Discussion
Breast cancer is the most common invasive cancer in women and the second leading cause of death in women after lung cancer. Metastatic cancers, such as breast cancer, are usually resistant to chemotherapy, so researchers are finding new and effective compounds to treat these cancers. SUMOylation regulates essential cellular processes, many of which are often misregulated in human cancers. As the SUMO pathway is also misregulated in numerous cancers, it has been implicated as a contributing factor in the development and progression of these diseases [14,15]. Researchers have found that expression levels of individual SUMO pathway enzyme can be used as prognostic markers for cancers like prostate and cervical cancer [15,16].
In this study the expression of SENP1 gene in normal and tumor cell lines was studied and the effect of Thymoquinone on SENP1 gene expression inhibition was investigated. This gene has been shown alter expression patterns in many cancers. There have been many studies demonstrating the effective role of the SENP1 gene in tumor promotion in all types of cancers. SENPs are often expressed in a variety of cancers, such as prostate cancer, ovarian and breast cancer, and they reduce the effects of chemotherapy. Therefore, SENPs may be effective targets for new cancer therapies. A new study showed that SENP1-shRNA significantly downregulates SENP1 expression in lymphoma cell lines and induces cell apoptosis. This indicates that SENP1 is a potential aim of targeted gene therapy [17]. Qiao and partners developed a group of small SENP-based benzodiazepine inhibitors and showed that they effectively inhibited the proliferation of prostate cancer cells [18]. In addition, Uno et al. affected a specific non-peptide SENP1 antagonist, GN6958, on cervical cancer cell lines [19]. In 2016, Wu and colleagues identified a new SENP1 inhibitor, Momordin IC. Momordin IC is a natural triterpenoid tropenoid, and it reduces SENP1 expression and stability [20]. As the studies indicate many of the published data on SENP proteases are based on overexpression of SENP family members. Most of the studies of SENP deactivation was based on the use of siRNA or CRISPR, making it difficult to distinguish between direct and indirect effects of SENP deactivation [15]. Currently reported SENP inhibitors have low activity and specificity for the SENP subgroup [21]. The availability of chemical inhibitors is also a prerequisite for targeting SENPs in human diseases, including cancer, where high expression of SENPs has been observed [15].
Despite significant advances in the production of synthetic drugs, medicinal plants are still considered as one of the important strategies for the treatment of cancer. People usually choose natural antioxidants since they believe that herbal remedies have no significant side effects. Due to the growing concern about the side effects of chemical drugs and the ineffectiveness of a number of them for long-term usage, the use of natural alternatives or complementary remedies has become increasingly accepted [22]. Nigella sativa is a flavoring medicinal plant widely used as a supplement and also as a treatment for many disorders. Experimental studies have shown the beneficial and mechanistic effects of thymoquinone action against many diseases. Thymoquinone is an active ingredient and a phytochemical compound found in the canola plant. For the anticancer impacts of thymoquinone, various mechanisms such as effect on free radicals, affect on enzyme activity, inhibition of cell proliferation, antioxidant activity, and induction of apoptosis in cancer cells have been reported [23,24].
Several recent studies have also revealed the impresses of thymoquinone on MCF7 cell lines in different molecular pathways:
Interpretation of the analysis of the experiments showed that the cytochrome P450 gene and the UDP glucuronosyl transferase were significantly up-regulated in the estrogen metabolic pathway. The anionic amino acid light chain transporter gene was up-regulated 15-fold in the interferon pathway, which has been reported to be involved in the development of chemotherapy. Interferon-induced protein with tetra-tricyclic peptide repeats, G1P3 protein, interferon regulatory factor 9 (IRF9), (ISGF3, 2'-5'-oligoddenylate synthetase 1, OAS1, and signal transducer and activator of STAT1 transcription genes all after treatment Caspase-10 gene, activated by cysteine peptidase apoptosis, and tyrosine phosphatase and myocyte-factor enhancer protein were up-regulated in MAPK and p38 MAPK pathways, suggesting that thymoquinone is involved in metabolic pathways. Estrogen and interferon target specific genes [25]. In another finding, thymoquinone was used as a radiosensitizer to evaluate its ability to migrate and invade in the irradiated MCF7 cell lines by evaluating the relevant properties. This study confirms the ability of thymoquinone to repair TGF-� in radiation migration and invasion. Moreover, the results indicated that epithelial markers of E-cadherin and cytokeratin 19 were up-regulated [26]. In another study, the ability of thymoquinone to express p53 tumor suppressor gene and induce apoptosis in MCF-7 breast cancer cell line was reported. Thymoquinone can increase p53 expression in MCF7 cell line by regulating time-dependent regulation and induce apoptosis in MCF-7 cells [27]. Another study was undertaken to investigate the combined effect of thymoquinone and tamoxifen on the survival and apoptosis of human breast cancer cell lines. The consequences revealed that within 24 hours of treatment, tamoxifen and all doses of thymoquinone, alone or in combination, significantly reduced the viability of both MCF7 and MDA-MB-231 cells [28].
Conclusion
The outcomes of this assay represented that the toxicity of Thymoquinone on MCF-7 cell line was higher than those of healthy cells and had a selective effect. Evaluation of alteration and diminution of SENP1 gene expression by Thymoquinone in breast cancer cell line exhibited that Thymoquinone has inhibitory efficacy on SENP1 gene. Furthermore according to docking results, the amount of free energy of the bond between Thymoquinone and protein SENP1 is -54 and with dimer of Thymoquinone is -80. Based on the results of the Docking, it can be concluded that the TQ and its dimer, can interfere with the important amino acids in the enzymatic cavity to prevent the SENP1 protease. Therefore, the inhibition of SENP1 gene and protein with Thymoquinone can be considered as a therapeutic target for the treatment of the breast cancer.
Acknowledgments
This study was supported by National Institute of Genetic Engineering and Biotechnology, Tehran, Iran.
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